FENS 2020 VIRTUAL FORUM
MITOCHONDRIA-NUCLEUS CROSSTALK TO RESCUE AGEING-IMPAIRED NEUROGENESIS
DEVELOPMENT AND STEM CELLS
Sónia Sá Santos, Beatriz Estremores, Márcia Costa, João Moreira, Cecília Rodrigues, Susana Solá
Introduction: The decline in adult neurogenesis throughout life contributes to ageing-associated cognitive deficits and neurodegeneration. Mitochondria bioenergetics and oxidative stress are suggested to significantly impact neural stem cells (NSCs) fate. The mitochondrial deacetylase Sirtuin 3 (SIRT3) is a central player in mitochondrial metabolism and oxidative protection. Aim: To further dissect how SIRT3 regulates NSC ageing. Methods: Chronic ageing in mouse NS cell lines was induced with tert-butyl hydroperoxide (tBHP). NSC viability, late senescence (SA-β-GAL), telomerase activity and expression of stemness and differentiation markers (qRT-PCR) were evaluated. SIRT3 and the mitochondrial fatty acid oxidation enzyme, long-chain acyl-CoA dehydrogenase (LCAD) were modulated through overexpression plasmid and siRNA transfection, respectively. SIRT3-mediated deacetylation of LCAD and superoxide dismutase 2 (SOD2), a mtROS-detoxifying enzyme, were assessed by immunoprecipitation and western blot assays. Results: tBHP treatment decreased NSC viability, differentiation and proliferation potentials, and increased senescence. Importantly, SIRT3 overexpression revert changes in differentiation and stemness potential of tBHP-treated NSCs. SIRT3-induced changes in aged NSCs occurred concomitantly with increased activation of SOD2 and telomerase reverse transcriptase, a key catalytic subunit of the anti-ageing enzyme telomerase. Interestingly, LCAD silencing was shown to decrease NSC differentiation potential. Finally, SIRT3 physically interacts with LCAD, especially when the former is upregulated, indicating LCAD as a SIRT3 target in aged NSCs. Conclusions: Our work reinforces the role of mitochondrial activity in regulating age-related NSC fate outcomes, suggesting that targeting mitochondrial oxidative state and metabolism could be a promising strategy to arrest neurogenesis decline and cognitive dysfunction during ageing.
26858637: Sá Santos S, Santos SM, Pinto AR, Ramu VG, Heras M, Bardaji E, Tavares I, Castanho MA
Amidated and Ibuprofen-Conjugated Kyotorphins Promote Neuronal Rescue and Memory Recovery in Cerebral Hypoperfusion Dementia Model.
Chronic brain ischemia is a prominent risk factor for neurological dysfunction and progression for dementias, including Alzheimer’s disease (AD). In rats, permanent bilateral common carotid artery occlusion (2VO) causes a progressive neurodegeneration in the hippocampus, learning deficits and memory loss as it occurs in AD. Kyotorphin (KTP) is an endogenous antinociceptive dipeptide whose role as neuromodulator/neuroprotector has been suggested. Recently, we designed two analgesic KTP-derivatives, KTP-amide (KTP-NH2) and KTP-NH2 linked to ibuprofen (IbKTP-NH2) to improve KTP brain targeting. This study investigated the effects of KTP-derivatives on cognitive/behavioral functions (motor/spatial memory/nociception) and hippocampal pathology of female rats in chronic cerebral hypoperfusion (2VO-rat model). 2VO-animals were treated with KTP-NH2 or IbKTP-NH2 for 7 days at weeks 2 and 5 post-surgery. After behavioral testing (week 6), coronal sections of hippocampus were H&E-stained or immunolabeled for the cellular markers GFAP (astrocytes) and NFL (neurons). Our findings show that KTP-derivatives, mainly IbKTP-NH2, enhanced cognitive impairment of 2VO-animals and prevented neuronal damage in hippocampal CA1 subfield, suggesting their potential usefulness for the treatment of dementia.
Front Aging Neurosci, 2016; 8
28127286: Perazzo J, Castanho MA, Sá Santos S
Pharmacological Potential of the Endogenous Dipeptide Kyotorphin and Selected Derivatives.
The endogenous peptide kyotorphin (KTP) has been extensively studied since it was discovered in 1979. The dipeptide is distributed unevenly over the brain but the majority is concentrated in the cerebral cortex. The putative KTP receptor has not been identified yet. As many other neuropeptides, KTP clearance is mediated by extracellular peptidases and peptide transporters. From the wide spectrum of biological activity of KTP, analgesia was by far the most studied. The mechanism of action is still unclear, but researchers agree that KTP induces Met-enkephalins release. More recently, KTP was proposed as biomarker of Alzheimer disease. Despite all that, KTP limited pharmacological value prompted researchers to develop derivatives more lipophilic and therefore more prone to cross the blood-brain barrier (BBB), and also more resistant to enzymatic degradation. Conjugation of KTP with functional molecules, such as ibuprofen, generated a new class of compounds with additional biological properties. Moreover, the safety profile of these derivatives compared to opioids and their efficacy as neuroprotective agents greatly increases their pharmacological value.
Front Pharmacol, 2016; 7
MOLECULAR AND CELLULAR CHARACTERIZATION OF THE LATE RADIAL GLIA CELLS AND THE EFFECT OF VITAMIN C ON THEIR DIFFERENTIATION PROCESS
Natalia Saldivia1, Katterine Salazar1, Adriana Tienda2, Fiona E Harrison2, Francisco Nualart1
1Chile , 2United States of America
Introduction. The main stem cell in cerebral cortex development is the radial glia cell (RGc). RGcs remain during postnatal (PN) development, termed later RGcs (lRGcs). Few studies have analyzed the molecules that regulate the lRGc function. Here, we have analyzed the (i) proteins present in lRGcs, (ii) distribution/expression of vitamin C (Vc) transporters (SVCT2 and GLUT1), (iii) in vivo formation of neurons from lRGc and (iv) effects of SVCT2 overexpression. Materials-Methods. PN1-20 lRGcs from wild-type and SVCT2+/- mice were analyzed by immunohistochemistry. Scanning electron microscopy was used for morphological analysis. SVCT2 distribution and expression in the cerebral cortex was analyzed by laser microdissection and qrt-PCR. GFP-adenoviral labeling was used to define normal lRGc distribution. PN1 rats were electroporated with a GFP-SVCT2 plasmid, and lRGcs were analyzed in subsequent days. Results. lRGcs are highly present in the first postnatal week, exhibiting a polarized morphology, both in wild-type and in SVCT2 +/- brains. SVCT2 expression decreased as development proceeds and the expression of GLUT1 begins to appear. Migratory neurons are present after lRGc-GFP-adenovirus labeling. Remarkably, SVCT2-GFP overexpression generates cells with rounded morphology that migrate from the ventricular zone and are positive for neuronal markers. Discussion. Vc, through SVCT2, may regulate genes expression that modulate pluripotency and differentiation mechanisms in stem cells (e.g., Nanog). Thus, it may promote the neuronal differentiation of lRGcs. Support: FONDECYT 1190848, FONDECYT 1181243 and CMA BIOBIO PIA-Conicyt ECM-12, UCO1866, JM I01 CX001610.
31594969: Ulloa V, Saldivia N, Ferrada L, Salazar K, Martínez F, Silva-Alvarez C, Magdalena R, Oviedo MJ, Montecinos H, Torres-Vergara P, Cifuentes M, Nualart F
Basal Sodium-Dependent Vitamin C Transporter 2 polarization in choroid plexus explant cells in normal or scorbutic conditions.
Vitamin C is incorporated into the cerebrospinal fluid (CSF) through choroid plexus cells. While the transfer of vitamin C from the blood to the brain has been studied functionally, the vitamin C transporter, SVCT2, has not been detected in the basolateral membrane of choroid plexus cells. Furthermore, it is unknown how its expression is induced in the developing brain and modulated in scurvy conditions. We concluded that SVCT2 is intensely expressed in the second half of embryonic brain development and postnatal stages. In postnatal and adult brain, SVCT2 is highly expressed in all choroidal plexus epithelial cells, shown by colocalization with GLUT1 in the basolateral membranes and without MCT1 colocalization, which is expressed in the apical membrane. We confirmed that choroid plexus explant cells (in vitro) form a sealed epithelial structure, which polarized basolaterally, endogenous or overexpressed SVCT2. These results are reproduced in vivo by injecting hSVCT2wt-EYFP lentivirus into the CSF. Overexpressed SVCT2 incorporates AA (intraperitoneally injected) from the blood to the CSF. Finally, we observed in Guinea pig brain under scorbutic condition, that normal distribution of SVCT2 in choroid plexus may be regulated by peripheral concentrations of vitamin C. Additionally, we observed that SVCT2 polarization also depends on the metabolic stage of the choroid plexus cells.
Sci Rep, 2019; 9
28942474: Salazar K, Martínez F, Pérez-Martín M, Cifuentes M, Trigueros L, Ferrada L, Espinoza F, Saldivia N, Bertinat R, Forman K, Oviedo MJ, López-Gambero AJ, Bonansco C, Bongarzone ER, Nualart F
SVCT2 Expression and Function in Reactive Astrocytes Is a Common Event in Different Brain Pathologies.
Ascorbic acid (AA), the reduced form of vitamin C, acts as a neuroprotector by eliminating free radicals in the brain. Sodium/vitamin C co-transporter isoform 2 (SVCT2) mediates uptake of AA by neurons. It has been reported that SVCT2 mRNA is induced in astrocytes under ischemic damage, suggesting that its expression is enhanced in pathological conditions. However, it remains to be established if SVCT expression is altered in the presence of reactive astrogliosis generated by different brain pathologies. In the present work, we demonstrate that SVCT2 expression is increased in astrocytes present at sites of neuroinflammation induced by intracerebroventricular injection of a GFP-adenovirus or the microbial enzyme, neuraminidase. A similar result was observed at 5 and 10 days after damage in a model of traumatic injury and in the hippocampus and cerebral cortex in the in vivo kindling model of epilepsy. Furthermore, we defined that cortical astrocytes maintained in culture for long periods acquire markers of reactive gliosis and express SVCT2, in a similar way as previously observed in situ. Finally, by means of second harmonic generation and 2-photon fluorescence imaging, we analyzed brain necropsied material from patients with Alzheimer’s disease (AD), which presented with an accumulation of amyloid plaques. Strikingly, although AD is characterized by focalized astrogliosis surrounding amyloid plaques, SVCT2 expression at the astroglial level was not detected. We conclude that SVCT2 is heterogeneously induced in reactive astrogliosis generated in different pathologies affecting the central nervous system (CNS).
Mol. Neurobiol., 2018; 55
27733818: Stanic K, Saldivia N, Förstera B, Torrejón M, Montecinos H, Caprile T
Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development.
Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons.
Front Neuroanat, 2016; 10
26074785: Vera A, Recabal A, Saldivia N, Stanic K, Torrejón M, Montecinos H, Caprile T
Interaction between SCO-spondin and low density lipoproteins from embryonic cerebrospinal fluid modulates their roles in early neurogenesis.
During early stages of development, encephalic vesicles are composed by a layer of neuroepithelial cells surrounding a central cavity filled with embryonic cerebrospinal fluid (eCSF). This fluid contains several morphogens that regulate proliferation and differentiation of neuroepithelial cells. One of these neurogenic factors is SCO-spondin, a giant protein secreted to the eCSF from early stages of development. Inhibition of this protein in vivo or in vitro drastically decreases the neurodifferentiation process. Other important neurogenic factors of the eCSF are low density lipoproteins (LDL), the depletion of which generates a 60% decrease in mesencephalic explant neurodifferentiation. The presence of several LDL receptor class A (LDLrA) domains (responsible for LDL binding in other proteins) in the SCO-spondin sequence suggests a possible interaction between both molecules. This possibility was analyzed using three different experimental approaches: (1) Bioinformatics analyses of the SCO-spondin region, that contains eight LDLrA domains in tandem, and of comparisons with the LDL receptor consensus sequence; (2) Analysis of the physical interactions of both molecules through immunohistochemical colocalization in embryonic chick brains and through the immunoprecipitation of LDL with anti-SCO-spondin antibodies; and (3) Analysis of functional interactions during the neurodifferentiation process when these molecules were added to a culture medium of mesencephalic explants. The results revealed that LDL and SCO-spondin interact to form a complex that diminishes the neurogenic capacities that both molecules have separately. Our work suggests that the eCSF is an active signaling center with a complex regulation system that allows for correct brain development.
