Alpha-Synuclein Expression in the Oligodendrocyte Lineage: an In Vitro and In Vivo Study Using Rodent and Human Models.
ABSTRACT: In this study, we sought evidence for alpha-synuclein (ASYN) expression in oligodendrocytes, as a possible endogenous source of ASYN to explain its presence in glial inclusions found in multiple system atrophy (MSA) and Parkinson's disease (PD). We identified ASYN in oligodendrocyte lineage progenitors isolated from the rodent brain, in oligodendrocytes generated from embryonic stem cells, and in induced pluripotent stem cells produced from fibroblasts of a healthy individual and patients diagnosed with MSA or PD, in cultures in vitro. Notably, we observed a significant decrease in ?SYN during oligodendrocyte maturation. Additionally, we show the presence of transcripts in PDGFR?/CD140a(+) cells and SOX10(+) oligodendrocyte lineage nuclei isolated by FACS from rodent and human healthy and diseased brains, respectively. Our work identifies ASYN in oligodendrocyte lineage cells, and it offers additional in vitro cellular models that should provide significant insights of the functional implication of ASYN during oligodendrocyte development and disease.
Project description:The mechanisms underlying the specification of oligodendrocyte fate from multipotent neural progenitor cells (NPCs) in developing human brain are unknown. In this study, we sought to identify antigens sufficient to distinguish NPCs free from oligodendrocyte progenitor cells (OPCs). We investigated the potential overlap of NPC and OPC antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFαR antigens. Surprisingly, we found that CD133, but not A2B5, was capable of enriching for OLIG2 expression, Sox10 enhancer activity, and oligodendrocyte potential. As a subpopulation of CD133-positive cells expressed CD140a, we asked whether CD133 enriched bone fide NPCs regardless of CD140a expression. We found that CD133(+)CD140a(-) cells were highly enriched for neurosphere initiating cells and were multipotent. Importantly, when analyzed immediately following isolation, CD133(+)CD140a(-) NPCs lacked the capacity to generate oligodendrocytes. In contrast, CD133(+)CD140a(+) cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Gene expression analysis further confirmed the stem cell nature of CD133(+)CD140a(-) cells. As human CD133(+) cells comprised both NPCs and OPCs, CD133 expression alone cannot be considered a specific marker of the stem cell phenotype, but rather comprises a heterogeneous mix of glial restricted as well as multipotent neural precursors. In contrast, CD133/CD140a-based FACS permits the separation of defined progenitor populations and the study of neural stem and oligodendrocyte fate specification in the human brain.
Project description:Human oligodendrocyte progenitor cell (OPC) specification and differentiation occurs slowly and limits the potential for cell-based treatment of demyelinating disease. In this study, using FACS-based isolation and microarray analysis, we identified a set of transcription factors expressed by human primary CD140a(+)O4(+) OPCs relative to CD133(+)CD140a(-) neural stem/progenitor cells (NPCs). Among these, lentiviral overexpression of transcription factors ASCL1, SOX10, and NKX2.2 in NPCs was sufficient to induce Sox10 enhancer activity, OPC mRNA, and protein expression consistent with OPC fate; however, unlike ASCL1 and NKX2.2, only the transcriptome of SOX10-infected NPCs was induced to a human OPC gene expression signature. Furthermore, only SOX10 promoted oligodendrocyte commitment, and did so at quantitatively equivalent levels to native OPCs. In xenografts of shiverer/rag2 animals, SOX10 increased the rate of mature oligodendrocyte differentiation and axon ensheathment. Thus, SOX10 appears to be the principle and rate-limiting regulator of myelinogenic fate from human NPCs.
Project description:Multiple system atrophy (MSA) is characterized by the presence of distinctive glial cytoplasmic inclusions (GCIs) within oligodendrocytes that contain the neuronal protein alpha-synuclein (aSyn) and the oligodendroglia-specific phosphoprotein TPPP/p25?. However, the role of oligodendroglial aSyn and p25? in the formation of aSyn-rich GCIs remains unclear. To address this conundrum, we have applied human aSyn (haSyn) pre-formed fibrils (PFFs) to rat wild-type (WT)-, haSyn-, or p25?-overexpressing oligodendroglial cells and to primary differentiated oligodendrocytes derived from WT, knockout (KO)-aSyn, and PLP-haSyn-transgenic mice. HaSyn PFFs are readily taken up by oligodendroglial cells and can recruit minute amounts of endogenous aSyn into the formation of insoluble, highly aggregated, pathological assemblies. The overexpression of haSyn or p25? accelerates the recruitment of endogenous protein and the generation of such aberrant species. In haSyn PFF-treated primary oligodendrocytes, the microtubule and myelin networks are disrupted, thus recapitulating a pathological hallmark of MSA, in a manner totally dependent upon the seeding of endogenous aSyn. Furthermore, using oligodendroglial and primary cortical cultures, we demonstrated that pathology-related S129 aSyn phosphorylation depends on aSyn and p25? protein load and may involve different aSyn "strains" present in oligodendroglial and neuronal synucleinopathies. Importantly, this hypothesis was further supported by data obtained from human post-mortem brain material derived from patients with MSA and dementia with Lewy bodies. Finally, delivery of haSyn PFFs into the mouse brain led to the formation of aberrant aSyn forms, including the endogenous protein, within oligodendroglia and evoked myelin decompaction in WT mice, but not in KO-aSyn mice. This line of research highlights the role of endogenous aSyn and p25? in the formation of pathological aSyn assemblies in oligodendrocytes and provides in vivo evidence of the contribution of oligodendroglial aSyn in the establishment of aSyn pathology in MSA.
