DNAJB6, a Key Factor in Neuronal Sensitivity to Amyloidogenesis.

CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.

The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma.

Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with platelet-derived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.

Alpha-Synuclein Toxicity on Protein Quality Control, Mitochondria and Endoplasmic Reticulum.

Parkinson's disease (PD) is characterized by the presence of insoluble protein clusters containing α-synuclein. Impairment of mitochondria, endoplasmic reticulum, autophagy and intracellular trafficking proper function has been suggested to be caused by α-synuclein toxicity, which is also associated with the higher levels of ROS found in the aged brain and in PD. Oxidative stress leads to protein oligomerization and aggregation that impair autophagy and mitochondrial dynamics leading to a vicious cycle of organelles damage and neurodegeneration. In this review we focused on the role of α-synuclein dysfunction as a cellular stressor that impairs mitochondria, endoplasmic reticulum, autophagy and cellular dynamics culminating with dopaminergic depletion and the pathogenesis of PD.

Participation of perforin in mediating dopaminergic neuron loss in MPTP-induced Parkinson's disease in mice.

Both resident innate and peripheral immune aberrations have been demonstrated to influence Parkinson's disease (PD) progression. However, it is still enigmatic how and which immune components are lethal to the dopaminergic neuron in PD. We now show that levels of perforin, a pore-forming protein expressed in cytotoxic immune cells, was significantly increased in the serum of wild-type mice 4 weeks after injection of MPTP, a toxin used to induce PD-like symptoms. We demonstrate that perforin-deficiency attenuated the acute striatal dopamine reduction by 33%, ablated microglia activation 3 days post MPTP-injection; and retarded dopaminergic neuron death 4 weeks post MPTP-injection. Our study suggests that perforin plays a role in dopaminergic neuron loss in PD.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Multipotent stem cell factor UGS148 is a marker for tanycytes in the adult hypothalamus.

The present study describes for the first time the neural expression and distribution of UGS148, a protein encoded by the RIKEN cDNA63330403K07 gene that has been shown to be prominently and characteristically expressed in neural stem cells (NSCs). Based on its molecular structure, UGS148 is an intracellular protein expected to be involved in intracellular sorting, trafficking, exocytosis and membrane insertion of proteins. We demonstrate that UGS148 is highly expressed in embryonic NSCs as well as, albeit at low level, in the adult neurogenic niches, the subventricular zone and the hippocampal dentate gyrus. Interestingly, the highest expression level of UGS148 in the adult mouse brain was observed specifically in the neurogenic cells lining the third ventricle, the tanycytes. Our in vitro studies show the involvement of UGS148 in the regulation of the proliferation of NSCs.

Human oligodendrocytes in remyelination research.

Studies on myelination and oligodendrocyte development are inevitably linked with demyelinating conditions such as multiple sclerosis (MS), leukodystrophies or spinal cord injury (SCI). Chronic loss of myelin, subsequently leading to neurodegeneration, is the ultimate cause of severe and permanent disability. Thus, fast restoration of myelin (remyelination) is essential for circumventing demyelination-caused pathologies. Implantation of exogenous remyelinating cells has been considered as a potential remyelination strategy. Researchers have examined a variety of cell types endowed with myelin-forming capacity (oligodendrocytes, Schwann cells, olfactory ensheathing cells etc.) in vitro and in vivo for their potential application as myelin restoring cell grafts. This review gives a summary of studies on the generation and testing of pure suspensions of human oligodendrocytes as a clinically relevant, efficient cellular tool for treating myelin pathology. We start with a brief overview of the current knowledge on the development of human oligodendrocytes from the late stages of embryogenesis up to the early postnatal stage. Insight in the specific extrinsic and intrinsic factors regulating normal oligodendrogenesis is crucial in order to achieve and maintain a sufficient population of engraftable functional oligodendrocytes in vitro. We discuss potential sources of human oligodendrocytes, including novel oligodendrocyte generation strategies employing induced pluripotent stem cells (iPSCs) and direct conversion technology. Finally, we provide a systematic overview of (the outcome of) experimental studies, in which human oligodendrocytes were tested for their (re)myelination capacity and efficiency.