Front Neuroanat, 2015; 9
28194645: Cifuentes M, Baeza V, Arrabal PM, Visser R, Grondona JM, Saldivia N, Martínez F, Nualart F, Salazar K
Expression of a Novel Ciliary Protein, IIIG9, During the Differentiation and Maturation of Ependymal Cells.
IIIG9 is the regulatory subunit 32 of protein phosphatase 1 (PPP1R32), a key phosphatase in the regulation of ciliary movement. IIIG9 localization is restricted to cilia in the trachea, fallopian tube, and testicle, suggesting its involvement in the polarization of ciliary epithelium. In the adult brain, IIIG9 mRNA has only been detected in ciliated ependymal cells that cover the ventricular walls. In this work, we prepared a polyclonal antibody against rat IIIG9 and used this antibody to show for the first time the ciliary localization of this protein in adult ependymal cells. We demonstrated IIIG9 localization at the apical border of the ventricular wall of 17-day-old embryonic (E17) and 1-day-old postnatal (PN1) brains and at the level of ependymal cilia at 10- and 20-day-old postnatal (PN10-20) using temporospatial distribution analysis and comparing the localization with a ciliary marker. Spectral confocal and super-resolution Structured Illumination Microscopy (SIM) analysis allowed us to demonstrate that IIIG9 shows a punctate pattern that is preferentially located at the borders of ependymal cilia in situ and in cultures of ependymocytes obtained from adult rat brains. Finally, by immunogold ultrastructural analysis, we showed that IIIG9 is preferentially located between the axoneme and the ciliary membrane. Taken together, our data allow us to conclude that IIIG9 is localized in the cilia of adult ependymal cells and that its expression is correlated with the process of ependymal differentiation and with the maturation of radial glia. Similarly, its particular localization within ependymal cilia suggests a role of this protein in the regulation of ciliary movement.
Mol. Neurobiol., 2018; 55
SOXD GENES IN THE CONTROL OF DENTATE GYRUS DEVELOPMENT
Cristina Medina Menéndez1, Lingling Li1, Aixa Morales García1, Pilar Jurado1, Rafael López-San Segundo1, Eva Monserrat1, Véronique Lefebvre2
1Spain , 2United States of America
During the development of the dentate gyrus (DG), both at embryonic and postnatal stages, neural progenitors proliferate and generate mature granule neurons. In an unique way, in the adult DG, a subpolulation of progenitors is retained as adult neural stem cells with radial glial like morphology (RGLs) and continue to produce new granule neurons throughout adult life. However, the mechanisms by which embryonic neural progenitors expand and transform into adult neural stem cells remain unclear. We have previously characterized that SoxD transcription factors Sox5/Sox6 are highly expressed in adult RGLs in the SGZ and that they are required for the activation of quiescent RGLs during adult neurogenesis. More interestingly, both Sox5/Sox6 are also expressed in embryonic neural progenitors during the development of the DG. Using Sox5fl/fl and Sox6fl/fl mice crossed to a transgenic Nestin-cre line, we have found that the absence of Sox5 results in a clear reduction in the number of proliferating progenitors (Ki67+) and radial glial cells from P5 to P14. However, an increased number of proliferating Ki67+ and intermediate progenitors Tbr2+ cells was observed from P30 to P90 in Sox5 conditional mutants, in part due to higher activation of RGLs at P30. The excess of RGLs proliferation causes a reduction of the RGL pool by P90 in Sox5 mutants. These results suggest that Sox5 controls the proliferation of DG progenitors during development and that could be involved in the transition from embryonic neural progenitor into adult RGLs during the establishment of the adult neurogenic niche of DG.
31825616: Zaldivar-Diez J, Li L, Garcia AM, Zhao WN, Medina-Menendez C, Haggarty SJ, Gil C, Morales AV, Martinez A
Benzothiazole-Based LRRK2 Inhibitors as Wnt Enhancers and Promoters of Oligodendrocytic Fate.
Leucine rich repeat kinase 2 (LRRK2) is an enigmatic enzyme and a relevant target for Parkinson’s disease (PD). However, despite the significant amount of research done in the past decade, the precise function of LRRK2 remains largely unknown. Moreover, the therapeutic potential of its inhibitors is in its infancy with the first clinical trial having just started. In the present work, the molecular mechanism of LRRK2 in the control of neurogenesis or gliogenesis was investigated. We designed and synthesized novel benzothiazole-based LRRK2 inhibitors and showed that they can modulate the Wnt/β-catenin signaling pathway. Furthermore, compounds and were able to promote neural progenitors proliferation and drive their differentiation toward neuronal and oligodendrocytic cell fates. These results suggest potential new avenues for the application of LRRK2 inhibitors in demyelinating diseases in which oligodendrocyte cell-death is one of the pathological features.
J. Med. Chem., 2020; 63
ANALYSIS OF PRIMARY CILIA FROM SUBVENTRICULAR ZONE NEURAL STEM CELLS
Katja Baur, Sara Monaco, Claudia Mandl, Gabriele Hölzl-Wenig, Francesca Ciccolini
Primary cilia are tiny cellular organelles which have recently emerged as having vital roles in cellular signalling and cell cycle control. Neural stem cells (NSCs) of the subventricular zone (SVZ) in the brain depend on primary cilia for signals governing activation, quiescence, and maintenance. The molecular mechanisms underlying these functions are however not extensively studied. Consistent with previous observations, we found that most ciliated cells in the adult as well as in the neonatal SVZ represent NSCs expressing Nestin and are in contact with the apical side of the niche, whereas basal cells rarely present a primary cilium. Here we employed flow cytometry for purification and molecular characterization of NSC primary cilia. Primary cilia were identified by adenylate cyclase type III (AC3) immunoreactivity. We found that in prenatal mice, AC3+ particles showed high co-localization with C-X-C motif chemokine receptor 4 (CXCR4) and platelet-derived growth factor receptor alpha (PDGFRα), which both massively decreased co-expression after birth, being barely present in primary cilia of 8 weeks old animals and virtually undetectable those of 24 weeks old animals. In mass spectrometric analysis, FACS-sorted AC3+ particles showed enrichment in ciliary alpha tubulin as well as actin binding protein cofilin 1. Since actin and cofilin regulate cilia length in vitro, this indicates such a mechanism in NSCs. These findings provide first evidence that NSC cilia can effectively sorted by flow cytometry and that FACS-sorted cilia are suited for mass spectrometric analysis.
CELL PROGENY OF GFAP+ PROGENITORS CELLS
Ana Cristina Ojalvo Sanz, Rebeca Sánchez-González, Laura López-Mascaraque
Radial Glial Cells are considered as neural progenitor cells (NPCs) of most neural cells, since during development generate neurons, astrocytes, NG2-glia and oligodendrocytes. A low percentage of embryonic cortical progenitors appears to be lineage multipotent, suggesting a lineage unipotential progenitor diversity. However, other studies display a cell potential diversity depending on the spatio-temporal pattern. That reinforces the idea of heterogeneity of NPCs. NG2-glia is another remarkable cell-type that could act as precursors. NG2-cells also known as oligodendrocyte-precursor cells, displayed different degrees of differentiation and maturation properties depending on brain areas. Further, in lesioned brains generated reactive astrocytes. Our previous data showed that NG2-glia give rise to astrocytes, NG2-glia and oligodendrocytes, with distinct spatio-temporal patterns. Additionally, NG2 progenitor cells from E16 onwards are preferentially committed to oligodendroglial lineage. To decipher the complete cell-progeny of active GFAP progenitors, we targeted those progenitors in the SVZ with the multicolor genetic tool, UbC-(GFAP-PB)-StarTrack. This technique, based on UbC-StarTrack, allows tracing cell lineages from single progenitors with a permanent and inheritable label of their entire progeny. We exchanged the CMV promoter in the piggyback transposase by the GFAP-promoter, introducing the plasmids only in those progenitors with the GFAP-promoter active at the electroporation time. After in vivo electroporation, with UbC-(GFAP-PB)-StarTrack mixture, at different embryonic ages in brain mouse, we analyzed their progeny at short and long-term periods. This allowed tracking and identifying different neural cell populations. Our findings provide fundamental aspects of the lineage heterogeneity and cell fate of specific progenitors. Supported by Grant BFU2016-75207-R (MINECO).
31202640: Yanguas-Casás N, Ojalvo-Sanz AC, Martínez-Vázquez A, Goneau MF, Gilbert M, Nieto-Sampedro M, Romero-Ramírez L
Neurostatin and other O-acetylated gangliosides show anti-neuroinflammatory activity involving the NFκB pathway.
In many neuropathologies activated microglia and macrophages cause neurotoxicity and prolong the inflammatory response. We have previously characterized the glycosphingolipid Neurostatin (Nst), which potentially reduces these detrimental mechanisms. Nst, isolated from mammalian brain, is the GD1b ganglioside with O-acetylation of the outer sialic acid residue. Using the enzyme sialate-O-acetyltransferase (SOAT), we obtained several O-acetylated gangliosides and O-propionylated GD1b (PrGD1b). In the present study we investigated the anti-inflammatory effects of these compounds. Nst and other O-acetylated gangliosides reduced nitrite production in microglial cells which were activated with lipopolysaccharide (LPS), but did not affect nitrite production after their stimulation with interferon gamma (IFNγ). Structure-activity relationship analysis showed that Nst was the most active ganglioside as inhibitor of nitrite production. Its ceramide moiety is essential for this, and both, the O-acetylation and the monosaccharide chain are important for the anti-inflammatory activity of the gangliosides. We also found that Nst reduced iNOS, IL-6 and IL-12 transcription in LPS-induced microglia, likely by inhibiting nuclear localization of NFκB. In co-cultures, Nst reduced neuronal cell death caused by LPS-activated microglia. In vivo, Nst diminished microglia activation in a mouse model of acute neuroinflammation. We propose that Nst and other O-acetylated gangliosides are neuroprotective regulators of microglia activity under both physiological and pathological conditions.
Toxicol. Appl. Pharmacol., 2019; 377
MOSAIC ANALYSIS WITH DOUBLE MARKERS REVEALS ONTOGENY OF THE SUPERIOR COLLICULUS AT SINGLE CELL RESOLUTION
Giselle Cheung, Carmen Streicher, Simon Hippenmeyer
The superior colliculus (SC) is a midbrain structure essential for sensory integration mediating motor responses important for behaviors like attention and arousal. SC dysfunction has been associated with neurodevelopmental diseases like autism. SC is a laminated structure with layer-specific neuronal subtypes and connections with other regions. While much work has focused on the physiological properties of the SC, its intrinsic organization and formation throughout development is still largely unknown. In order to determine the developmental principles of SC formation, we performed lineage analysis to 1) establish the ontogeny of different SC cell-types and 2) define a framework of SC formation at single cell resolution. We used the Mosaic Analysis with Double Markers (MADM) technique which relies on Cre recombinase activity to differentially and fluorescently label daughter cells and their progenies providing unambiguous lineage information of clones as well as their birthdates and division patterns. Here we utilized inducible CreER expression under the Fzd10 promotor to specifically target SC progenitor cells. Systematic clonal analysis was performed on SC clones originating from single progenitor cells labeled at precise embryonic time points. Our results show that in the mature SC (P28), clonally-related cells were distributed throughout different SC layers and the periaqueductal gray. Furthermore, adult SC clones consist of various subtypes of morphologically-distinct neurons and glia. Ultimately, our work on cell-type composition of neuronal clones will define the precise ontogeny of all SC cell-types over development.
31479508: Cheung G, Cousin MA
Synaptic vesicle generation from activity-dependent bulk endosomes requires a dephosphorylation-dependent dynamin-syndapin interaction.
