Alpha-Synuclein: The Spark That Flames Dopaminergic Neurons, In Vitro and In Vivo Evidence.

Mitochondria, α-syn fibrils and the endo-lysosomal system are key players in the pathophysiology of Parkinson's disease. The toxicity of α-syn is amplified by cell-to-cell transmission and aggregation of endogenous species in newly invaded neurons. Toxicity of α-syn PFF was investigated using primary cultures of dopaminergic neurons or on aged mice after infusion in the SNpc and combined with mild inhibition of GBA. In primary dopaminergic neurons, application of α-syn PFF induced a progressive cytotoxicity associated with mitochondrial dysfunction, oxidative stress, and accumulation of lysosomes suggesting that exogenous α-syn reached the lysosome (from the endosome). Counteracting the α-syn endocytosis with a clathrin inhibitor, dopaminergic neuron degeneration was prevented. In vivo, α-syn PFF induced progressive neurodegeneration of dopaminergic neurons associated with motor deficits. Histology revealed progressive aggregation of α-syn and microglial activation and accounted for the seeding role of α-syn, injection of which acted as a spark suggesting a triggering of cell-to-cell toxicity. We showed for the first time that a localized SNpc α-syn administration combined with a slight lysosomal deficiency and aging triggered a progressive lesion. The cellular and animal models described could help in the understanding of the human disease and might contribute to the development of new therapies.

Composite Fibrin and Carbon Microfibre Implant to Modulate Postraumatic Inflammation after Spinal Cord Injury.

Poor functional recovery after spinal cord injury (SCI) drives the development of novel strategies to manage this devastating condition. We recently showed promising immunomodulatory and pro-regenerative actions of bio-functionalized carbon microfibres (MFs) implanted in a rodent model of SCI. In order to maximize tissue repair while easing MF implantation, we produced a composite implant based on the embedding of several MFs within a fibrin hydrogel. We used intravital imaging of fluorescent reporter mice at the early stages and spinal sections of the same animals 3 months later to characterize the neuroinflammatory response to the implant and its impact on axonal regeneration. Whereas fibrin alone was inert in the first week, its enzymatic degradation drove the chronic activation of microglial cells and axonal degeneration within 3 months. However, the presence of MFs inside the fibrin hydrogel slowed down fibrin degradation and boosted the early recruitment of immune cells. Noteworthy, there was an enhanced contribution of monocyte-derived dendritic cells (moDCs), preceding a faster transition toward an anti-inflammatory environment with increased axonal regeneration over 3 months. The inclusion of MF here ensured the long-term biocompatibility of fibrin hydrogels, which would otherwise preclude successful spinal cord regeneration.

Contribution of Intravital Neuroimaging to Study Animal Models of Multiple Sclerosis.

Multiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fibers, axonopathy, and inflammatory responses leading to consequent sensory-motor and cognitive deficits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which offers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals.

Neuroblasts contribute to oligodendrocytes generation upon demyelination in the adult mouse brain.

After demyelinating insult, the neuronal progenitors of the adult mouse sub-ventricular zone (SVZ) called neuroblasts convert into oligodendrocytes that participate to the remyelination process. We use this rare example of spontaneous fate conversion to identify the molecular mechanisms governing these processes. Using in vivo cell lineage and single cell RNA-sequencing, we demonstrate that SVZ neuroblasts fate conversion proceeds through formation of a non-proliferating transient cellular state co-expressing markers of both neuronal and oligodendrocyte identities. Transition between the two identities starts immediately after demyelination and occurs gradually, by a stepwise upregulation/downregulation of key TFs and chromatin modifiers. Each step of this fate conversion involves fine adjustments of the transcription and translation machineries as well as tight regulation of metabolism and migratory behaviors. Together, these data constitute the first in-depth analysis of a spontaneous cell fate conversion in the adult mammalian CNS.

Deletion of YWHAE in a patient with periventricular heterotopias and pronounced corpus callosum hypoplasia.