PET imaging of demyelination and remyelination in the cuprizone mouse model for multiple sclerosis: a comparison between [11C]CIC and [11C]MeDAS.

Multiple Sclerosis (MS) is a neurodegenerative disease characterized by demyelinated lesions. PET imaging using specific myelin radioligands might solve the lack of a specific imaging tool for diagnosing and monitoring demyelination and remyelination in MS patients. In recent years, a few tracers have been developed for in vivo PET imaging of myelin, but they have not been fully evaluated yet. In this study, we compared [(11)C]CIC and [(11)C]MeDAS as PET tracers for monitoring demyelination and remyelination in cuprizone-fed mice. The ex vivo biodistribution of [(11)C]CIC showed decreased tracer uptake in mice fed with 0.2% cuprizone diet for 5 weeks, as compared to control mice. However, tracer uptake did not increase again after normal diet was restored for 5 weeks (remyelination). Surprisingly, in vivo PET imaging with [(11)C]CIC in cuprizone-fed mice revealed a significant reduction in whole brain tracer uptake after 5 weeks of remyelination. No correlation between ex vivo biodistribution and in vivo imaging data was found for [(11)C]CIC (r(2)=0.15, p=0.11). However, a strong correlation was found for [(11)C]MeDAS (r(2)=0.88, p<0.0001). [(11)C]MeDAS ex vivo biodistribution revealed significant decreased brain uptake in the demyelination group, as compared to controls and increased the tracer uptake after 5 weeks of remyelination. [(11)C]MeDAS images showed a low background signal and clear uptake in the brain white matter and spinal cord. Taken together, the results of this comparative study between [(11)C]CIC and [(11)C]MeDAS clearly show that [(11)C]MeDAS is the preferred PET tracer to monitor myelin changes in the brain and spinal cord in vivo.

PET imaging of focal demyelination and remyelination in a rat model of multiple sclerosis: comparison of [11C]MeDAS, [11C]CIC and [11C]PIB.

PURPOSE: In this study, we compared the ability of [(11)C]CIC, [(11)C]MeDAS and [(11)C]PIB to reveal temporal changes in myelin content in focal lesions in the lysolecithin rat model of multiple sclerosis. Pharmacokinetic modelling was performed to determine the best method to quantify tracer uptake. METHODS: Sprague-Dawley rats were stereotactically injected with either 1 % lysolecithin or saline into the corpus callosum and striatum of the right brain hemisphere. Dynamic PET imaging with simultaneous arterial blood sampling was performed 7 days after saline injection (control group), 7 days after lysolecithin injection (demyelination group) and 4 weeks after lysolecithin injection (remyelination group). RESULTS: The kinetics of [(11)C]CIC, [(11)C]MeDAS and [(11)C]PIB was best fitted by Logan graphical analysis, suggesting that tracer binding is reversible. Compartment modelling revealed that all tracers were fitted best with the reversible two-tissue compartment model. Tracer uptake and distribution volume in lesions were in agreement with myelin status. However, the slow kinetics and homogeneous brain uptake of [(11)C]CIC make this tracer less suitable for in vivo PET imaging. [(11)C]PIB showed good uptake in the white matter in the cerebrum, but [(11)C]PIB uptake in the cerebellum was low, despite high myelin density in this region. [(11)C]MeDAS distribution correlated well with myelin density in different brain regions. CONCLUSION: This study showed that PET imaging of demyelination and remyelination processes in focal lesions is feasible. Our comparison of three myelin tracers showed that [(11)C]MeDAS has more favourable properties for quantitative PET imaging of demyelinated and remyelinated lesions throughout the CNS than [(11)C]CIC and [(11)C]PIB.

PET imaging of glucose metabolism, neuroinflammation and demyelination in the lysolecithin rat model for multiple sclerosis.