Activity-dependent bulk endocytosis generates synaptic vesicles (SVs) during intense neuronal activity via a two-step process. First, bulk endosomes are formed direct from the plasma membrane from which SVs are then generated. SV generation from bulk endosomes requires the efflux of previously accumulated calcium and activation of the protein phosphatase calcineurin. However, it is still unknown how calcineurin mediates SV generation. We addressed this question using a series of acute interventions that decoupled the generation of SVs from bulk endosomes in rat primary neuronal culture. This was achieved by either disruption of protein-protein interactions via delivery of competitive peptides, or inhibition of enzyme activity by known inhibitors. SV generation was monitored using either a morphological horseradish peroxidase assay or an optical assay that monitors the replenishment of the reserve SV pool. We found that SV generation was inhibited by, (i) peptides that disrupt calcineurin interactions, (ii) an inhibitor of dynamin I GTPase activity and (iii) peptides that disrupt the phosphorylation-dependent dynamin I-syndapin I interaction. Peptides that disrupted syndapin I interactions with eps15 homology domain-containing proteins had no effect. This revealed that (i) calcineurin must be localized at bulk endosomes to mediate its effect, (ii) dynamin I GTPase activity is essential for SV fission and (iii) the calcineurin-dependent interaction between dynamin I and syndapin I is essential for SV generation. We therefore propose that a calcineurin-dependent dephosphorylation cascade that requires both dynamin I GTPase and syndapin I lipid-deforming activity is essential for SV generation from bulk endosomes.
J. Neurochem., 2019; 151
23426665: Cheung G, Cousin MA
Synaptic vesicle generation from activity-dependent bulk endosomes requires calcium and calcineurin.
Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high-frequency stimulation in central nerve terminals. ADBE generates endosomes direct from the plasma membrane, meaning that high concentrations of calcium will be present in their interior due to fluid phase uptake from the extracellular space. Morphological and fluorescent assays were used to track the generation of SVs from bulk endosomes in primary neuronal culture. This process was functionally uncoupled from both SV exocytosis and plasma membrane retrieval events by intervening only after SV fusion and endocytosis were completed. Either intracellular (BAPTA-AM) or intra-endosomal (Rhod-dextran) calcium chelation inhibited SV generation from bulk endosomes, indicating that calcium efflux from this compartment is critical for this process. The V-type ATPase antagonist bafilomycin A1 also arrested SV generation from bulk endosomes, indicating endosomal acidification may be required for calcium efflux. Finally, pharmacological inhibition of the calcium-dependent protein phosphatase calcineurin blocked endosomal SV generation, identifying it as a key downstream effector in this process. These results reveal a novel and key role for the fluid phase uptake of extracellular calcium and its subsequent efflux in the SV lifecycle.
J. Neurosci., 2013; 33
22539861: Cheung G, Cousin MA
Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.
Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.
J. Neurosci., 2012; 32
26346563: Cheung G, Sibille J, Zapata J, Rouach N
Activity-Dependent Plasticity of Astroglial Potassium and Glutamate Clearance.
Recent evidence has shown that astrocytes play essential roles in synaptic transmission and plasticity. Nevertheless, how neuronal activity alters astroglial functional properties and whether such properties also display specific forms of plasticity still remain elusive. Here, we review research findings supporting this aspect of astrocytes, focusing on their roles in the clearance of extracellular potassium and glutamate, two neuroactive substances promptly released during excitatory synaptic transmission. Their subsequent removal, which is primarily carried out by glial potassium channels and glutamate transporters, is essential for proper functioning of the brain. Similar to neurons, different forms of short- and long-term plasticity in astroglial uptake have been reported. In addition, we also present novel findings showing robust potentiation of astrocytic inward currents in response to repetitive stimulations at mild frequencies, as low as 0.75 Hz, in acute hippocampal slices. Interestingly, neurotransmission was hardly affected at this frequency range, suggesting that astrocytes may be more sensitive to low frequency stimulation and may exhibit stronger plasticity than neurons to prevent hyperexcitability. Taken together, these important findings strongly indicate that astrocytes display both short- and long-term plasticity in their clearance of excess neuroactive substances from the extracellular space, thereby regulating neuronal activity and brain homeostasis.
Neural Plast., 2015; 2015
25408635: Cheung G, Chever O, Rouach N
Connexons and pannexons: newcomers in neurophysiology.
Connexin hemichannels are single membrane channels which have been traditionally thought to work in pairs to form gap junction channels across two opposing cells. In astrocytes, gap junction channels allow direct intercellular communication and greatly facilitate the transmission of signals. Recently, there has been growing evidence demonstrating that connexin hemichannels, as well as pannexin channels, on their own are open in various conditions. They allow bidirectional flow of ions and signaling molecules and act as release sites for transmitters like ATP and glutamate into the extracellular space. While much attention has focused on the function of connexin hemichannels and pannexons during pathological situations like epilepsy, inflammation, neurodegeneration or ischemia, their potential roles in physiology is often ignored. In order to fully understand the dynamic properties and roles of connexin hemichannels and pannexons in the brain, it is essential to decipher whether they also have some physiological functions and contribute to normal cerebral processes. Here, we present recent studies in the CNS suggesting emerging physiological functions of connexin hemichannels and pannexons in normal neuronal activity and behavior. We also discuss how these pioneer studies pave the way for future research to extend the physiological relevance of connexons and pannexons, and some fundamental issues yet to be addressed.
Front Cell Neurosci, 2014; 8
PHARMALOGICAL WNT/B-CATENIN ACTIVATION PROMOTES CELLULAR REGENERATION WITHIN THE NEONATAL CORTEX WHILE PRESERVING STEM CELL MAINTENANCE
Louis Foucault, Diane Angonin, Guillaume Marcy, Vanessa Donega, Laurent Bezin, Olivier Raineteau
Germinal activity persists throughout life within the subventricular zone (SVZ) of the postnatal forebrain, due to the presence of quiescent neural stem cells that gradually reactivate throughout life. Accumulating evidences point at a role for these cells during tissue repair following premature brain injuries (Fagel et al., 2006; Salmaso et al., 2014), and suggest their amenability to pharmacological manipulations (Azim et al., 2017 Scafidi, 2014). The extent of this repair and its long-term consequences on forebrain germinal activity however remain to be explored. We used chronic neonatal hypoxia as a rodent model of premature brain injury, to investigate the contribution of SVZ NSCs to cellular regeneration within the cortex. Our results reveal an increased proliferation and production of TBR2+ & OLIG2+ progenitors within the dorsal SVZ following chronic hypoxia, which was paralleled by an activation of the Wnt canonical pathway. Fate mapping of SVZ NSCs demonstrates their contribution to de novo oligodendrogenesis and cortical neurogenesis following hypoxia, while confirming a delay of oligodendrocytes maturation. Remarkably, a pharmacological activation of the Wnt/β-catenin pathway by intranasal administration of a Gsk3β inhibitor following hypoxia, promotes neurogenesis and oligodendrogenesis as well as their specification and maturation. Importantly, labeling of NSCs in different states of activation, demonstrates that pharmacological NSCs activation have no adverse effects on the reservoir of SVZ NSCs and on their long-term germinal activity. Altogether, our work highlights the potential of pharmacological approaches to promote cellular regeneration within the neonatal forebrain, while demonstrating no detrimental long-term effect on forebrain germinal activity.
30282727: Benito N, Gaborieau E, Sanz Diez A, Kosar S, Foucault L, Raineteau O, De Saint Jan D
A Pool of Postnatally Generated Interneurons Persists in an Immature Stage in the Olfactory Bulb.
Calretinin (CR)-expressing periglomerular (PG) cells are the most abundant interneurons in the glomerular layer of the olfactory bulb. They are predominately generated postnatally from the septal and dorsal subventricular zones that continue producing them well into adulthood. Yet, little is known about their properties and functions. Using transgenic approaches and patch-clamp recording in mice of both sexes we show that CR(+) PG cells of both septal and dorsal origin have homogeneous morphological and electrophysiological properties. However, unlike other PG cells, these axonless neurons express a surprisingly small repertoire of voltage-activated channels and do not fire or fire at most a single and often small action potential. Moreover, they are not innervated by olfactory sensory neurons and receive little synaptic inputs from mitral or tufted cells at excitatory synapses where NMDA receptors predominate. These membrane and synaptic properties, that resemble those of newborn immature neurons not yet integrated in the network, persist over time and limit the recruitment of CR(+) PG cells by afferent inputs that strongly drive local network activity. Together, our results show that postnatally generated CR(+) PG cells continuously supply a large pool of neurons with unconventional properties. These data also question the contribution of CR(+) PG cells in olfactory bulb computation. Calretinin-expressing PG cells are by far the most abundant interneurons in the glomerular layer of the olfactory bulb. They are continuously produced during postnatal life, including adulthood, from neural stem cells located in the subventricular zones. Surprisingly, unlike other postnatally generated newborn neurons that quickly integrate into preexisting olfactory bulb networks, calretinin-expressing PG cells retain immature properties that limit their recruitment in local network activity for weeks, if not months, as if they would never fully mature. The function of this so far unsuspected pool of latent neurons is still unknown.
J. Neurosci., 2018; 38
PAX6: A TRANSCRIPTIONAL GUARDIAN OF GLUTAMATERGIC FATE SPECIFICATION IN THE DEVELOPING NEOCORTEX
Kai Boon Tan, Zrinko Kozic, Daniel Dobolyi, Martine Manuel, John Mason, David Price
During forebrain development, the transcription factor PAX6 is highly expressed by progenitors in the dorsal telencephalon (dTel) i.e. the primitive cerebral cortex with a sharp boundary at the pallial-subpallial boundary, thereby establishing the dorso-ventral patterning of the forebrain and regulating the generation of cortical glutamatergic neurons. Strikingly, removal of Pax6 led to a diversion away from the glutamatergic identity in a subset of cortical progenitors indicated by ectopic gene expression. We postulate that PAX6 confers glutamatergic fate in progenitors by preventing them from responding to signaling cues such as SHH that can induce abberant fates. In the present study, we used the transgenic mouse model with Pax6 conditionally deleted in the cortex using a tamoxifen-inducible Emx1-CreERT2 transgene combined with a floxed Pax6 and an EGFP constructs. Single-cell transcriptome revealed multiple ectopic leanages in cortical progenitors with morphogen-regulated transcriptional signatures upon Pax6 deletion. We also undertook a candidate approach to investigate how attenuation of signaling cues by surgical and pharmacological means using in vitro slice culture affect the magnitude of ectopic gene expression in the cortical progenitors. We demonstrated that attenuation of interneuron migration into the cortex and inhibition of SHH signaling pathway in slice culture substatially reduced aberrant gene expression in the cortical progenitors. Our findings suggest that ventral cues from vTel such as SHH possess ventralizing effect on cortical progenitors, this is consistent with a requirement for PAX6 to resist such effects in order to safeguard glutamatergic fate.
PSA DEPLETION INDUCES THE DIFFERENTIATION OF IMMATURE NEURONS IN THE PIRIFORM CORTEX OF ADULT MICE
Simona Coviello1, Maria Bellés-Esteller1, Yaiza Gramuntell-Roch1, Patrycja Klimczak1, Bruno Benedetti2, Sebastien Couillard-Despres2, Juan Nacher1
1Spain , 2Austria
In adult rodents a population of cells that exhibit characteristics of immature neurons resides in the piriform cortex (PCX). In mammals with complex cerebral cortices, including humans, the immature neurons have a more widespread distribution. The majority of these cells are generated during embryonic development, but nevertheless remain in an “undifferentiated stage” until adulthood. Then, they progressively mature and incorporate to the circuitry as excitatory neurons. These immature neurons express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), which is widely involved in neuronal plasticity. This molecule isolates the cells from the surrounding circuitry when they are in their most immature stage (tangled cells) and probably further influences some of their maturational events. In order to deepen the knowledge over these immature neurons, we have studied their phenotype in human cortical samples and have explored the impact of PSA depletion in a transgenic mouse model to understand the role of this molecule in immature neurons differentiation. We have used different antibodies to study the phenotype of these immature cells in both humans and rodents and have injected intracerebrally the enzyme Endoneuraminidase-N, which degrades specifically PSA, in the PCX of adult mice. Our results confirm the presence of an immature neuronal population in the human neocortex, which shares many features with that found in the rodent PCX. We have also gathered data providing evidence that the depletion of PSA promotes the entry into the final stages of development of these immature neurons in adult mice.
31647533: Benedetti B, Dannehl D, König R, Coviello S, Kreutzer C, Zaunmair P, Jakubecova D, Weiger TM, Aigner L, Nacher J, Engelhardt M, Couillard-Després S
Functional Integration of Neuronal Precursors in the Adult Murine Piriform Cortex.