BACKGROUND: Malformations of cortical development are not rare and cause a wide spectrum of neurological diseases based on the affected region in the cerebral cortex. A significant proportion of these malformations could have a genetic basis. However, genetic studies are limited because most cases are sporadic and mendelian forms are rare. METHODS: In order to identify new genetic causes in patients presenting defects of cortical organisation, array based comparative genomic hybridisation was performed in a cohort of 100 sporadic cases with various types of cortical malformations in search for inframicroscopic chromosomal rearrangements. RESULTS: In one patient presenting with periventricular nodular heterotopias and pronounced corpus callosum hypoplasia, a small (400 kb) 17p13.3 deletion involving the YWHAE gene was identified. It is shown that YWHAE is the only brain expressed gene in the deleted region and that the other genes in the interval are unlikely to contribute to the brain malformation phenotype of this patient. CONCLUSION: Most 17p13.3 deletions reported to date are large, such as the deletions causing Miller-Dieker syndrome, and involve several genes implicated in various steps of brain development. Haploinsufficiency of the mouse orthologue of YWHAE causes a defect of neuronal migration. However, the human counterpart of this phenotype was not known. The case described here represents the smallest reported deletion involving the YWHAE gene and could represent the human counterpart of the abnormal cortical organisation phenotype presented by the Ywhae heterozygous knockout mouse.

Intravital Assessment of Cells Responses to Conducting Polymer-Coated Carbon Microfibres for Bridging Spinal Cord Injury.

The extension of the lesion following spinal cord injury (SCI) poses a major challenge for regenerating axons, which must grow across several centimetres of damaged tissue in the absence of ordered guidance cues. Biofunctionalized electroconducting microfibres (MFs) that provide biochemical signals, as well as electrical and mechanical cues, offer a promising therapeutic approach to help axons overcome this blind journey. We used poly(3,4-ethylenedioxythiophene)-coated carbon MFs functionalized with cell adhesion molecules and growth factors to bridge the spinal cord after a partial unilateral dorsal quadrant lesion (PUDQL) in mice and followed cellular responses by intravital two-photon (2P) imaging through a spinal glass window. Thy1-CFP//LysM-EGFP//CD11c-EYFP triple transgenic reporter animals allowed real time simultaneous monitoring of axons, myeloid cells and microglial cells in the vicinity of the implanted MFs. MF biocompatibility was confirmed by the absence of inflammatory storm after implantation. We found that the sprouting of sensory axons was significantly accelerated by the implantation of functionalized MFs after PUDQL. Their implantation produced better axon alignment compared to random and misrouted axon regeneration that occurred in the absence of MF, with a most striking effect occurring two months after injury. Importantly, we observed differences in the intensity and composition of the innate immune response in comparison to PUDQL-only animals. A significant decrease of immune cell density was found in MF-implanted mice one month after lesion along with a higher ratio of monocyte-derived dendritic cells whose differentiation was accelerated. Therefore, functionalized carbon MFs promote the beneficial immune responses required for neural tissue repair, providing an encouraging strategy for SCI management.

Longitudinal Intravital Microscopy Reveals Axon Degeneration Concomitant With Inflammatory Cell Infiltration in an LPC Model of Demyelination.

Demyelination and axon degeneration are major events in all neurodegenerative diseases, including multiple sclerosis. Intoxication of oligodendrocytes with lysophosphatidylcholine (LPC) is often used as a selective model of focal and reversible demyelination thought to have no incidence for neurons. To characterize the cascade of cellular events involved in LPC-induced demyelination, we have combined intravital coherent antistoke Raman scattering microscopy with intravital two-photon fluorescence microscopy in multicolor transgenic reporter mice. Moreover, taking advantage of a unique technique of spinal glass window implantation, we here provide the first longitudinal description of cell dynamics in the same volume of interest over weeks after insults. We have detected several patterns of axon-myelin interactions and classified them in early and advanced events. Unexpectedly, we have found that oligodendrocyte damages are followed by axon degeneration within 2 days after LPC incubation, and this degeneration is amplified after the recruitment of the peripheral proinflammatory cells at day 4. Beyond day 7, the recovery of axon number and myelin takes 3 more weeks postlesion and involves a new wave of anti-inflammatory innate immune cells at day 14. Therefore, recurrent imaging over several weeks suggests an important role of peripheral immune cells in regulating both the axonal and oligodendroglial fates and thereby the remyelination status. Better understanding the recruitment of peripheral immune cells during demyelinating events should help to improve diagnosis and therapy.