BACKGROUND: Injection of lysolecithin in the central nervous system results in demyelination accompanied by local activation of microglia and recruitment of monocytes. Positron-emission tomography (PET) imaging, using specific tracers, may be an adequate technique to monitor these events in vivo and therefore may become a tool for monitoring disease progression in multiple sclerosis (MS) patients. OBJECTIVES: The objective of this paper is to evaluate the potential of PET imaging in monitoring local lesions, using [(11)C]MeDAS, [(11)C]PK11195 and [(18)F]FDG as PET tracers for myelin density, microglia activation and glucose metabolism, respectively. METHODS: Sprague-Dawley rats were stereotactically injected with either 1% lysolecithin or saline in the corpus callosum and striatum of the right brain hemisphere. PET imaging was performed three days, one week and four weeks after injection. Animals were terminated after PET imaging and the brains were explanted for (immuno)histochemical analysis. RESULTS: PET imaging was able to detect local demyelination induced by lysolecithin in the corpus callosum and striatum with [(11)C]MeDAS and concomitant microglia activation and monocyte recruitment with [(11)C]PK11195. [(18)F]FDG imaging demonstrated that glucose metabolism was maintained in the demyelinated lesions. CONCLUSION: PET imaging with multiple tracers allows simultaneous in vivo monitoring of myelin density, neuroinflammation and brain metabolism in small MS-like lesions, indicating its potential to monitor disease progression in MS patients.

Differentiation of induced pluripotent stem cells into functional oligodendrocytes.

The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.

Bioluminescence imaging of Olig2-neural stem cells reveals improved engraftment in a demyelination mouse model.

A major issue in the potential application of neural stem cell (NSC)-based cell replacement therapy for demyelinating diseases is the question of the survival, functional behavior, and stability of implanted NSC-derived oligodendrocyte precursor cells (OPCs) over an extended period. To address this issue, we employed bioluminescence imaging (BLI) as a noninvasive longitudinal in vivo monitoring technique and followed the fate of NSCs isolated from luciferase-green fluorescent protein-actin transgenic mice after stereotactic implantation in the demyelinated corpus callosum of cuprizone-fed mice. We compared normal NSCs with NSCs that were primed to become OPCs by the induction of Olig2 overexpression (Olig2-NSCs). BLI, validated by immunohistochemistry, revealed that, after a steep cell loss after implantation during the first 3 weeks, approximately 10% of the Olig2-NSCs stably survived for 2 months after implantation, in contrast to <1% of the normal NSCs. Immunohistochemistry, at the light and electron microscopic levels, revealed that the majority of the surviving Olig2-NSCs had differentiated into an oligodendrocytic cell lineage and contributed to remyelination of axons in the corpus callosum. The number of axons remyelinated by the implanted cells, however, was a small fraction of the total number of axons remyelinated by endogenous oligodendrocytes. Apparently, most of the implanted NSCs did not survive the transition into an inappropriate non-neurogenic niche, compressed by surrounding host tissue, in hostile, inflammatory conditions created by activated microglia. Only the ones that managed to differentiate rapidly into a mature neural cell type and become functionally integrated survived.

Epigenetic mechanisms facilitating oligodendrocyte development, maturation, and aging.

The process of oligodendrocyte differentiation is regulated by a dynamic interaction between a genetic and an epigenetic program. Recent studies, addressing nucleosomal histone modifications have considerably increased our knowledge regarding epigenetic regulation of gene expression during oligodendrocyte development and aging. These results have generated new hypotheses regarding the mechanisms underlying the decreased efficiency of endogenous remyelination in response to demyelinating injuries with increasing age. In this review, we present an overview of the epigenetic mechanisms regulating gene expression at specific stages of oligodendrocyte differentiation and maturation as well as the changes that occur with aging.

The cuprizone animal model: new insights into an old story.

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease that affects the central nervous system and represents the most common neurological disorder in young adults in the Western hemisphere. There are several well-characterized experimental animal models that allow studying potential mechanisms of MS pathology. While experimental allergic encephalomyelitis is one of the most frequently used models to investigate MS pathology and therapeutic interventions, the cuprizone model reflects a toxic experimental model. Cuprizone-induced demyelination in animals is accepted for studying MS-related lesions and is characterized by degeneration of oligodendrocytes rather than by a direct attack on the myelin sheet. The present article reviews recent data concerning the cuprizone model and its relevance for MS. Particular focus is given to the concordance and difference between human MS patterns (types I-IV lesions) and cuprizone-induced histopathology, including a detailed description of the sensitive brain regions extending the observations to different white and grey matter structures. Similarities between pattern III lesions and cuprizone-induced demyelination and dissimilarities, such as inflamed blood vessels or the presence of CD3+ T cells, are outlined. We also aim to distinguish acute and chronic demyelination under cuprizone including processes such as spontaneous remyelination during acute demyelination. Finally, we point at strain and gender differences in this animal model and highlight the contribution of some growth factors and cytokines during and after cuprizone intoxication, including LIF, IGF-1, and PDGFalpha.