The extent of functional maturation and integration of nonproliferative neuronal precursors, becoming neurons in the adult murine piriform cortex, is largely unexplored. We thus questioned whether precursors eventually become equivalent to neighboring principal neurons or whether they represent a novel functional network element. Adult brain neuronal precursors and immature neurons (complex cells) were labeled in transgenic mice (DCX-DsRed and DCX-CreERT2 /flox-EGFP), and their cell fate was characterized with patch clamp experiments and morphometric analysis of axon initial segments. Young (DCX+) complex cells in the piriform cortex of 2- to 4-month-old mice received sparse synaptic input and fired action potentials at low maximal frequency, resembling neonatal principal neurons. Following maturation, the synaptic input detected on older (DCX-) complex cells was larger, but predominantly GABAergic, despite evidence of glutamatergic synaptic contacts. Furthermore, the rheobase current of old complex cells was larger and the maximal firing frequency was lower than those measured in neighboring age-matched principal neurons. The striking differences between principal neurons and complex cells suggest that the latter are a novel type of neuron and new coding element in the adult brain rather than simple addition or replacement for preexisting network components.
Cereb. Cortex, 2020; 30
REMOVAL OF PAX6 FROM CORTICAL PROGENITORS RENDERS THEM ABNORMALLY LIKELY TO SWITCH FATE IN RESPONSE TO SONIC HEDGEHOG ACTIVITY.
Maizatul Fazilah Abd Razak, John Mason, David Price
The paired-box protein PAX6 is a transcription factor that acts as a master regulator during neurodevelopment. In mice, it is expressed by neural progenitors in the cortical ventricular zone (VZ) where it regulates neurogenesis and fate specification. The complex processes of neurodevelopment are also regulated by morphogens such as Sonic Hedgehog (Shh) that promotes ventralization of the ventral telencephalon (vTel), through diffusion to form a concentration gradient. Strikingly, when Pax6 is deleted, a subset of cortical progenitor cells that would normally become glutamatergic commit into a GABAergic fate, expressing ventral markers such as Gad67, Mash1, and Dlx1. Previous studies have shown that when wild-type cortical cells are treated with substances that activates Shh signalling, they can be made to express genes such as Gsx2 which are associated with the generation of interneurons. Based on these findings, we asked what makes cortical progenitor cells change their fate when Pax6 is deleted? This study aimed to investigate the sensitivity of cortical progenitor cells in Pax6 mutant mice to Shh signalling. To answer this question, we exposed cultured cortical progenitors to increasing concentrations of Shh agonist, developed for the protein Smoothened, which is a key part of the Hedgehog signalling pathway. A significant increase of Gsx2+ cells was observed in parallel with the increase of Shh agonist concentrations. This finding suggests that cortical progenitors are vulnerable to Shh when Pax6 is deleted. This indicates that Pax6 may safeguard cortical fate by suppressing pro-GABAergic cues originating from vTel during cortical development.
ELUCIDATING THE ROLE OF AUTOPHAGY DURING REPROGRAMMING OF GLIA INTO NEURON BY CRISPR SCREENING
Aida Platero1, Stella Krämer2, Oliver Baker1, Benedikt Berninger1
1United Kingdom , 2Germany
The transition from glia to neuron during direct reprogramming compiles a series of cellular events that will determine the conversion success. The glial cells will undergo dramatic adjustments including morphological, metabolic and proteomic changes in order to rebuild their cell program. In this context of conversion, autophagy is expected to play a fundamental role. Here, we are developing an in vitro platform, using primary murine astrocyte cultures, CRISPR screening, transgenic mouse models and high image content to scrutinize our glia-to-neuron direct reprogramming paradigm.
24439383: Platero-Luengo A, González-Granero S, Durán R, Díaz-Castro B, Piruat JI, García-Verdugo JM, Pardal R, López-Barneo J
An O2-sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia.
Neural stem cells (NSCs) exist in germinal centers of the adult brain and in the carotid body (CB), an oxygen-sensing organ that grows under chronic hypoxemia. How stem cell lineage differentiation into mature glomus cells is coupled with changes in physiological demand is poorly understood. Here, we show that hypoxia does not affect CB NSC proliferation directly. Rather, mature glomus cells expressing endothelin-1, the O2-sensing elements in the CB that secrete neurotransmitters in response to hypoxia, establish abundant synaptic-like contacts with stem cells, which express endothelin receptors, and instruct their growth. Inhibition of glomus cell transmitter release or their selective destruction markedly diminishes CB cell growth during hypoxia, showing that CB NSCs are under the direct “synaptic” control of the mature O2-sensitive cells. Thus, glomus cells not only acutely activate the respiratory center but also induce NSC-dependent CB hypertrophy necessary for acclimatization to chronic hypoxemia.
Cell, 2014; 156
28129541: Wu J, Platero-Luengo A, Sakurai M, Sugawara A, Gil MA, Yamauchi T, Suzuki K, Bogliotti YS, Cuello C, Morales Valencia M, Okumura D, Luo J, Vilariño M, Parrilla I, Soto DA, Martinez CA, Hishida T, Sánchez-Bautista S, Martinez-Martinez ML, Wang H, Nohalez A, Aizawa E, Martinez-Redondo P, Ocampo A, Reddy P, Roca J, Maga EA, Esteban CR, Berggren WT, Nuñez Delicado E, Lajara J, Guillen I, Guillen P, Campistol JM, Martinez EA, Ross PJ, Izpisua Belmonte JC
Interspecies Chimerism with Mammalian Pluripotent Stem Cells.
Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
Cell, 2017; 168
27984723: Ocampo A, Reddy P, Martinez-Redondo P, Platero-Luengo A, Hatanaka F, Hishida T, Li M, Lam D, Kurita M, Beyret E, Araoka T, Vazquez-Ferrer E, Donoso D, Roman JL, Xu J, Rodriguez Esteban C, Nuñez G, Nuñez Delicado E, Campistol JM, Guillen I, Guillen P, Izpisua Belmonte JC
In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming.
Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.
Cell, 2016; 167
27919073: Kang E, Wu J, Gutierrez NM, Koski A, Tippner-Hedges R, Agaronyan K, Platero-Luengo A, Martinez-Redondo P, Ma H, Lee Y, Hayama T, Van Dyken C, Wang X, Luo S, Ahmed R, Li Y, Ji D, Kayali R, Cinnioglu C, Olson S, Jensen J, Battaglia D, Lee D, Wu D, Huang T, Wolf DP, Temiakov D, Belmonte JC, Amato P, Mitalipov S
Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations.
Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother’s oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.
Nature, 2016; 540
26866353: Navarro-Guerrero E, Platero-Luengo A, Linares-Clemente P, Cases I, López-Barneo J, Pardal R
Gene Expression Profiling Supports the Neural Crest Origin of Adult Rodent Carotid Body Stem Cells and Identifies CD10 as a Marker for Mesectoderm-Committed Progenitors.
Neural stem cells (NSCs) are promising tools for understanding nervous system plasticity and repair, but their use is hampered by the lack of markers suitable for their prospective isolation and characterization. The carotid body (CB) contains a population of peripheral NSCs, which support organ growth during acclimatization to hypoxia. We have set up CB neurosphere (NS) cultures enriched in differentiated neuronal (glomus) cells versus undifferentiated progenitors to investigate molecular hallmarks of cell classes within the CB stem cell (CBSC) niche. Microarray gene expression analysis in NS is compatible with CBSCs being neural crest derived-multipotent progenitor cells able to sustain CB growth upon exposure to hypoxia. Moreover, we have identified CD10 as a marker suitable for isolation of a population of CB mesectoderm-committed progenitor cells. CD10 + cells are resting in normoxia, and during hypoxia they are activated to proliferate and to eventually complete maturation into mesectodermal cells, thus participating in the angiogenesis necessary for CB growth. Our results shed light into the molecular and cellular mechanisms involved in CBSC fate choice, favoring a potential use of these cells for cell therapy. Stem Cells 2016;34:1637-1650.
Stem Cells, 2016; 34
SINGLE CELL AND SPATIAL TRANSCRIPTOMICS OF CELL TYPES AND LINEAGES IN THE MOUSE BRAIN
Michael Ratz, Leonie Von Berlin, Ludvig Larsson, Marcel Martin, Jakub Westholm, Joakim Lundeberg, Jonas Frisén
The mammalian brain contains a wide variety of neurons, astrocytes, oligodendrocytes and immune cells generated from (neural) stem cells during development. While single-cell RNA-seq revealed the existence of hundreds of molecularly distinct cell types, the clonal relationships among these cells as well as the strategies that build cellular diversity remain elusive. We report a method that allows for simultaneous cell type discovery and lineage reconstruction via single-cell RNA-seq of mouse brain cells. Our approach involves the delivery of a highly diverse lentiviral barcode library to label progenitors in the early developing brain followed by isolation and RNA-seq of transduced cells from the juvenile brain. We sequenced thousands of cells from discrete anatomical regions and established the clonal relationships among the identified cell types. Interestingly, microglia formed the largest clones and cells from identical microglia clones were distributed across multiple forebrain regions. Comparing transcriptome profiles of microglia revealed regional differences and suggests that the brain microenvironment drives a region-specific microglia phenotype within the same clone. Our results yield novel insights into the lineage and regional potential of various progenitor cells and the approach is widely applicable to simultaneously profile cell types and lineages in different tissues during development, homeostasis and disease.
THALAMOCORTICAL AXONS REGULATE NEUROGENESIS IN PRIMARY SENSORY AREAS OF THE MOUSE NEOCORTEX.
Timothy Monko, Jeff Stolley, Samantha Dabruzzi, Peter Goncharov, Stephen Salton, Yasushi Nakagawa
United States of America
The area-specific axonal projections from the mammalian thalamus to the neocortex have an instructive role in shaping unique cellular organization in the target cortical areas. It is unknown if thalamocortical axons only play a role on postmitotic cortical neurons or if they also regulate embryonic neurogenesis. Here we found that mutant mice that lack the majority of thalamocortical axons have a reduced number of superficial layer neurons in primary somatosensory and visual cortex. In addition, there were fewer radial glia and intermediate progenitor cells in the embryonic primary sensory cortex of these mice. Thalamocortical axons increased the divisions of progenitor cells to contribute to increased superficial layer neurons. Finally, we have identified VGF, a neuropeptide expressed specifically in sensory nuclei of the embryonic thalamus, as a molecule that mediates the role of thalamic afferents. This work provides insight into non-local molecular regulatory mechanisms coordinating neurogenesis in the mammalian brain, building upon well established work in zebrafish and invertebrates. Our work reveals that extrinsic molecular cues, such as the polypeptide VGF, derived from sensory thalamic projections selectively modulate neurogenesis in the embryonic neocortex.
ABERRANT DIFFERENTIATION OF NEURAL PROGENITOR CELLS OF RATS GENETICALLY PRONE TO AUDIOGENIC EPILEPSY
Alexandra Naumova, Elena Chernigovskaya, Ekaterina Oleinik, Vera Bachteeva, Margarita Glazova
Epilepsy is associated with altered neurogenesis in brain of human and epileptic animals. We hypothesized that aberrant proliferation and differentiation of neural progenitors can underlie the development of hereditary epilepsy. The aim of the present work was to analyze glutamatergic differentiation of neural progenitor cells (NPC) of Krushinsky-Molodkina (KM) rats with inherited audiogenic epilepsy. Methods. NPC were isolated from brains of Wistar and KM rat embryos (E18-19) and cultured according with the standard protocol. Glutamatergic differentiation was stimulated by specific neurotrophins. Differentiation was analyzed by immunofluorescent detection of vesicular glutamate transporters 1/2 (VGLUT1/2), glutamate decarboxylases 65/67 (GAD65/67), and doublecortin. Activity of Akt, glycogen synthase kinase 3b (GSK3b), extracellular signal-regulated kinases 1/2 (ERK1/2), and protein kinase A (PKA) that participate in the regulation of differentiation was evaluated by Western blot analysis. Results. We showed that control (unstimulated) KM NPC culture was mainly presented by VGLUT1/2-positive glutamatergic neurons with prevalence of immature doublecortin-positive cells, while unstimulated Wistar NPC culture contained both glutamatergic and GAD65/67-positive GABAergic neurons. After neurotrophin stimulation Wistar NPC demonstrated expected glutamatergic differentiation that was accompanied with activation of PKA as compared with control Wistar NPC. However, the similar stimulation of KM NPC did not affect the numbers of glutamate- or GABAergic cells but resulted in activation of ERK1/2 and Akt/GSK3b-signaling cascade. Conclusion. Our results indicate the genetically determined aberrations of mechanisms that regulate the differentiation of glutamatergic neurons in KM rats. We suppose that revealed alterations are involved in epileptogenesis. This study was supported by the RFBR 19-015-00070.