Molecular characterization of a 1p36 chromosomal duplication and in utero interference define ENO1 as a candidate gene for polymicrogyria.

While chromosome 1p36 deletion syndrome is one of the most common terminal subtelomeric microdeletion syndrome, 1p36 microduplications are rare events. Polymicrogyria (PMG) is a brain malformation phenotype frequently present in patients with 1p36 monosomy. The gene whose haploinsufficiency could cause this phenotype remains to be identified. We used high-resolution arrayCGH in patients with various forms of PMG in order to identify chromosomal variants associated to the malformation and characterized the genes included in these regions in vitro and in vivo. We identified the smallest case of 1p36 duplication reported to date in a patient presenting intellectual disability, microcephaly, epilepsy, and perisylvian polymicrogyria. The duplicated segment is intrachromosomal, duplicated in mirror and contains two genes: enolase 1 (ENO1) and RERE, both disrupted by the rearrangement. Gene expression analysis performed using the patient cells revealed a reduced expression, mimicking haploinsufficiency. We performed in situ hybridization to describe the developmental expression profile of the two genes in mouse development. In addition, we used in utero electroporation of shRNAs to show that Eno1 inactivation in the rat causes a brain development defect. These experiments allowed us to define the ENO1 gene as the most likely candidate to contribute to the brain malformation phenotype of the studied patient and consequently a candidate to contribute to the malformations of the cerebral cortex observed in patients with 1p36 monosomy.

Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulfate production.

Myelin destruction is followed by resident glia activation and mobilization of endogenous progenitors (OPC) which participate in myelin repair. Here we show that in response to demyelination, mature oligodendrocytes (OLG) bordering the lesion express Ndst1, a key enzyme for heparan sulfates (HS) synthesis. Ndst1+ OLG form a belt that demarcates lesioned from intact white matter. Mice with selective inactivation of Ndst1 in the OLG lineage display increased lesion size, sustained microglia and OPC reactivity. HS production around the lesion allows Sonic hedgehog (Shh) binding and favors the local enrichment of this morphogen involved in myelin regeneration. In MS patients, Ndst1 is also found overexpressed in oligodendroglia and the number of Ndst1-expressing oligodendroglia is inversely correlated with lesion size and positively correlated with remyelination potential. Our study suggests that mature OLG surrounding demyelinated lesions are not passive witnesses but contribute to protection and regeneration by producing HS.

Promoting Myelin Repair through In Vivo Neuroblast Reprogramming.

Demyelination is frequently observed in a variety of CNS insults and neurodegenerative diseases. In rodents, adult neural stem cells can generate oligodendrocytes and participate to myelin repair. However, these cells mainly produce migratory neuroblasts that differentiate in the olfactory bulb. Here, we show that, in the demyelination context, a small subset of these neuroblasts can spontaneously convert into myelinating oligodendrocytes. Furthermore, we demonstrate that the contribution of neuroblasts to myelin repair can be improved by in vivo forced expression of two transcription factors: OLIG2 and SOX10. These factors promote directed fate conversion of endogenous subventricular zone neuroblasts into mature functional oligodendrocytes, leading to enhanced remyelination in a cuprizone-induced mouse model of demyelination. These findings highlight the unexpected plasticity of committed neuroblasts and provide proof of concept that they could be targeted for the treatment of demyelinated lesions in the adult brain.

