MAB326R Sigma-AldrichAnti-CNPase Antibody, clone 11-5B

The critical period is a distinct time-window during the neonatal stage when animals display elevated sensitivity to certain environmental stimuli, and particular experiences can have profound and long-lasting effects on behaviors. Increasing evidence suggests that disruption of neuronal activity during the critical period contributes to autistic phenotype, although the pathogenic mechanism is largely unknown. Herein we show that extracellular signal-regulated protein kinases (ERKs) play important roles in proper formation of neural circuits during the critical period. Transient blockade of ERKs phosphorylation at postnatal day 6 (P6) by intraperitoneal injection of blood-brain barrier-penetrating MEK inhibitor, α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327) caused significant increase of apoptosis in the forebrain. Furthermore, this induced long-term deleterious effects on brain functioning later in adulthood, resulting in social deficits, impaired memory and reduced long-term potentiation (LTP). Conversely, blockade of ERK phosphorylation at P14 no longer induced apoptosis, nor behavioral deficits, nor the reduced LTP. Thus, surprisingly, these effects of ERKs are strongly age-dependent, indicating that phosphorylation of ERKs during the critical period is absolutely required for proper development of brain functioning. This study provides novel insight into the mechanistic basis for neurodevelopment disorders: various neurodevelopment disorders might be generally linked to defects in ERKs signaling during the critical period.

Glioblastoma (GBM) is the most common and deadly malignant brain cancer, with a median survival of less than 2 years. GBM displays a cellular complexity that includes brain tumour-initiating cells (BTICs), which are considered as potential key targets for GBM therapies. Here we show that the transcription factors FOXG1 and Groucho/TLE are expressed in poorly differentiated astroglial cells in human GBM specimens and in primary cultures of GBM-derived BTICs, where they form a complex. FOXG1 knockdown in BTICs causes downregulation of neural stem/progenitor and proliferation markers, increased replicative senescence, upregulation of astroglial differentiation genes and decreased BTIC-initiated tumour growth after intracranial transplantation into host mice. These effects are phenocopied by Groucho/TLE knockdown or dominant inhibition of the FOXG1:Groucho/TLE complex. These results provide evidence that transcriptional programmes regulated by FOXG1 and Groucho/TLE are important for BTIC-initiated brain tumour growth, implicating FOXG1 and Groucho/TLE in GBM tumourigenesis.

Stereology is the study of estimating geometric quantities. When successfully applied, the combination of immunohistochemistry (IHC) and stereology eliminates intra- and interobserver variability for cell type identification.We propose a method to validate existing antibody based cell type markers for stereological application. Comparison was made on the 100-days-old Göttingen minipig (G-mini) neocortex between estimates of total neuron number derived from Giemsa staining using morphological criteria and immunohistochemistry-based cell counting with NeuN. The mean total neuron numbers estimated by the two staining methods were not significantly different. Estimated quantities, including glial cell number, neocortical volume, cell densities and glial-to-neuron ratio were also presented. Additionally, we assessed other commonly used glial markers and discussed how to evaluate the advantages and disadvantages of these markers for stereological estimation of cell number.The concordance in quantitative estimates of total neuron number derived from NeuN- and Giemsa-stained sections provides evidence for the sensitivity and specificity of NeuN as a neuronal marker in the G-mini. Although time-consuming, quantitative validation of IHC should always be considered in stereological studies if there is doubt of the sensitivity, specificity, or reproducibility of cell type markers. Inaccurate staining may cause both over- and underestimation of the total cell number and inflict considerable limitation when analyzing the results.

Amyotrophic lateral sclerosis (ALS) is a paralytic and usually fatal disorder caused by motor-neuron degeneration in the brain and spinal cord. Most cases of ALS are sporadic but about 5-10% are familial. Mutations in superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP, also known as TDP43) and fused in sarcoma (FUS, also known as translocated in liposarcoma (TLS)) account for approximately 30% of classic familial ALS. Mutations in several other genes have also been reported as rare causes of ALS or ALS-like syndromes. The causes of the remaining cases of familial ALS and of the vast majority of sporadic ALS are unknown. Despite extensive studies of previously identified ALS-causing genes, the pathogenic mechanism underlying motor-neuron degeneration in ALS remains largely obscure. Dementia, usually of the frontotemporal lobar type, may occur in some ALS cases. It is unclear whether ALS and dementia share common aetiology and pathogenesis in ALS/dementia. Here we show that mutations in UBQLN2, which encodes the ubiquitin-like protein ubiquilin 2, cause dominantly inherited, chromosome-X-linked ALS and ALS/dementia. We describe novel ubiquilin 2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia cases with or without UBQLN2 mutations. Ubiquilin 2 is a member of the ubiquilin family, which regulates the degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Therefore, our findings link abnormalities in ubiquilin 2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, indicating a common pathogenic mechanism that can be exploited for therapeutic intervention.