30168065: Krutetskaya ZI, Milenina LS, Naumova AA, Butov SN, Antonov VG, Nozdrachev AD
Amitriptyline Attenuates Ca Responses Induced by Glutoxim and Molixan in Macrophages.
Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor agonist, tricyclic antidepressant amitriptyline, significantly inhibits glutoxim- and molixan-induced Ca-responses in rat peritoneal macrophages. The results suggest possible involvement of sigma-1 receptors in the signaling cascade induced by glutoxim or molixan and leading to intracellular Ca concentration increase in macrophages.
Dokl. Biochem. Biophys., 2018; 481
30008101: Krutetskaya ZI, Milenina LS, Naumova AA, Butov SN, Antonov VG, Nozdrachev AD
Sigma-1 Receptor Antagonist Haloperidol Attenuates Store-Dependent Ca Entry in Macrophages.
Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with sigma-1 receptor antagonist haloperidol leads to a significant inhibition of the store-dependent Ca entry induced by endoplasmic Ca-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of the sigma-1 receptor in the regulation of storedependent Ca entry in macrophages.
Dokl. Biochem. Biophys., 2018; 480
29536309: Krutetskaya ZI, Milenina LS, Naumova AA, Butov SN, Antonov VG, Nozdrachev AD
Trifluoperazine Attenuates Store-Dependent Ca Entry in Macrophages.
Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with the calsequestrin inhibitor neuroleptic trifluoperazine leads to a significant inhibition of the store-dependent Ca entry induced by endoplasmic Ca-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest calsequestrin involvement in the regulation of the store-dependent Ca entry in macrophages.
Dokl. Biochem. Biophys., 2018; 478
29536308: Krutetskaya ZI, Milenina LS, Naumova AA, Butov SN, Antonov VG, Nozdrachev AD
Phospholipase A Inhibitors Modulate the Effect of Trifluoperazine on the Intracellular Ca Concentration in Macrophages.
Using Fura-2AM microfluorimetry, it was shown for the first time that phospholipase A inhibitors 4-bromophenacyl bromide and glucocorticosteroids prednisolone and dexamethasone attenuate Ca responses induced by neuroleptic trifluoperazine in macrophages. The results suggest the involvement of phospholipase A and arachidonic acid metabolism cascade in the effect of trifluoperazine on intracellular Ca concentration in macrophages.
Dokl. Biochem. Biophys., 2018; 478
28726103: Krutetskaya ZI, Milenina LS, Naumova AA, Butov SN, Antonov VG, Nozdrachev AD
The effect of chlorpromazine on intracellular Ca concentration in macrophages.
Using Fura-2AM microfluorimetry, it was shown for the first time that neuroleptic chlorpromazine causes intracellular Ca concentration increase in macrophages due to Ca mobilization from intracellular Ca stores and subsequent Ca entry from the external medium. Chlorpromazine-induced Ca entry is inhibited by La and 2-aminoethoxydiphenyl borate and is associated with Ca store depletion.
Dokl. Biochem. Biophys., 2017; 474
COMPETING FUNCTIONS OF KAT3 TRANSCRIPTIONAL CO-ACTIVATORS: NEURONAL FATE MAINTENANCE VS ACTIVITY-DRIVE TRANSCRIPTION
Beatriz Del Blanco1, Michal Lipinski2, Sergio Niñerola1, Rafael Muñoz-Viana1, Alejandro Medrano-Fernández1, Luis Miguel Valor1, Angel Barco1
1Spain , 2United States of America
Competing functions of KAT3 transcriptional co-activators: neuronal fate maintenance vs activity-drive transcription Numerous studies document the importance of the paralog lysine acetyltransferases and transcriptional co-activators CBP and p300 (aka,KAT3) during neuronal development. However, its precise role in cognitive processes is not yet described. We have recently shown that both proteins play a joint role preserving cell identity during neuronal homeostasis. In that study, we also showed that KAT3 proteins are distributed in enhancers enriched at bHLH binding sites of neuronal genes to maintain histone acetylation and regulate the correct levels of transcription. We now show that during neuronal activation by kainic acid, KAT3 proteins are temporarily redistributed in the genome, leaving their basal function to regulate the transcriptional program necessary during synaptic activity and neuroadaptation processes. As a result, the neuronal identity program results transiently shutdown. These intriguing results point to a double function of KAT3 proteins in adult neurons. Supporting this theory, KAT3 redistribution in the genome led to the identification of different set of anchor transcription factors. While, the enrichment in transcription factor binding sites at KAT3 peaks in the basal state underscores CBP/p300 cooperation with bHLH transcriptional factors that act as terminal selectors, upon activity we detect de novo enrichment for binding sites of activity-regulated transcription factors such as AP1 and CREB. Our experiments reveal an intriguing competition for these two essential transcriptional programs in mature neurons that may have important implications in neuropathology.
30850733: Del Blanco B, Guiretti D, Tomasoni R, Lopez-Cascales MT, Muñoz-Viana R, Lipinski M, Scandaglia M, Coca Y, Olivares R, Valor LM, Herrera E, Barco A
CBP and SRF co-regulate dendritic growth and synaptic maturation.
The CREB-binding protein (CBP) exerts tight control of developmental processes. Here, we investigated the consequences of its selective ablation in newborn neurons. Mice in which CBP was eliminated during neuronal differentiation showed perinatal death and defective diaphragm innervation. Adult-born neurons also showed impaired growth and maturation after inducible and restricted CBP loss in dentate gyrus neuroprogenitors. Consistent with these in vivo findings, cultured neurons displayed impaired outgrowth, immature spines, and deficient activity-dependent synaptic remodeling after CBP ablation. These deficits coincided with broad transcriptional changes affecting genes involved in neuronal growth and plasticity. The affected gene set included many predicted targets of both CBP and the serum response factor (SRF), an activity-regulated transcription factor involved in structural plasticity. Notably, increasing SRF activity in a CBP-independent manner ameliorated the transcriptional, synaptic, and growth defects. These results underscore the relevance of CBP-SRF interactions during neuronal outgrowth and synaptic maturation, and demonstrate that CBP plays an essential role in supporting the gene program underlying the last steps of neuronal differentiation, both during development and in the adult brain.
Cell Death Differ., 2019; 26
TESTING PLATINUM NANOPARTICLE-BASED MICROREACTORS TO OVERCOME EXCITOTOXICITY IN SUBVENTRICULAR ZONE-DERIVED CULTURES
Filipa F. Ribeiro, Adam Armada-Moreira, Ana M Sebastião, Sara Xapelli, Sandra Vaz
Glutamate excitotoxicity is a pathological process that contributes to the progression of neurodegenerative diseases and is associated to overactivation of glutamate receptors which triggers an intracellular cascade of neurotoxic events, including exacerbated production of hydrogen peroxide (H2O2) and ammonia toxicity, leading to cell death. Following cerebral damage, neurogenesis/gliogenesis is altered in neurogenic niches. The main neurogenic niches in the adult mammalian brain where constitutive neurogenesis takes place are the subventricular zone of the lateral ventricle and in the subgranular zone of the dentate gyrus (DG) in the hippocampus. Microreactors equipped with platinum nanoparticles (astrosomes) have been reported to ameliorate the eﬀects of excitotoxicity in neuroblastoma cell culture by scavenging H2O2 and ammonia. In this project we aim to evaluate the best microreactor size to generate the astrosomes, so that they can be the small as possible without being incorporated by cells. After choosing the microreactor size we want to study neurogenesis and gliogenesis promoted by excitotoxicity and the presence of astrosomes. Preliminary data suggest that microreactors with a diameter equal or less than 2.2 µm could be internalized by neurons and astrocytes, while microreactors with diameters 7.1 and 19.3 µm were not. To study neurogenesis and gliogenesis promoted by excitotoxicity and astrosomes, we applied different concentrations of either glutamate, H2O2 or ammonia and/or delivered the astrosomes to SVZ-derived cultures. Cell survival and proliferation, neuronal and astrocytic differentiation and morphology will be evaluated by immunohistochemistry and cell function by calcium signalling.
30546297: Ferreira FF, Ribeiro FF, Rodrigues RS, Sebastião AM, Xapelli S
Brain-Derived Neurotrophic Factor (BDNF) Role in Cannabinoid-Mediated Neurogenesis.
The adult mammalian brain can produce new neurons in a process called adult neurogenesis, which occurs mainly in the subventricular zone (SVZ) and in the hippocampal dentate gyrus (DG). Brain-derived neurotrophic factor (BDNF) signaling and cannabinoid type 1 and 2 receptors (CB1R and CB2R) have been shown to independently modulate neurogenesis, but how they may interact is unknown. We now used SVZ and DG neurosphere cultures from early (P1-3) postnatal rats to study the CB1R and CB2R crosstalk with BDNF in modulating neurogenesis. BDNF promoted an increase in SVZ and DG stemness and cell proliferation, an effect blocked by a CB2R selective antagonist. CB2R selective activation promoted an increase in DG multipotency, which was inhibited by the presence of a BDNF scavenger. CB1R activation induced an increase in SVZ and DG cell proliferation, being both effects dependent on BDNF. Furthermore, SVZ and DG neuronal differentiation was facilitated by CB1R and/or CB2R activation and this effect was blocked by sequestering endogenous BDNF. Conversely, BDNF promoted neuronal differentiation, an effect abrogated in SVZ cells by CB1R or CB2R blockade while in DG cells was inhibited by CB2R blockade. We conclude that endogenous BDNF is crucial for the cannabinoid-mediated effects on SVZ and DG neurogenesis. On the other hand, cannabinoid receptor signaling is also determinant for BDNF actions upon neurogenesis. These findings provide support for an interaction between BDNF and endocannabinoid signaling to control neurogenesis at distinct levels, further contributing to highlight novel mechanisms in the emerging field of brain repair.
Front Cell Neurosci, 2018; 12
29053799: Dias RB, Rodrigues TM, Rombo DM, Ribeiro FF, Rodrigues J, McGarvey J, Orcinha C, Henley JM, Sebastião AM
Erythropoietin Induces Homeostatic Plasticity at Hippocampal Synapses.
The cytokine erythropoietin (EPO) is the master regulator of erythropoiesis. Intriguingly, many studies have shown that the cognitive performance of patients receiving EPO for its hematopoietic effects is enhanced, which prompted the growing interest in the use of EPO-based strategies to treat neuropsychiatric disorders. EPO plays key roles in brain development and maturation, but also modulates synaptic transmission. However, the mechanisms underlying the latter have remained elusive. Here, we show that acute (40-60 min) exposure to EPO presynaptically downregulates spontaneous and afferent-evoked excitatory transmission, without affecting basal firing of action potentials. Conversely, prolonged (3 h) exposure to EPO, if followed by a recovery period (1 h), is able to elicit a homeostatic increase in excitatory spontaneous, but not in evoked, synaptic transmission. These data lend support to the emerging view that segregated pathways underlie spontaneous and evoked neurotransmitter release. Furthermore, we show that prolonged exposure to EPO facilitates a form of hippocampal long-term potentiation that requires noncanonical recruitment of calcium-permeable AMPA receptors for its maintenance. These findings provide important new insight into the mechanisms by which EPO enhances neuronal function, learning, and memory.
Cereb. Cortex, 2018; 28
28848435: Rodrigues RS, Ribeiro FF, Ferreira F, Vaz SH, Sebastião AM, Xapelli S
Interaction between Cannabinoid Type 1 and Type 2 Receptors in the Modulation of Subventricular Zone and Dentate Gyrus Neurogenesis.
Neurogenesis in the adult mammalian brain occurs mainly in two neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus (DG). Cannabinoid type 1 and 2 receptors (CBR and CBR) have been shown to differently modulate neurogenesis. However, low attention has been given to the interaction between CBR and CBR in modulating postnatal neurogenesis (proliferation, neuronal differentiation and maturation). We focused on a putative crosstalk between CBR and CBR to modulate neurogenesis and cultured SVZ and DG stem/progenitor cells from early postnatal (P1-3) Sprague-Dawley rats. Data showed that the non-selective cannabinoid receptor agonist WIN55,212-2 promotes DG cell proliferation (measured by BrdU staining), an effect blocked by either CBR or CBR selective antagonists. Experiments with selective agonists showed that facilitation of DG cell proliferation requires co-activation of both CBR and CBR. Cell proliferation in the SVZ was not affected by the non-selective receptor agonist, but it was enhanced by CBR selective activation. However, either CBR or CBR selective antagonists abolished the effect of the CBR agonist in SVZ cell proliferation. Neuronal differentiation (measured by immunocytochemistry against neuronal markers of different stages and calcium imaging) was facilitated by WIN55,212-2 at both SVZ and DG. This effect was mimicked by either CBR or CBR selective agonists and blocked by either CBR or CBR selective antagonists, cross-antagonism being evident. In summary, our findings indicate a tight interaction between CBR and CBR to modulate neurogenesis in the two major neurogenic niches, thus contributing to further unraveling the mechanisms behind the action of endocannabinoids in the brain.
