Erik Boddeke

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.

Induction of a common microglia gene expression signature by aging and neurodegenerative conditions: a co-expression meta-analysis

IntroductionMicroglia are tissue macrophages of the central nervous system that monitor brain homeostasis and react upon neuronal damage and stress. Aging and neurodegeneration induce a hypersensitive, pro-inflammatory phenotype, referred to as primed microglia. To determine the gene expression signature of priming, the transcriptomes of microglia in aging, Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) mouse models were compared using Weighted Gene Co-expression Network Analysis (WGCNA).ResultsA highly consistent consensus transcriptional profile of up-regulated genes was identified, which prominently differed from the acute inflammatory gene network induced by lipopolysaccharide (LPS). Where the acute inflammatory network was significantly enriched for NF-κB signaling, the primed microglia profile contained key features related to phagosome, lysosome, antigen presentation, and AD signaling. In addition, specific signatures for aging, AD, and ALS were identified.ConclusionMicroglia priming induces a highly conserved transcriptional signature with aging- and disease-specific aspects.

Effects of histone deacetylation inhibition on neuronal differentiation of embryonic mouse neural stem cells

Neural stem cells (NSCs) are multipotent cells that have the capacity for self-renewal and for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. The molecular mechanisms regulating gene transcription resulting in NSC differentiation and cell lineage specification are slowly being unraveled. An important mechanism in transcriptional regulation is modulation of chromatin by histone acetylation and deacetylation, allowing or blocking the access of transcriptional factors to DNA sequences. The precise involvement of histone acetyltransferases and histone deacetylases (HDACs) in the differentiation of NSCs into mature functional neurons is still to be revealed. In this in vitro study we have investigated the effects of the HDAC inhibitor trichostatin A (TSA) on the differentiation pattern of embryonic mouse NSCs during culture in a minimal, serum-free medium, lacking any induction or growth factor. We demonstrated that under these basic conditions TSA treatment increased neuronal differentiation of the NSCs and decreased astrocyte differentiation. Most strikingly, electrophysiological recordings revealed that in our minimal culture system only TSA-treated NSC-derived neurons developed normal electrophysiological membrane properties characteristic for functional, i.e. excitable and firing, neurons. Furthermore, TSA-treated NSC-derived neurons were characterized by an increased elongation and arborization of the dendrites. Our study shows that chromatin structure modulation by HDACs plays an important role in the transcriptional regulation of the neuronal differentiation of embryonic NSCs particularly as far as the development of functional properties are concerned. Manipulation of HDAC activity may be an important tool to generate specific neuronal populations from NSCs for transplantation purposes.

Cultured rat microglia express functional beta-chemokine receptors.

We have investigated the functional expression of the beta-chemokine receptors CCR1 to 5 in cultured rat microglia. RT-PCR analysis revealed constitutive expression of CCR1, CCR2 and CCR5 mRNA. The beta-chemokines MCP-1 (1-30 nM) as well as RANTES and MIP-1alpha (100-1000 nM) evoked calcium transients in control and LPS-treated microglia. Whereas, the response to MCP-1 was dependent on extracellular calcium the response to RANTES was not. The effect of MCP-1 but not that of RANTES was inhibited by the calcium-induced calcium release inhibitor ryanodine. Calcium responses to MCP-1- and RANTES were observed in distinct populations of microglia.

Olig2 overexpression induces the in vitro differentiation of neural stem cells into mature oligodendrocytes