Project description:Recent studies demonstrate that astroglial cells can be directly converted into functional neurons or oligodendrocytes. Here, we report that a single transcription factor Sox10 could reprogram astrocytes into oligodendrocyte-like cells, in vivo. For transdifferentiation, Sox10-GFP expressing viral particles were injected into cuprizone-induced demyelinated mice brains after which we assessed for the presence of specific oligodendrocyte lineage cell markers by immunohistofluorescence (IHF). As control, another group of demyelinated mice received GFP expressing viral particles. After 3 weeks, the majority of transduced (GFP+) cells in animals which received control vector were astrocytes, while in animals which received Sox10-GFP vector, the main population of GFP+ cells were positive for oligodendrocyte lineage markers. We also extracted primary astrocytes from mouse pups and purified them. Primary astrocytes were transduced in vitro and then transplanted into demyelinated brains for later fate mapping. After three weeks, in vitro transduced and then transplanted astrocytes showed oligodendrocyte progenitor and mature oligodendrocyte markers. Further confirmation was done by transduction of astrocytes with lentiviral particles that expressed Sox10 and GFP and their culture in the oligodendrocyte progenitor medium. The induced cells expressed oligodendrocyte progenitor cells (iOPCs) markers. Our findings showed the feasibility of reprogramming of astrocytes into oligodendrocyte-like cells in vivo, by using a single transcription factor, Sox10. This finding suggested a master regulatory role for Sox10 which enabled astrocytes to change their fate to OPC-like cells and establish an oligodendroglial phenotype. We hope this approach lead to effective myelin repair in patients suffering from myelination deficit.
Project description:The oligodendrocyte lineage genes (Olig1/2), encoding basic helix-loop-helix transcription factors, were first identified in screens for master regulators of oligodendrocyte development. OLIG1 is important for differentiation of oligodendrocyte precursors into myelin-forming oligodendrocytes during development and is thought to play a crucial role in remyelination during multiple sclerosis. However, it is still unclear how OLIG1 interacts with its transcriptional cofactors and DNA targets. OLIG1 was reportedly restricted to mammals, but we demonstrate here that zebrafish and other teleosts also possess an OLIG1 homolog. In zebrafish, as in mammals, Olig1 is expressed in the oligodendrocyte lineage. Olig1 associates physically with another myelin-associated transcription factor, Sox10, and the Olig1/Sox10 complex activates mbp (myelin basic protein) transcription via conserved DNA sequence motifs in the mbp promoter region. In contrast, Olig2 does not bind to Sox10 in zebrafish, although both OLIG1 and OLIG2 bind SOX10 in mouse.
Project description:Cells of the oligodendrocyte lineage, which form the myelinating glia of the vertebrate central nervous system, undergo a stepwise developmental progression entailing specification from neuroepithelial precursors, proliferation, migration to expand and distribute the population, and differentiation to ensheath axons with myelin. Understanding the genetic mechanisms that regulate each of these steps during development is important, because this might lead to therapies to promote remyelination following neural injury or disease. Genetic studies in mice indicated that the Sox10 transcription factor is required during the differentiation stage to promote myelin gene expression. However, whether Sox10 also promotes other features of oligodendroctye differentiation remained unknown. In this study, we used time-lapse imaging to investigate the behavior and fates of oligodendrocyte lineage cells in zebrafish embryos and larvae that lacked Sox10 function. This revealed that the myelinating subset of oligodendrocyte progenitor cells (OPCs) migrates, divides, and wraps axons normally, but then dies. Nonmyelinating oligodendrocyte progenitors divided more frequently, maintaining a normal population size. New oligodendrocytes produced by these progenitors wrapped axons and survived, but did not express myelin genes at high levels. We conclude that, in addition to promoting myelin gene expression, Sox10 function is necessary for the survival of myelinating oligodedrocytes subsequent to axon wrapping but is not required for the survival of nonmyelinating OPCs.