Front Pharmacol, 2017; 8
28534273: Soares R, Ribeiro FF, Xapelli S, Genebra T, Ribeiro MF, Sebastião AM, Rodrigues CMP, Solá S
Tauroursodeoxycholic Acid Enhances Mitochondrial Biogenesis, Neural Stem Cell Pool, and Early Neurogenesis in Adult Rats.
Although neurogenesis occurs in restricted regions of the adult mammalian brain, neural stem cells (NSCs) produce very few neurons during ageing or after injury. We have recently discovered that the endogenous bile acid tauroursodeoxycholic acid (TUDCA), a strong inhibitor of mitochondrial apoptosis and a neuroprotective in animal models of neurodegenerative disorders, also enhances NSC proliferation, self-renewal, and neuronal conversion by improving mitochondrial integrity and function of NSCs. In the present study, we explore the effect of TUDCA on regulation of NSC fate in neurogenic niches, the subventricular zone (SVZ) of the lateral ventricles and the hippocampal dentate gyrus (DG), using rat postnatal neurospheres and adult rats exposed to the bile acid. TUDCA significantly induced NSC proliferation, self-renewal, and neural differentiation in the SVZ, without affecting DG-derived NSCs. More importantly, expression levels of mitochondrial biogenesis-related proteins and mitochondrial antioxidant responses were significantly increased by TUDCA in SVZ-derived NSCs. Finally, intracerebroventricular administration of TUDCA in adult rats markedly enhanced both NSC proliferation and early differentiation in SVZ regions, corroborating in vitro data. Collectively, our results highlight a potential novel role for TUDCA in neurologic disorders associated with SVZ niche deterioration and impaired neurogenesis.
Mol. Neurobiol., 2018; 55
26068054: Ribeiro FF, Neves-Tomé R, Assaife-Lopes N, Santos TE, Silva RF, Brites D, Ribeiro JA, Sousa MM, Sebastião AM
Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons.
Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague-Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration.
Brain Struct Funct, 2016; 221
METFORMIN-PRODUCED ROS TARGET CDK5/P35/DRP1 AND AKT SIGNALING PATHWAYS IN THE REGULATION OF NEURONAL DIFFERENTIATION IN NEUROBLASTOMA
Thunwa Binlateh, Supita Tanasawet, Pilaiwanwadee Hutamekalin
Besides being an anti-diabetic drug, metformin has also become of interest in field of differentiation. However, the implication and mechanisms underlying metformin action on differentiation potential is still controversial. Here, we showed that metformin induced neuronal differentiation in neuroblastoma as accompanied by the significant increase of neurite length and upregulation of MAP2, β-tubulin-III and TH. Analysis of the mechanism of metformin action found that metformin significantly generated intracellular ROS levels. Pretreatment with ROS scavenger (NAC) prevented neuronal differentiation in metformin treatment. Determining the target of metformin-produced ROS revealed that Cdk5/p35 and Akt served as the downstream of ROS. Cdk5/p35 was suppressed during metformin-mediated neuronal differentiation while pretreatment with NAC rescued its expression with prevention of neuronal differentiation. Pharmacological inhibition of Cdk5 using roscovitine notably reduced p-Drp1 expression and also promoted neuronal differentiation, suggested that Cdk5 regulated Drp1 during mediation of neuronal differentiation. Furthermore, activation of Akt activity was observed in metformin-mediated neuronal differentiation, however, pretreatment with NAC and Akt inhibitor perifosine blocked its activity and inhibited neuronal differentiation. Additionally, metformin substantially decreased Erk1/2 activity during induction of neuronal differentiation. NAC pretreatment did not influence Erk1/2 activity in metformin-treated cells. PD98095 inactivation of Erk1/2 activity induced neuronal differentiation with inhibition of cell proliferation, indicated the role of Erk1/2 in growth arrest-associated differentiation. Taken together, these finding revealed the promising effect of metformin on promoting neuronal differentiation which relies on ROS-targeted Cdk5/p35/Drp1 and Akt signaling pathways, and Erk1/2-related growth arrest-associated differentiation.
30911317: Binlateh T, Tanasawet S, Rattanaporn O, Sukketsiri W, Hutamekalin P
Metformin Promotes Neuronal Differentiation via Crosstalk between Cdk5 and Sox6 in Neuroblastoma Cells.
Metformin has recently emerged as a key player in promotion of neuroblastoma differentiation and neurite outgrowth. However, molecular mechanisms of how metformin promotes cellular differentiation have not yet been fully elucidated. In this study, we investigated how metformin promotes cell differentiation, via an inhibition of cell proliferation, by culturing SH-SY5Y neuroblastoma cells with or without metformin. Pretreatment with reactive oxygen species (ROS) scavenger, NAC, revealed that ROS plays a crucial role in induction of cell differentiation. Cell differentiation was observed under various morphological criteria: extension of neuritic processes and neuronal differentiation markers. Treatment with metformin significantly increased neurite length, number of cells with neurite, and expression of neuronal differentiation markers, -tubulin III and tyrosine hydroxylase (TH) compared with untreated control. Further investigation found that metformin significantly decreased Cdk5 but increased Sox6 during cell differentiation. Analysis of the mechanism underlying these changes using Cdk5 inhibitor, roscovitine, indicated that expressions of Cdk5 and Sox6 corresponded to metformin treatment. These results suggested that metformin produces neuronal differentiation via Cdk5 and Sox6. In addition, phosphorylated Erk1/2 was decreased while phosphorylated Akt was increased in metformin treatment. Taken together, these findings suggest that metformin promotes neuronal differentiation via ROS activation through Cdk5/Sox6 crosstalk, relating to Erk1/2 and Akt signaling.
Evid Based Complement Alternat Med, 2019; 2019
PRENATAL EXPOSURE TO ROSUVASTATIN CHANGES HISTONE METHYLATION PATTERNS IN THE NEWBORN RAT BRAIN
Karolina Fábián-Dulka, Melinda Szabó, Noemi Lajko, Istvan Belecz, Zsofia Hoyk, Karoly Gulya
Rosuvastatin is primarily used for the treatment of high cholesterol levels. While it has beneficial effects on inflammation and may reduce the risk of certain cancers, there are a few known side effects of the drug. Because of its potentially harmful, but not well documented, effects on the embryo, it is contraindicated during pregnancy. Our aim was to demonstrate whether rosuvastatin would elicit molecular epigenetic events, measured as histone methylations, in the brains of the newborn rats whose mothers had been treated with the drug. Pregnant female Sprague-Dawley rats were given daily oral doses of rosuvastatin from the 11. day of pregnancy for 10 days (or until delivery). On postnatal day 1 the cerebral hemispheres of control and rosuvastatin-treated rats were removed and either homogenized for Western blots or embedded in paraffin for fluorescent immunohistochemistry. Several antibodies that recognize different methylation sites for H2A, H2B, H3 and H4 histones were quantified in Western blots. Data were expressed as percent of control values. Prenatal rosuvastatin administration induced a general increase in histone methylation as compared to controls. Significant changes were detected in histone methylations that are known to result in transcriptional activation. For example, larger increases for H3K4me1 and H3K4me3 (134 and 128% of controls, respectively), while a smaller one (119%) for H2AK118me1 were measured. These observations were corroborated by multicolor immunofluorescence stainings on cerebral tissue sections that indicated that most of the histone methylation increase could be localized to neurons. This work was supported by GINOP 2.3.2-15-2016-00030 and 2.3.2-15-2016-00034.
TOPOGRAPHICAL SURFACES FOR BIOMECHANICAL CONTROL OF NEURAL STEM CELLS BEHAVIOUR
Miguel Esteban, Judith Estengre Pérez, Jaime J Hernández, María T Alameda, Sergio Dávila, Silvia García-López, Marta P Pereira, Alberto Martinez-Serrano, Isabel Rodriguez
Aims: The use of stem cells in medicine is of great interest for its huge therapeutic potential. There are numerous evidences that mechanical stimulus directs cellular behaviour and fate  and as such, there is a considerable interest in stablishing the relationship between the mechanical stimulus and the cell response obtained. Hence, this work examines cellular behaviour of immortalized human neural stem cells (hNS1) when cultured on dense high aspect ratio (HAR) polystyrene nanopillars with the final goal of guiding hNS1 fate commitment. Methods: The topography was prepared by nanoimprint lithography. O2 plasma treatment and poly-l-lysine coating were applied on substrates to improve cell adhesion. Immunofluorescence and electron microscopy were used to characterise cell morphology. Metabolic assays were used to assess cell viability and proliferation on substrates. Glial fibrillary acidic protein (GFAP), Doublecortin (DCX) and microtubule associated protein (MAP2) expression were used to assess differentiation. Results: It was found that HAR nanopillars had an influence over the hNS1 proliferation increasing population doubling time in comparison with controls. The hNS1 morphology was affected by topographical features as cells presented more round shape as well as a smaller cell projected area while attached to the nanopillars. The HAR nanopillars guided the orientation of hNS1 along the square pillar lattice where cytoplasmic projections along the perpendicular lattice directions were often exhibited. Conclusions: Our results suggest that HAR surface nanotopographies module neural stem cell behaviour in proliferation and differentiation conditions. References  Shin el al., Cell Stem Cell 18 (2016) 16-19
31546411: Santos-Coquillat A, Esteban-Lucia M, Martinez-Campos E, Mohedano M, Arrabal R, Blawert C, Zheludkevich ML, Matykina E
PEO coatings design for Mg-Ca alloy for cardiovascular stent and bone regeneration applications.
Four bioactive PEO (plasma electrolytic oxidation) coatings were generated on Mg0.8Ca alloy using a Ca/P-based electrolyte and adding Si or Fas necessary. Surface characteristics, chemical composition and ion liberation of the coatings were characterized using SEM/EDS (Scanning Electron Microscopy/Energy Dispersive X-ray spectroscopy), X-ray diffraction, optical profilometry and ICP-OES (inductively coupled plasma optical emission spectrometry). Direct biocompatibility studies were performed by seeding premyoblastic, endothelial and preosteoblastic cell lines over the coatings. Biocompatibility of the coatings was also evaluated with respect to murine endothelial, preosteoblastic, preosteoclastic and premyoblastic cell cultures using extracts obtained by the immersion degradation of the PEO-coated specimens. The coatings reduced the degradation of magnesium alloy and released Mg Ca, P, Si and F. Of all the studied compositions, the Si-containing PEO coating exhibited the optimal characteristics for use in all potential applications, including bone regeneration and cardiovascular applications. Coatings with high F content negatively influenced the endothelial cells. RAW 264.7, MC3T3 and co-culture differentiation studies using extracts of PEO coated Mg0.8Ca demonstrated improved osteoclastogenesis and osteoblastogenesis processes compared to bare alloy.
Mater Sci Eng C Mater Biol Appl, 2019; 105
STRIATAL NEURONS DERIVED FROM HUNTINGTON’S DISEASE PATIENT’S IPSC EXHIBIT MITOCHONDRIAL DYSFUNCTION AND ELECTROPHYSIOLOGICAL IMPAIRMENT
Margarida Beatriz1, Rita Vilaça1, Ricardo J. Rodrigues1, Thorsten Schlaeger2, George Daley2, Cristina Januário1, Carla Lopes1, Ana Cristina Rego1
1Portugal , 2United States of America
Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG expansion at the HTT gene that encodes for a misfolded protein. Several mechanisms have been implicated in HD, including mitochondrial dysfunction and synaptic impairment, leading to the loss of medium spiny neurons. This work aims to characterize mitochondrial function and dynamics in human neural stem cells (NSC) and striatal-like neurons (Ne) derived from symptomatic HD patient induced-pluripotent stem cells (iPSC). Fibroblasts from 2 HD patients and 2 controls were reprogrammed into iPSC. Mitochondrial function and dynamics were evaluated along the differentiation process from neural progenitors until mature Ne with 80 days in culture. HD-NSC exhibited a tendency for diminished maximal respiration and increased proton leak, when compared to controls, and increased levels of mitochondrial and cellular reactive oxygen species. Mitochondrial morphological analysis showed reduced area and complexity in HD-NSC. During striatal patterning and maturation, cultures expressed GABAergic markers (GAD65/67 and DARPP-32), with an increase in MAP2+/bIII-tubulin+ ratio, as well as a tendency for increasing the yield of DARPP-32+ neurons, particularly in control cells. The presence of SMI-32 neuronal marker assured full cell maturation. HD-Ne displayed decreased mitochondrial mean velocity, compared to controls. Lower frequency and amplitude of mIPSC were verified in HD-Ne as well as lower GABAR-mediated current density in comparison with controls. Taken together, mitochondria from HD-NSC presented altered mitochondrial function and morphology, while striatal-like HD-Ne exhibited electrophysiological impairment and reduced mitochondrial movement, constituting good human cell models for studying HD early pathogenesis.