Although there is growing evidence for a role of excess intracellular cations, particularly calcium ions, in neuronal and glial cell injury in multiple sclerosis, as well as in non-inflammatory neurological conditions, the molecular mechanisms involved are not fully determined. We previously showed that the acid-sensing ion channel 1 which, when activated under the acidotic tissue conditions found in inflammatory lesions opens to allow influx of sodium and calcium ions, contributes to axonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, the extent and cellular distribution of acid-sensing ion channel 1 expression in neurons and glia in inflammatory lesions is unknown and, crucially, acid-sensing ion channel 1 expression has not been determined in multiple sclerosis lesions. Here we studied acute and chronic experimental autoimmune encephalomyelitis and multiple sclerosis spinal cord and optic nerve tissues to describe in detail the distribution of acid-sensing ion channel 1 and its relationship with neuronal and glial damage. We also tested the effects of amiloride treatment on tissue damage in the mouse models. We found that acid-sensing ion channel 1 was upregulated in axons and oligodendrocytes within lesions from mice with acute experimental autoimmune encephalomyelitis and from patients with active multiple sclerosis. The expression of acid-sensing ion channel 1 was associated with axonal damage as indicated by co-localization with the axonal injury marker beta amyloid precursor protein. Moreover, blocking acid-sensing ion channel 1 with amiloride protected both myelin and neurons from damage in the acute model, and when given either at disease onset or, more clinically relevant, at first relapse, ameliorated disability in mice with chronic-relapsing experimental autoimmune encephalomyelitis. Together these findings suggest that blockade of acid-sensing ion channel 1 has the potential to provide both neuro- and myelo-protective benefits in multiple sclerosis.

Cyclic GMP (cGMP), produced in response to either nitric oxide (NO) or certain peptides, controls important neuronal functions. NG108-15 cells were used to characterize the expression of NO- and cGMP-generating proteins and to identify potential alterations associated with neuronal differentiation (neurite outgrowth). We find that these cells contain exclusively neuronal NO synthase (nNOS) isoforms as well as both NO- (soluble guanylyl cyclase, sGC) and natriuretic peptide- (natriuretic peptide receptor-A, NPR-A) responsive cGMP-producing enzymes. The sGC beta(1) subunit (unlike protein phosphatase 2A subunits) is highly membrane-associated. Membrane concentrations of NPR-A and nNOS, but not sGC beta(1) protein are up-regulated with neuronal differentiation. Intriguingly, the rate of hormone-induced cGMP production by NPR-A is significantly diminished in differentiated cells. These findings support roles for NPR-A, the common receptor of atrial (ANP) and B-type (BNP) natriuretic peptide in mature neurons and provide evidence for pronounced changes in neuronal submembrane cGMP signalling during neuronal differentiation.

Lack of appropriate tools and techniques to study fate and functional integration of newly generated neurons has so far hindered understanding of neurogenesis' relevance under physiological and pathological conditions. Current analyses are either dependent on mitotic labeling, for example BrdU-incorporation or retroviral infection, or on the detection of transient immature neuronal markers. Here, we report a transgenic mouse model (DCX-CreERT2) for time-resolved fate analysis of newly generated neurons. This model is based on the expression of a tamoxifen-inducible Cre recombinase under the control of a doublecortin (DCX) promoter, which is specific for immature neuronal cells in the CNS.In the DCX-CreERT2 transgenic mice, expression of CreERT2 was restricted to DCX+ cells. In the CNS of transgenic embryos and adult DCX-CreERT2 mice, tamoxifen administration caused the transient translocation of CreERT2 to the nucleus, allowing for the recombination of loxP-flanked sequences. In our system, tamoxifen administration at E14.5 resulted in reporter gene activation throughout the developing CNS of transgenic embryos. In the adult CNS, neurogenic regions were the primary sites of tamoxifen-induced reporter gene activation. In addition, reporter expression could also be detected outside of neurogenic regions in cells physiologically expressing DCX (e.g. piriform cortex, corpus callosum, hypothalamus). Four weeks after recombination, the vast majority of reporter-expressing cells were found to co-express NeuN, revealing the neuronal fate of DCX+ cells upon maturation.This first validation demonstrates that our new DCX-CreERT2 transgenic mouse model constitutes a powerful tool to investigate neurogenesis, migration and their long-term fate of neuronal precursors. Moreover, it allows for a targeted activation or deletion of specific genes in neuronal precursors and will thereby contribute to unravel the molecular mechanisms controlling neurogenesis.