Differentiation induction of neural stem cells (NSCs) into oligodendrocytes during embryogenesis is the result of a complex interaction between local induction factors and intracellular transcription factors. At the early stage of differentiation, in particular, the helix‐loop‐helix transcription factors Olig1 and Olig2 have been shown to be essential for oligodendrocyte lineage determination. In view of the possible application of NSCs as a source for remyelinating cell transplants in demyelinating diseases (e.g., multiple sclerosis), in vitro procedures need to be developed to drive the oligodendrocyte differentiation process. Mere culture in medium supplemented with major embryonic oligodendrogenic induction factors, such as Sonic hedgehog, results in oligodendrocyte differentiation of only about 10% of NSCs. We previously showed that induction of Olig1 expression by gene transfection could indeed initiate the first stage of oligodendrocyte differentiation in NSCs, but appeared to be unable to generate fully mature, functional oligodendrocytes. In this study, we transfected NSCs isolated from the embryonic mouse brain with the Olig2 gene and found that the introduced overexpression of Olig2 could induce the development of fully mature oligodendrocytes expressing the transcription factor Nkx2.2 and all major myelin‐specific proteins. Moreover, Olig2‐transfected NSCs, in contrast to nontransfected NSCs, developed into actively remyelinating oligodendrocytes after transplantation into the corpus callo‐sum of long‐term cuprizonefed mice, an animal model for demyelination. Our results show that transfection of genes encoding for oligodendrogenic transcription factors can be an efficient way to induce the differentiation of NSCs into functional oligodendrocytes.

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.

What is microglia neurotoxicity (Not)

Microglia most likely appeared early in evolution as they are not only present in vertebrates, but are also found in nervous systems of various nonvertebrate organisms. Mammalian microglia are derived from a specific embryonic, self‐renewable myeloid cell population that is throughout lifetime not replaced by peripheral myeloid cells. These phylogenic and ontogenic features suggest that microglia serve vital functions. Yet, microglia often are described as neurotoxic cells, that actively kill (healthy) neurons. Since it is from an evolutionary point of view difficult to understand why an important and vulnerable organ like the brain should host numerous potential killers, we here review the concept of microglia neurotoxicity. On one hand it is discussed that most of our understanding about how microglia kill neurons is based on in vitro experiments or correlative staining studies that suffer from the difficulty to discriminate microglia and peripheral myeloid cells in the diseased brain. On the other hand it is described that a more functional approach by mutating, inactivating or deleting microglia is seldom associated with a beneficial outcome in an acute injury situation, suggesting that microglia are normally important protective elements in the brain. This might change in chronic disease or the aged brain, where; however, it remains to be established whether microglia simply lose their protective capacities or whether microglia become truly neurotoxic cells. GLIA 2014;62:841–854

Mutations in potassium channel kcnd3 cause spinocerebellar ataxia type 19

To identify the causative gene for the neurodegenerative disorder spinocerebellar ataxia type 19 (SCA19) located on chromosomal region 1p21‐q21.

Functional expression of the fractalkine (CX3C) receptor and its regulation by lipopolysaccharide in rat microglia

Functional expression of CX3CR1, a recently discovered receptor for the chemokine fractalkine, was investigated in cultured rat microglia. Reverse transcriptase polymerase chain reaction (PCR) experiments show abundant expression of fractalkine receptor mRNA in microglia. mRNA expression of fractalkine was undetectable in astrocytes and microglia but was very strong in cortical neurons. Incubation of microglia with lipopolysaccharide (100 ng/ml) transiently suppressed expression of fractalkine receptor mRNA. Fractalkine induced a concentration-dependent (10(-10)-10(-8) M) and, at high concentrations, oscillatory mobilization of intracellular Ca2+ in microglia The concentration-response curve of fractalkine was shifted to the right after 12 h incubation with lipopolysaccharide. It is concluded that treatment with endotoxin downregulates expression of fractalkine receptor mRNA in rat microglia and suppresses the functional response to fractalkine.

Involvement of chemokines in pain

It is well established that neuroinflammation plays an important role in neurodegenerative diseases like Alzheimers disease, stroke, traumatic brain- and spinal cord injury and demyelinating diseases. Likewise, it has been suggested that neuroinflammation plays an important role in nociception and hyperalgesia. Most research concerning inflammatory aspects of pain has concerned the effects of proinflammatory cytokines, prostaglandins and growth factors. Recently, it has been suggested that chemokines play a role in inflammatory pain. Chemokines do not only attract blood leukocytes to the site of injury but also contribute directly to nociception.