Nhej1 Deficiency Causes Abnormal Development of the Cerebral Cortex.

DNA double-strand breaks (DSBs) frequently occur in rapidly dividing cells such as proliferating progenitors during central nervous system development. If they cannot be repaired, these lesions will cause cell death. The non-homologous end joining (NHEJ) DNA repair pathway is the only pathway available to repair DSBs in post-mitotic neurons. The non-homologous end joining factor 1 (Nhej1) protein is a key component of the NHEJ pathway. Nhej1 interacts with Xrcc4 and Lig4 to repair DSBs. Loss of function of Xrcc4 or Lig4 is embryonic lethal in the mouse while the loss of Nhej1 is not. Surprisingly, the brains of Nhej1-deficient mice appear to be normal although NHEJ1 deficiency in humans causes severe neurological dysfunction and microcephaly. Here, we studied the consequences of Nhej1 dysfunction for the development of the cerebral cortex using in utero electroporation of inactivating small hairpin RNAs (shRNAs) in the developing rat brain. We found that decreasing Nhej1 expression during neuronal migration phases causes severe neuronal migration defects visualized at embryonic stages by an accumulation of heterotopic neurons in the intermediate zone. Knocked-down cells die by 7 days after birth and the brain regions where RNA interference was achieved are structurally abnormal, suffering from a reduction of the width of the external cortical layers. These results indicate that the Nhej1 protein is necessary for proper rat cortical development. Neurons unable to properly repair DNA DSBs are unable to reach their final destination during the development and undergo apoptosis, leading to an abnormal cortical development.

Oligodendrogenesis in the normal and pathological central nervous system.

Oligodendrocytes (OLGs) are generated late in development and myelination is thus a tardive event in the brain developmental process. It is however maintained whole life long at lower rate, and myelin sheath is crucial for proper signal transmission and neuronal survival. Unfortunately, OLGs present a high susceptibility to oxidative stress, thus demyelination often takes place secondary to diverse brain lesions or pathologies. OLGs can also be the target of immune attacks, leading to primary demyelination lesions. Following oligodendrocytic death, spontaneous remyelination may occur to a certain extent. In this review, we will mainly focus on the adult brain and on the two main sources of progenitor cells that contribute to oligodendrogenesis: parenchymal oligodendrocyte precursor cells (OPCs) and subventricular zone (SVZ)-derived progenitors. We will shortly come back on the main steps of oligodendrogenesis in the postnatal and adult brain, and summarize the key factors involved in the determination of oligodendrocytic fate. We will then shed light on the main causes of demyelination in the adult brain and present the animal models that have been developed to get insight on the demyelination/remyelination process. Finally, we will synthetize the results of studies searching for factors able to modulate spontaneous myelin repair.

Disruption of the ATP8A2 gene in a patient with a t(10;13) de novo balanced translocation and a severe neurological phenotype.

Mental retardation is a frequent condition that is clinically and genetically highly heterogeneous. One of the strategies used to identify new causative genes is to take advantage of balanced chromosomal rearrangements in affected patients. We characterized a de novo t(10;13) balanced translocation in a patient with severe mental retardation and major hypotonia. We found that the balanced translocation is molecularly balanced. The translocation breakpoint disrupts the coding sequence of a single gene, called ATP8A2. The ATP8A2 gene is not ubiquitously expressed, but it is highly expressed in the brain. In situ hybridization performed in mouse embryos at different stages of development with the mouse homologue confirms this observation. A total of 38 patients with a similar phenotype were screened for mutations in the ATP8A2 gene but no mutations were found. The balanced translocation identified in this patient disrupts a single candidate gene highly expressed in the brain. Although this chromosomal rearrangement could be the cause of the severe phenotype of the patient, we were not able to identify additional cases. Extensive screening in the mentally retarded population will be needed to determine if ATP8A2 haploinsufficiency or dysfunction causes a neurological phenotype in humans.