NUCLEAR L1 TRANSCRIPTS REGULATE CHROMATIN STATE DURING NEURONAL DEVELOPMENT AND FUNCTIONS
Damiano Mangoni, Stefano Gustincich, Devid Damiani, Alessandro Simi
Aims: Long Interspersed Nuclear Elements 1 (L1s) are transposons which maintained the ability to increase their genomic copy number by a mechanism called retrotransposition. In the brain, L1 retrotransposition is supposed to be important for generating genomic diversity and increasing the plasticity of neuronal networks. We hypothesized that L1s may have a retrotransposition-independent role with L1 transcripts acting as an epigenetic factor that regulates chromatin organization and promotes the activation of transcriptional programs required for proper development and functionalities of neurons. Methods and Results: we found that both the expression and the half-life of L1 transcripts increase along neuronal maturation and occur in parallel with a decreased methylation of L1 promoter and expression of DNA methyltransferases 1 and 3b, lower levels of H3K9me3 and higher recruitment of H3K4me3. We investigated the cellular localization of L1 transcripts by subcellular RNA fractionation and we found that the majority of L1 transcripts are nuclear and chromatin-associated, with chromatinic L1s increasing at the late stages of neuronal development, suggesting that L1 transcripts may play a role for chromatin organization. Ongoing experiments are aimed to assess how L1 silencing affects chromatin accessibility and the transcriptional landscape of mouse developing neurons and the development of the mouse cerebral cortex by in utero electroporation. Conclusions: our findings outline that L1 transcripts are chromatin components that can have a role in the epigenomic regulation of neuronal development and could be useful to better understand how L1 dysregulations can affect cognitive and behavioral pathologies.
PGC-1 ALPHA DIRECTED NEURONAL DIFFERENTIATION REVERSES THE INHIBITORY EFFECTS OF CYPERMETHRIN ON HIPPOCAMPAL NEUROGENESIS
Anuradha Yadav, Rajnish Kumar Chaturvedi
The ability of NSCs to proliferate and generate new neurons is essential to maintain critical functions of the brain. Several studies have highlighted that environmental toxicants disrupt the process of neurogenesis and enhance neuronal degeneration resulting in cognitive disabilities. Cypermethrin is a type II pyrethroid insecticide widely used to control a broad range of insect pests. Despite beneficial roles of cypermethrin, its uncontrolled and repetitive use has lead to unintended effects in non-target organisms. In the present study, we evaluated the effects of cypermethrin exposure on neurogenesis and mitochondrial dynamics. We observed that cypermethrin treatment led to a considerable decrease in the expression of neurogenic and mitochondrial proteins resulting in compromised mitochondrial functions and dysregulated neurogenesis in the hippocampus. But we were clueless about the co-dependency/co-regulation of these two processes. Therefore, to understand the involvement of mitochondrial proteins in regulating neurogenesis we carried out genetic inhibition and overexpression of PGC-1α (key mitochondrial biogenesis protein) in both primary NSCs cultures and rat hippocampus. Interestingly, we found that knockdown of PGC-1α decreased NSC proliferation and neuronal differentiation. Furthermore, inhibition of PGC-1α in conjunction with cypermethrin treatment aggravated cypermethrin toxicity. Conversely, overexpression of PGC-1α via AdPGC-1α and nicotinamide rescued the inhibitory effects of cypermethrin on neurogenesis by replenishing the NSCs pool and improving cognitive functions in cypermethrin exposed rats. Overall, these results illustrate that PGC-1α has a key involvement in regulating the NSCs self-renewal, proliferation and switch of stem cell fate, and can serve as a potential target to ameliorate cypermethrin induced neurotoxicity.
30471306: Seth B, Yadav A, Tandon A, Shankar J, Chaturvedi RK
Carbofuran hampers oligodendrocytes development leading to impaired myelination in the hippocampus of rat brain.
During the mammalian brain development, oligodendrocyte progenitor cells (OPCs) are generated from neuroepithelium and migrate throughout the brain. Myelination is a tightly regulated process which involves time framed sequential events of OPCs proliferation, migration, differentiation and interaction with axons for functional insulated sheath formation. Myelin is essential for efficient and rapid conduction of electric impulses and its loss in the hippocampus of the brain may result in impaired memory and long-term neurological deficits. Carbofuran, a carbamate pesticide is known to cause inhibition of hippocampal neurogenesis and memory dysfunctions in rats. Nonetheless, the effects of carbofuran on OPCs proliferation, fate determination, maturation/differentiation and myelination potential in the hippocampus of the rat brain are still completely elusive. Herein, we investigated the effects of sub-chronic exposure of carbofuran during two different time periods including prenatal and adult brain development in rats. We observed carbofuran hampers OPCs proliferation (BrdU incorporation) and oligodendroglial differentiation in vitro. Similar effects of carbofuran were also observed in the hippocampus region of the brain at both the time points. Carbofuran exposure resulted in reduced expression of key genes and proteins involved in the regulation of oligodendrocyte development and functional myelination. It also affects the survival of oligodendrocytes by inducing apoptotic cell death. The ultrastructural analysis of myelin architecture clearly depicted carbofuran-mediated negative effects on myelin compaction and g-ratio alteration. Conclusively, our study demonstrated that carbofuran alters myelination potential in the hippocampus, which leads to cognitive deficits in rats.
Neurotoxicology, 2019; 70
28982980: Seth B, Yadav A, Agarwal S, Tiwari SK, Chaturvedi RK
Inhibition of the transforming growth factor-β/SMAD cascade mitigates the anti-neurogenic effects of the carbamate pesticide carbofuran.
The widely used carbamate pesticide carbofuran causes neurophysiological and neurobehavioral deficits in rodents and humans and therefore poses serious health hazards around the world. Previously, we reported that gestational carbofuran exposure has detrimental effects on hippocampal neurogenesis, the generation of new neurons from neural stem cells (NSC), in offspring. However, the underlying cellular and molecular mechanisms for carbofuran-impaired neurogenesis remain unknown. Herein, we observed that chronic carbofuran exposure from gestational day 7 to postnatal day 21 altered expression of genes and transcription factors and levels of proteins involved in neurogenesis and the TGF-β pathway ( TGF-β; SMAD-2, -3, and -7; and SMURF-2) in the rat hippocampus. We found that carbofuran increases TGF-β signaling ( increased phosphorylated SMAD-2/3 and reduced SMAD-7 expression) in the hippocampus, which reduced NSC proliferation because of increased p21 levels and reduced cyclin D1 levels. Moreover, the carbofuran-altered TGF-β signaling impaired neuronal differentiation (BrdU/DCX and BrdU/NeuN cells) and increased apoptosis and neurodegeneration in the hippocampus. Blockade of the TGF-β pathway with the specific inhibitor SB431542 and via SMAD-3 siRNA prevented carbofuran-mediated inhibition of neurogenesis in both hippocampal NSC cultures and the hippocampus, suggesting the specific involvement of this pathway. Of note, both and studies indicated that TGF-β pathway attenuation reverses carbofuran’s inhibitory effects on neurogenesis and associated learning and memory deficits. These results suggest that carbofuran inhibits NSC proliferation and neuronal differentiation by altering TGF-β signaling. Therefore, we conclude that TGF-β may represent a potential therapeutic target against carbofuran-mediated neurotoxicity and neurogenesis disruption.
J. Biol. Chem., 2017; 292
27514452: Agarwal S, Yadav A, Chaturvedi RK
Peroxisome proliferator-activated receptors (PPARs) as therapeutic target in neurodegenerative disorders.
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors and they serve to be a promising therapeutic target for several neurodegenerative disorders, which includes Parkinson disease, Alzheimer’s disease, Huntington disease and Amyotrophic Lateral Sclerosis. PPARs play an important role in the downregulation of mitochondrial dysfunction, proteasomal dysfunction, oxidative stress, and neuroinflammation, which are the major causes of the pathogenesis of neurodegenerative disorders. In this review, we discuss about the role of PPARs as therapeutic targets in neurodegenerative disorders. Several experimental approaches suggest potential application of PPAR agonist as well as antagonist in the treatment of neurodegenerative disorders. Several epidemiological studies found that the regular usage of PPAR activating non-steroidal anti-inflammatory drugs is effective in decreasing the progression of neurodegenerative diseases including PD and AD. We also reviewed the neuroprotective effects of PPAR agonists and associated mechanism of action in several neurodegenerative disorders both in vitro as well as in vivo animal models.
Biochem. Biophys. Res. Commun., 2017; 483
24606805: Yadav A, Agarwal S, Tiwari SK, Chaturvedi RK
Mitochondria: prospective targets for neuroprotection in Parkinson’s disease.
Parkinson’s disease is the second most common neurodegenerative disorder characterized by persistent loss of dopaminergic neurons in the SN and clinically associated with cognitive, behavioral and motor deficits. There is an enormous amount of data that provides convincing evidence about the prime involvement of mitochondria in the onset and progression of neurodegeneration. Several studies have also emphasized that accumulation of toxic protein and their aggregates in mitochondria lead to energy deficits, excessive ROS generation, mutations in mitochondrial genome and proteins regulating mitochondrial homeostasis, and impaired mitochondrial dynamics in animal models of PD and patients. Here we discuss about the bioenergetic agents, which have been tested for reducing the mitochondrial dysfunction and associated disease pathology in cellular and animal models of PD and PD patients with encouraging outcomes. We also provide a succinct overview of current therapeutic implications of PGC-1α, SIRT, AMPK, and Nrf2-ARE as salutary targets to overcome the deleterious effects posed by mitochondrial dysfunction in the onset and progression of PD.
Curr. Pharm. Des., 2014; 20
24467380: Tiwari SK, Agarwal S, Seth B, Yadav A, Nair S, Bhatnagar P, Karmakar M, Kumari M, Chauhan LK, Patel DK, Srivastava V, Singh D, Gupta SK, Tripathi A, Chaturvedi RK, Gupta KC
Curcumin-loaded nanoparticles potently induce adult neurogenesis and reverse cognitive deficits in Alzheimer’s disease model via canonical Wnt/β-catenin pathway.
Neurogenesis, a process of generation of new neurons, is reported to be reduced in several neurodegenerative disorders including Alzheimer’s disease (AD). Induction of neurogenesis by targeting endogenous neural stem cells (NSC) could be a promising therapeutic approach to such diseases by influencing the brain self-regenerative capacity. Curcumin, a neuroprotective agent, has poor brain bioavailability. Herein, we report that curcumin-encapsulated PLGA nanoparticles (Cur-PLGA-NPs) potently induce NSC proliferation and neuronal differentiation in vitro and in the hippocampus and subventricular zone of adult rats, as compared to uncoated bulk curcumin. Cur-PLGA-NPs induce neurogenesis by internalization into the hippocampal NSC. Cur-PLGA-NPs significantly increase expression of genes involved in cell proliferation (reelin, nestin, and Pax6) and neuronal differentiation (neurogenin, neuroD1, neuregulin, neuroligin, and Stat3). Curcumin nanoparticles increase neuronal differentiation by activating the Wnt/β-catenin pathway, involved in regulation of neurogenesis. These nanoparticles caused enhanced nuclear translocation of β-catenin, decreased GSK-3β levels, and increased promoter activity of the TCF/LEF and cyclin-D1. Pharmacological and siRNA-mediated genetic inhibition of the Wnt pathway blocked neurogenesis-stimulating effects of curcumin. These nanoparticles reverse learning and memory impairments in an amyloid beta induced rat model of AD-like phenotypes, by inducing neurogenesis. In silico molecular docking studies suggest that curcumin interacts with Wif-1, Dkk, and GSK-3β. These results suggest that curcumin nanoparticles induce adult neurogenesis through activation of the canonical Wnt/β-catenin pathway and may offer a therapeutic approach to treating neurodegenerative diseases such as AD, by enhancing a brain self-repair mechanism.