Demyelination is prominent in experimental autoimmune encephalomyelitis (EAE). The receptor p75 and its high affinity ligand proNGF are required for oligodendrocyte death after injury. We hypothesize that bone marrow stromal cells (BMSCs) provide therapeutic benefit in EAE mice by reducing proNGF/p75 expression. PBS or BMSCs (2 x 10(circumflex)6) were administered intravenously on the day of EAE onset. Neurological function and demyelination areas were measured. Immunohistochemical staining was used to measure apoptotic oligodendrocytes, expression of proNGF and p75, and the relationship between proNGF and p75 in neural cells. proNGF was used to treat oligodendrocytes in culture with or without BMSCs. EAE mice exhibited neurological function deficit and demyelination, and expression of proNGF and p75 was increased. BMSC treatment improved functional recovery, reduced demyelination area and apoptotic oligodendrocytes, decreased expression of proNGF and p75 compared with PBS treatment. proNGF(+) cells colocalized with neural cell markers, while p75 colocalized with an oligodendrocytic marker, and proNGF colocalized with p75. proNGF induced apoptosis of oligodendrocytes in vitro, and p75 antibody blocked this apoptotic activity. BMSCs reduced p75 expression and apoptotic activity in oligodendrocytes with proNGF treatment. BMSC treatment benefits on EAE mice may be fostered by decreasing the cellular expression of proNGF and p75, thereby reducing oligodendrocyte death.

Periventricular leukomalacia (PVL) is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells.To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na+- and K+- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies.Surviving IFN-gamma and TNF-alpha treated cells continued to maintain voltage-activated Na+- and K+ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-gamma and TNF-alpha on cell survival, A2B5-immunostaining and expression of myelin basic protein. The most potent corticosteroid tested, dexamethasone, was shown to counteract cytokine effects on membrane surface extension and capacitance. Furthermore, coapplication of dexamethasone blocked the cytokine-induced downregulation of the inwardly rectifying potassium current in 80% of the precursor cells and restored the cytokine-blocked down-regulation of the voltage activated Na+- and K+ currents during subsequent differentiation.Our results show that treatment of oligodendrocyte precursors with the inflammatory cytokines TNF-alpha and IFN-gamma block the differentiation of oligodendrocyte precursors at the level of the differentiation of the voltage-gated ion currents. Co-treatment with corticosteroids at the time of cytokine application restores to a considerable extent survival and differentiation of oligodendrocytes at the level of morphological, myelin protein as well as ion current maturation suggesting the option for a functional restoration of cytokine-damaged immature oligodendrocytes.

Reproducible visualization of neurons and glia in human brain is essential for quantitative studies of the cellular changes in neurological disease. However, immunohistochemistry in human brain specimens is often compromised because of prolonged fixation. To select cell lineage-specific antibodies for quantitative studies of neurons and the major types of glia, we used 29 different antibodies, different epitope retrieval methods, and different detection systems to stain tissue arrays of formalin-fixed human brain. The screening pointed at CD45/leukocyte common antigen (LCA), CD68(KP1), 2',3' cyclic nucleotide phosphatase (CNPase), glial fibrillary acidic protein (GFAP), HLA-DR, Ki67, neuronal nuclei (NeuN), p25alpha-antigen, and S100beta as candidates for future cell counting purposes, because these markers visualized specific neuronal and glial cell bodies. However, significant negative correlation between staining result and formalin fixation was observed by blinded scoring of staining for CD45/LCA, CNPase, GFAP, and NeuN in brain specimens fixed by immersion and stored up to 10 years in 4% formalin solution at room temperature, independent of donor sex and postmortem interval. In contrast, improved preservation of NeuN and CNPase staining, and full preservation of GFAP and CD45/LCA staining in tissue fixed by perfusion and stored for up to 3 years in 0.1% paraformaldehyde solution at 4C, indicated that immunohistochemistry can be performed in well-preserved biobank material.