Project description:The mechanism that causes neural stem cells in the central nervous system to switch from neurogenesis to gliogenesis is poorly understood. Here we analyzed spinal cord development of mice in which the transcription factor Sox9 was specifically ablated from neural stem cells by the CRE/loxP recombination system. These mice exhibit defects in the specification of oligodendrocytes and astrocytes, the two main types of glial cells in the central nervous system. Accompanying an early dramatic reduction in progenitors of the myelin-forming oligodendrocytes, there was a transient increase in motoneurons. Oligodendrocyte progenitor numbers recovered at later stages of development, probably owing to compensatory actions of the related Sox10 and Sox8, both of which overlap with Sox9 in the oligodendrocyte lineage. In agreement, compound loss of Sox9 and Sox10 led to a further decrease in oligodendrocyte progenitors. Astrocyte numbers were also severely reduced in the absence of Sox9 and did not recover at later stages of spinal cord development. Taking the common origin of motoneurons and oligodendrocytes as well as V2 interneurons and some astrocytes into account, stem cells apparently fail to switch from neurogenesis to gliogenesis in at least two domains of the ventricular zone, indicating that Sox9 is a major molecular component of the neuron-glia switch in the developing spinal cord.
Project description:Oligodendrocyte precursor cells (OPCs) are lineage-restricted progenitors generally limited in vivo to producing oligodendrocytes. Mechanisms controlling genesis of OPCs are of interest because of their importance in myelin development and their potential for regenerative therapies in multiple sclerosis and dysmyelinating syndromes. We show here that the SoxE transcription factors (comprising Sox8, 9, and 10) induce multipotent neural precursor cells (NPCs) from the early postnatal subventricular zone (SVZ) to become OPCs in an autonomous manner. We performed a chromatin immunoprecipitation-based bioinformatic screen and identified Suppressor of Fused (Sufu) as a direct target of repression by Sox10. In vitro, overexpression of Sufu blocked OPC production, whereas RNAi-mediated inhibition augmented OPC production. Furthermore, mice heterozygous for Sufu have increased numbers of OPCs in the telencephalon during development. We conclude that Sox10 acts to restrict the potential of NPCs toward the oligodendrocyte lineage in part by regulating the expression of Sufu.
Project description:The mechanisms underlying the specification of oligodendrocyte fate from multipotent neural progenitor cells (NPCs) in developing human brain are unknown. In this study, we sought to identify antigens sufficient to distinguish NPCs free from oligodendrocyte progenitor cells (OPCs). We investigated the potential overlap of NPC and OPC antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFaR antigens. Surprisingly, we found that CD133, but not A2B5, was capable of enriching for OLIG2 expression, Sox10 enhancer activity, and oligodendrocyte potential. As a subpopulation of CD133- positive cells expressed CD140a, we asked whether CD133 enriched bone fide NPCs regardless of CD140a expression. We found that CD133+CD140a- cells were highly enriched for neurosphere initiating cells and were multipotent. Importantly, when analyzed immediately following isolation, CD133+CD140a- NPCs lacked the capacity to generate oligodendrocytes. In contrast, CD133+CD140a+ cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Gene expression analysis further confirmed the stem cell nature of CD133+CD140a- cells. As human CD133+ cells comprised both NPCs and OPCs, CD133 expression alone cannot be considered a specific marker of the stem cell phenotype, but rather comprises a heterogeneous mix of glial restricted as well as multipotent neural precursors. In contrast, CD133/CD140a-based FACS permits the separation of defined progenitor populations and the study of neural stem and oligodendrocyte fate specification in the human brain. 12 samples, 4 groups (FACS-sorted cell populations),3 replicates in each group, each replicate is from a separate patient sample
Project description:In the mature rodent brain, Sonic Hedgehog (Shh) signaling regulates stem and progenitor cell maintenance, neuronal and glial circuitry and brain repair. However, the sources and distribution of Shh mediating these effects are still poorly characterized. Here, we report in the adult mouse brain, a broad expression pattern of Shh recognized by the specific monoclonal C9C5 antibody in a subset (11-12%) of CC1+ mature oligodendrocytes that do not express carbonic anhydrase II. These cells express also Olig2 and Sox10, two oligodendrocyte lineage-specific markers, but not PDGFR?, a marker of oligodendrocyte progenitors. In agreement with oligodendroglial cells being a source of Shh in the adult mouse brain, we identify Shh transcripts by single molecule fluorescent in situ hybridization in a subset of cells expressing Olig2 and Sox10 mRNAs. These findings also reveal that Shh expression is more extensive than originally reported. The Shh-C9C5-associated signal labels the oligodendroglial cell body and decorates by intense puncta the processes. C9C5+ cells are distributed in a grid-like manner. They constitute small units that could deliver locally Shh to its receptor Patched expressed in GFAP+ and S100?+ astrocytes, and in HuC/D+ neurons as shown in PtcLacZ/+ reporter mice. Postnatally, C9C5 immunoreactivity overlaps the myelination peak that occurs between P10 and P20 and is down regulated during ageing. Thus, our data suggest that C9C5+CC1+ oligodendroglial cells are a source of Shh in the mouse postnatal brain.