Differentiation of neural stem cells into oligodendrocytes: involvement of the polycomb group protein Ezh2.

The mechanisms underlying the regulation of neural stem cell (NSC) renewal and maintenance of their multipotency are still not completely understood. Self-renewal of stem cells in general implies repression of genes that encode for cell lineage differentiation. Enhancer of zeste homolog 2 (Ezh2) is a Polycomb group protein involved in stem cell renewal and maintenance by inducing gene silencing via histone methylation and deacetylation. To establish the role of Ezh2 in the maintenance and differentiation of NSCs, we have examined the expression of Ezh2 in NSCs isolated from embryonic (embryonic day 14) mice during proliferation and differentiation in vitro. Our results show that Ezh2 is highly expressed in proliferating NSCs. In accordance with its suggested role as a transcription repressor, the expression of Ezh2 decreased when the NSCs differentiated into neurons and was completely suppressed during differentiation into astrocytes. Surprisingly, Ezh2 remained highly expressed in NSCs that differentiated into an oligodendrocytic cell lineage, starting from oligodendrocyte precursor cells (OPCs) up to the immature (premyelinating) oligodendrocyte stage. To further establish the role of Ezh2 in NSC differentiation, we silenced and induced overexpression of the Ezh2 gene in NSCs. High levels of Ezh2 in differentiating NSCs appeared to be associated with an increase in oligodendrocytes and a reduction in astrocytes, whereas low levels of Ezh2 led to completely opposite effects. The increase in the number of oligodendrocytes induced by enhanced expression of Ezh2 could be ascribed to stimulation of OPC proliferation although stimulation of oligodendrocyte differentiation cannot be excluded. Disclosure of potential conflicts of interest is found at the end of this article.

In vivo bioluminescent imaging of Schwann cells in a poly(DL-lactide-epsilon-caprolactone) nerve guide.

Nerve guides seeded with Schwann cells (SCs) promote axonal regeneration in peripheral nerve lesions. We examined the applicability of bioluminescent imaging (BLI) for monitoring the fate of SCs in nerve guides after implantation. Rat SCs were transfected with the firefly luciferase (Fluc) gene and subsequently seeded in nerve guides, which were implanted subcutaneously in rats. In vivo bioluminescence of transfected SCs (Fluc-SCs) was assessed with a BLI system. Scans were validated ex vivo using immunocytochemistry and electron microscopy. We found that BLI enables longitudinal in vivo monitoring of Fluc-SCs, given that proper access of luciferin to the cells is assured.

Association between NAT2 polymorphisms and the risk of schizophrenia in a Northern Chinese Han population.

The gene that encodes N-acetyltransferase 2 (NAT2), an enzyme that plays a crucial role in the metabolism of many drugs and xenobiotics, is located on chromosome 8p22, one of the most convictive susceptibility loci of schizophrenia. NAT2 genetic polymorphisms lead to various enzyme acetylation phenotypes. In the present study, six selected NAT2 exonic single nucleotide polymorphisms were genotyped in an independent case-control sample of a Northern Chinese Han population to verify the possible association between NAT2 and schizophrenia. Three (rs1801280T/341C, rs1799930/G590A, and rs1208/A803G) of the six single nucleotide polymorphisms showed significant allele frequency differences between the case and the control groups after rigorous Bonferroni correction. One protective fast-acetylation haplotype (NAT2*4) and two risk slow acetylation haplotypes (NAT2*5B and NAT2*6A) were discovered to be associated with schizophrenia. Our results indicate that NAT2 may be a susceptibility gene for schizophrenia in this Chinese Han population, and the risk haplotypes might cause the impairment of NAT2 in metabolizing neurotoxic substances.