ACS Nano, 2014; 8
THE ROLE OF ARTX IN NEURONAL NUCLEOLUS AND NUCLEAR CHROMATIN ARCHITECTURE
Dagmara Holm-Kaczmarek, Adriana Magalska
ATRX has been ﬁrst described through efforts to identify the genetic lesion that contributes to the α-thalassemia mental retardation X-linked (ATRX) syndrome. It is a chromatin remodeler helicase that mediates H3.3 deposition in heterochromatin formation at repetitive DNA elements. Initial immunoﬂuorescence studies of ATRX shows it localization to condensed DAPI-dense regions, suggesting that ATRX is primarily associated with heterochromatin. We aimed to investigate whether ATRX is necessary for global structural changes of chromatin fold in an activity dependent manner (in chemLTP model). In our in vitro studies on hippocampal rat neurons the knock down of ATRX alters chromatin condensation by enlargement of heterochromatin foci number as well as changes of volume. Such manipulations with ATRX results in abnormal dendritic tree morphology and disturbs chromatin modifications pattern. We found that reduction of ATRX expression by viral infection of siRNA led to a hypermethylation of H4K20me2/me3 and to the formation of an interesting ring pattern of H3K14Ac around nucleolus. The nucleolus as site of ribosome biogenesis holds a pivotal role in cell metabolism and under stress conditions ribosomal synthesis is being reduced. We observe interesting ATRX bodies inside nucleolus whose quantity increases after chemLTP induction and viral infection. That might be related to response to stress which could led to the conclusion that ATRX might be involved in ribosomal synthesis regulation.
PROX1 AFFECTS AXON OUTGROWTH AND NEURONAL MATURATION DURING CENTRAL NERVOUS SYSTEM DEVELOPMENT
Valeria Kaltezioti, Iosifina Foskolou, Matthieu Lavigne, Maria Fousteri, Marigoula Margarity, Panagiotis Politis
During central nervous system (CNS) development, proper and timely induction of axon elongation is critical for generating functional, mature neurons and neuronal networks. Despite the wealth of information on the action of extracellular cues, little is known about the intrinsic gene regulatory factors that control this developmental decision. Here we report the identification of Prox1, a homeobox transcription factor, as a key player in inhibiting axon elongation. Although Prox1 promotes acquisition of early neuronal identity and is expressed in nascent post-mitotic neurons, it is heavily down-regulated in the majority of terminally differentiated neurons, indicating a regulatory role in delaying axon outgrowth in newly formed neurons. Consistently, here we show that Prox1 is sufficient to inhibit neurite extension in Neuro2A and SH-SY5Y cells. Furthermore, shRNA-mediated knockdown of Prox1 in Neuro2A cells induces the extension of neurites. More importantly, Prox1 overexpression suppresses axon elongation in primary neuronal cultures from mouse embryonic forebrain and spinal cord. Mechanistically, RNA-Seq analysis reveals that Prox1 affects critical pathways for neuronal maturation and neurite extension. Interestingly, Prox1 strongly inhibits many components of Ca2+ signaling pathway, an important mediator of axon extension and neuronal maturation. In accordance, Prox1 is also sufficient to repress Ca2+ entry upon KCl-mediated depolarization and reduce CREB phosphorylation. Collectively, these observations suggest that Prox1 acts as a potent suppressor of axon elongation by inhibiting Ca2+ signaling pathway. This inhibitory action may provide the appropriate time window for nascent neurons to find the correct position in the CNS prior to initiation of axon elongation.
BOOSTING POSTNATAL OLIGODENDROGENESIS IN A MOUSE MODEL OF MULTIPLE SCLEROSIS
Joana Mateus, Marta Alonso Gomes, Rita Soares, Sara Raquel Landeira Nabais Paulo, Ângelo F Chora, Ana M Sebastião, Sara Xapelli
Under Multiple Sclerosis pathological conditions, oligodendrocyte precursor cells (OPCs) present in the brain parenchyma or derived from subventricular zone neural stem cells (SVZ-NSCs) can differentiate into oligodendrocytes (OLs), which migrate and partially remyelinate the lesioned areas. Previous data from our group demonstrated that activation of adenosine A2A receptors (A2AR) modulated SVZ-NSCs oligodendroglial differentiation, both in vitro and in vivo under physiological conditions. Hence, we aimed at understanding the role of A2AR in adult oligodendrogenesis derived from SVZ-NSCs in an in vivo mouse model of MS. For this, the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS was developed, and behavioural tests were performed to evaluate motor function. Cellular differentiation was assessed by immunohistochemistry assays for bromodeoxyuridine (BrdU) colocalization with oligodendrocytic markers in brain regions of interest. Western blot and ELISA assays were used for myelin protein levels and inflammatory cytokine quantification. Our results for EAE model characterization showed that motor impairment is proportional to the score of the disease and cellular and molecular data showed an increase in the levels of the pro-inflammatory cytokine TNFα (n=5, p<0.01). A significant increase in NG2+BrdU+ cells in the corpus callosum (CC) of EAE mice was observed (n=3, p<0.05), hinting at the migration of OPCs from the SVZ to the CC. Ongoing studies encompass the in vivo modulation of A2AR and assessing its effect on EAE phenotype and adult oligodendrogenesis, ultimately unveiling the modulation of adult oligodendrogenesis derived from SVZ-NSCs by A2AR as a putative therapy for MS.
30959794: Rodrigues RS, Lourenço DM, Paulo SL, Mateus JM, Ferreira MF, Mouro FM, Moreira JB, Ribeiro FF, Sebastião AM, Xapelli S
Cannabinoid Actions on Neural Stem Cells: Implications for Pathophysiology.
With the increase of life expectancy, neurodegenerative disorders are becoming not only a health but also a social burden worldwide. However, due to the multitude of pathophysiological disease states, current treatments fail to meet the desired outcomes. Therefore, there is a need for new therapeutic strategies focusing on more integrated, personalized and effective approaches. The prospect of using neural stem cells (NSC) as regenerative therapies is very promising, however several issues still need to be addressed. In particular, the potential actions of pharmacological agents used to modulate NSC activity are highly relevant. With the ongoing discussion of cannabinoid usage for medical purposes and reports drawing attention to the effects of cannabinoids on NSC regulation, there is an enormous, and yet, uncovered potential for cannabinoids as treatment options for several neurological disorders, specifically when combined with stem cell therapy. In this manuscript, we review in detail how cannabinoids act as potent regulators of NSC biology and their potential to modulate several neurogenic features in the context of pathophysiology.
Molecules, 2019; 24
ALTERED EPIGENETIC REGULATION AFFECTS OPCS PROLIFERATION AND DIFFERENTIATION IN THE RARE DEMYELINATING DISEASE AGC1 DEFICIENCY
Eleonora Poeta, Sabrina Petralla, Martina Masotti, Francesca De Chirico, Michele Protti, Marco Virgili, Federico Giorgi, Laura Mercolini, Francesco Lasorsa, Barbara Monti
AGC1 deficiency is a rare genetic demyelinating disease caused by mutations in the SLC25A12 gene encoding for mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), which leads to secondary hypomyelination and altered myelin formation in CNS, most likely due to a reduction of N-acetyl aspartate (NAA) levels. Aim: After confirming defects in proliferation and early differentiation in siAGC1 Oli-neu cells (where a reduction up to 40% of the carrier activity by shRNA was induced) and in AGC1+/- neurospheres (vs. controls), we studied whether these defects could correlate with an altered epigenetic regulation of the molecular mechanisms. Methods: We analyzed the expression of factors involved in proliferation/differentiation as well as histones post-translational modifications and HDACs expression through proliferation analysis, WB and/or immunostainings in presence or absence of specific HDACs or HATs activity inhibitors. Results: We observed lower acetylation and different HDAC isoforms expression in both siAGC1 Oli-neu and AGC1+/- neurospheres vs. controls, as well as a reduction in proliferation and a parallel induction of differentiation when cells treated with the specific HDACs or HATs inhibitors. Conclusions: A different epigenetic regulation of proliferation/differentiation mechanisms seems to affect both siAGC1 Oli-neu cells and AGC1+/- neurospheres, which could be responsible for reduction of proliferation and parallel induction of differentiation we observed vs. controls. We are going to perform the same analysis on NSCs derived from iPS of AGC1 deficiency patients to confirm our hypothesis on the molecular mechanisms underlying this disease.
31609608: Scheiner M, Dolles D, Gunesch S, Hoffmann M, Nabissi M, Marinelli O, Naldi M, Bartolini M, Petralla S, Poeta E, Monti B, Falkeis C, Vieth M, Hübner H, Gmeiner P, Maitra R, Maurice T, Decker M
Dual-Acting Cholinesterase-Human Cannabinoid Receptor 2 Ligands Show Pronounced Neuroprotection in Vitro and Overadditive and Disease-Modifying Neuroprotective Effects in Vivo.
We have designed and synthesized a series of 14 hybrid molecules out of the cholinesterase (ChE) inhibitor tacrine and a benzimidazole-based human cannabinoid receptor subtype 2 (hCBR) agonist and investigated them in vitro and in vivo. The compounds are potent ChE inhibitors, and for the most promising hybrids, the mechanism of human acetylcholinesterase (hAChE) inhibition as well as their ability to interfere with AChE-induced aggregation of β-amyloid (Aβ), and Aβ self-aggregation was assessed. All hybrids were evaluated for affinity and selectivity for hCBR and hCBR. To ensure that the hybrids retained their agonist character, the expression of cAMP-regulated genes was quantified, and potency and efficacy were determined. Additionally, the effects of the hybrids on microglia activation and neuroprotection on HT-22 cells were investigated. The most promising in vitro hybrids showed pronounced neuroprotection in an Alzheimer’s mouse model at low dosage (0.1 mg/kg, i.p.), lacking hepatotoxicity even at high dose (3 mg/kg, i.p.).
J. Med. Chem., 2019; 62
31514314: Petralla S, Peña-Altamira LE, Poeta E, Massenzio F, Virgili M, Barile SN, Sbano L, Profilo E, Corricelli M, Danese A, Giorgi C, Ostan R, Capri M, Pinton P, Palmieri F, Lasorsa FM, Monti B
Deficiency of Mitochondrial Aspartate-Glutamate Carrier 1 Leads to Oligodendrocyte Precursor Cell Proliferation Defects Both In Vitro and In Vivo.
Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 () gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate-aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor α (PDGFα) and Transforming Growth Factor βs (TGFβs). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency.
Int J Mol Sci, 2019; 20
31376562: Roldán-Peña JM, Romero-Real V, Hicke J, Maya I, Franconetti A, Lagunes I, Padrón JM, Petralla S, Poeta E, Naldi M, Bartolini M, Monti B, Bolognesi ML, López Ó, Fernández-Bolaños JG
Tacrine-O-protected phenolics heterodimers as multitarget-directed ligands against Alzheimer’s disease: Selective subnanomolar BuChE inhibitors.
Concerned by the devastating effects of Alzheimer’s disease, and the lack of effective drugs, we have carried out the design of a series of tacrine-phenolic heterodimers in order to tackle the multifactorial nature of the disease. Hybridization of both pharmacophores involved the modification of the nature (imino, amino, ether) and the length of the tether, together with the type (hydroxy, methoxy, benzyloxy), number and position of the substituents on the aromatic residue. Title compounds were found to be strong and selective inhibitors of human BuChE (from low nanomolar to subnanomolar range), an enzyme that becomes crucial in the more advanced stages of the disease. The lead compound, bearing an ether-type tether, had an IC50 value of 0.52 nM against human BuChE, and a selectivity index of 323, with an 85-fold increase of activity compared to parent tacrine; key interactions were analysed using molecular modelling. Moreover, it also inhibited the self-aggregation of Aβ42, lacking neurotoxicity up to 5 μM concentration, and showed neuroprotective activity in primary rat neurons in a serum and K+ deprivation model, widely employed for reproducing neuronal injury and senescence. Moreover, low hepatoxicity effects and complete stability under physiological conditions were found for that compound. So, overall, our lead compound can be considered as a promising multitarget-directed ligand against Alzheimer’s disease, and a good candidate for developing new drugs.
Eur J Med Chem, 2019; 181