Deficient p75 low-affinity neurotrophin receptor expression does alter the composition of cellular infiltrate in experimental autoimmune encephalomyelitis in C57BL/6 mice.

We have shown earlier that induction of experimental autoimmune encephalomyelitis (EAE)-a model for the human disease multiple sclerosis-in C57BL/6 wild-type mice resulted in the expression of the p75 low-affinity neurotrophin receptor (p75NTR) in endothelial cells in the CNS. In comparison to the clinical manifestation of EAE observed in wild-type C57BL/6 mice, C57BL/6 mice deficient for p75NTR (p75NTR knockout mice) developed a more severe or even lethal disease and concomitant increased levels of inflammation in the CNS. In order to elucidate the role of endothelial p75NTR in cellular infiltration under these pathological circumstances, we have performed a more detailed, quantitative examination of the composition of the cellular infiltrate invading the CNS in EAE wild-type and EAE p75NTR knockout mice. We compared spinal cords of EAE wild-type with those of EAE p75NTR knockout mice of the same clinical score (3.5) using immunohistochemical markers for the cell types present in the infiltratory cuffs in EAE: T-cells, B-cells, monocytes, microglia, resident and infiltrating macrophages and polymorphonuclear cells. Interestingly, we detected that the proportion of B-cells, cells of the monocyte-macrophage lineage and polymorphonuclear cells in the infiltratory cuff of EAE-p75NTR knockout mice was decreased at the account of the proportion of T-cells which appeared to be almost doubled in comparison to the EAE wild-type mice. The altered composition of the infiltrate in p75NTR deficient mice argues for an involvement of endothelial p75NTR in the interaction between the inflamed endothelium and the activated cells of the immune system, in particular the T-cells, in EAE.

The Therapeutic Potential of Induced Pluripotent Stem Cells After Stroke: Evidence from Rodent Models.

Stroke is the second most common cause of death and the leading cause of disability in the world. About 30% of the people that are affected by stroke die within a year; 25% of the patients that survive stroke remain in need of care after a year. Therefore, stroke is a major burden for health care costs. The most common subtype is ischemic stroke. This type is characterized by a reduced and insufficient blood supply to a certain part of the brain. Despite the high prevalence of stroke, the currently used therapeutic interventions are limited. No therapies that aim to restore damaged neuronal tissue or to promote recovery are available nowadays. Transplantation of stem cell-derived cells has been investigated as a potential regenerative and protective treatment. Embryonic stem cell (ESC)-based cell therapy in rodent models of stroke has been shown to improve functional outcome. However, the clinical use of ESCs still raises ethical questions and implantation of ESC-derived cells requires continuous immunosuppression. The groundbreaking detection of induced pluripotent stem cells (iPSCs) has provided a most promising alternative. This mini-review summarizes current literature in which the potential use of iPSC-derived cells has been tested in rodent models of stroke. iPSC-based cell therapy has been demonstrated to improve motor function, decrease stroke volume, promote neurogenesis and angiogenesis and to exert immunomodulatory, anti-inflammatory effects in the brain of stroke-affected rodents.

Comparison of Human Primary with Human iPS Cell-Derived Dopaminergic Neuron Grafts in the Rat Model for Parkinson's Disease.

Neuronal degeneration within the substantia nigra and the loss of the dopaminergic nigro-striatal pathway are the major hallmarks of Parkinson's disease (PD). Grafts of foetal ventral mesencephalic (VM) dopaminergic (DA) neurons into the striatum have been shown to be able to restore striatal dopamine levels and to improve overall PD symptoms. However, human foetus-derived cell grafts are not feasible for clinical application. Autologous induced pluripotent stem cell (iPS cell)-derived DA neurons are emerging as an unprecedented alternative. In this review, we summarize and compare the efficacy of human iPS cell-derived DA neuron grafts to restore normal behaviour in a rat model for PD with that of human foetal primary DA neurons. The differences we observed in the efficacy to restore normal function between the 2 types of DA neuron grafts could be ascribed to intrinsic properties of the iPS cell-derived DA neurons that critically affected survival and proper neurite extension in the striatum after implantation.