Mutant protein kinase Cgamma found in spinocerebellar ataxia type 14 is susceptible to aggregation and causes cell death

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease characterized by various symptoms including cerebellar ataxia. Recently, several missense mutations in the protein kinase Cgamma (gammaPKC) gene have been found in different SCA14 families. To elucidate how the mutant gammaPKC causes SCA14, we examined the molecular properties of seven mutant (H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L) gammaPKCs fused with green fluorescent protein (gammaPKC-GFP). Wild-type gammaPKC-GFP was expressed ubiquitously in the cytoplasm of CHO cells, whereas mutant gammaPKC-GFP tended to aggregate in the cytoplasm. The insolubility of mutant gammaPKC-GFP to Triton X-100 was increased and correlated with the extent of aggregation. gammaPKC-GFP in the Triton-insoluble fraction was rarely phosphorylated at Thr(514), whereas gammaPKC-GFP in the Triton-soluble fraction was phosphorylated. Furthermore, the stimulation of the P2Y receptor triggered the rapid aggregation of mutant gammaPKC-GFP within 10 min after transient translocation to the plasma membrane. Overexpression of the mutant gammaPKC-GFP caused cell death that was more prominent than wild type. The cytotoxicity was exacerbated in parallel with the expression level of the mutant. These results indicate that SCA14 mutations make gammaPKC form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.     

Cre‐mediated transgene activation in the developing and adult mouse brain

Summary: The neuron‐specific rat enolase (NSE) promoter was employed to establish transgenic mice expressing Cre recombinase in the central nervous system. Founders were crossed with dormant lacZ indicator mice and specificity as well as efficiency of Cre‐mediated transgene activation was determined by PCR and/or X‐gal staining. Whereas most transgenic lines exhibited Cre activity in early development resulting in widespread Cre activity, one line (NSE‐Cre26) expressed high levels of Cre in the developing and adult brain. With the exception of kidney, which showed occasionally low level of Cre activity, Cre recombination in double transgenics was restricted to the nervous system. Whole‐mount X‐gal staining of 9.5 dpc embryos indicated Cre‐mediated lacZ expression in forebrain, hindbrain, and along the midbrain flexure. A similar expression pattern was observed during later stages of embryogenesis (11.5–13.5 dpc). In adult mice, Cre recombinase was expressed in cerebral cortex and cerebellum and high levels of Cre‐mediated lacZ expression were observed in hippocampus, cortex, and septum. The NSE‐Cre26 transgenic mouse line thus provides a useful tool to specifically overexpress and/or inactivate genes in the developing and adult brain. genesis 31:118–125, 2001. © 2001 Wiley‐Liss, Inc.     

Expression of DNA sequences containing neuron specific enolase gene in Escherichia coli.

There is evidence that the gene for gamma-gamma enolase (neuron specific enolase, NSE) is regulated during cell differentiation and development, conserved in a variety of organisms and contains mRNA destabilizing sequences. In order to investigate further the mechanisms of these processes and to obtain large quantity of this protein, the NSE gene was isolated from neuroblastoma cells and cloned in E. coli using standard molecular biology techniques. The NSE gene expression was studied and the expressed protein (recombinant NSE) was characterized extensively. The recombinant NSE behaves like parental NSE in antisera specificity, resistance for chaotropic agents like urea, thermal stability at higher temperatures etc. The physical parameters like secondary structure, hydrophilicity, antigenic index and flexibility of the expressed protein were studied. The results of the present investigation collectively form the basis for initial investigations of how the expression of NSE gene is regulated. This is the first report where the recombinant NSE gene has been characterized so extensively.     

NO-cGMP mediated galanin expression in NGF-deprived or axotomized sensory neurons

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5' proximal promoter fragment (- 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF-deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above-mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF-deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF-deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant-negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO-cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up-regulation of galanin, and for DRG neuron survival and regeneration after axotomy.     

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 502–516, 1997     

Frequent expression of neuroendocrine markers in mucinous tubular and spindle cell carcinoma of the kidney

Mucinous tubular and spindle cell carcinoma (MTSCC) is a new tumorous entity which has been recently established. In this article, we examined the expression of neuroendocrine markers including neuron specific enolase (NSE), chromogranin A and synaptophysin in 16 cases of MTSCC using immunohistochemistry. The sex ratio (male: female) of the patients was 4:12. In normal kidney, distal tubules or collecting ducts were positive for NSE, but no structures were positive for chromogranin A or synaptophysin. All MTSCCs showed a positive reaction for NSE. Additionally, fifteen of sixteen neoplasms (93.8%) with MTSCC showed the expression of either chromogranin A or synaptophysin or both. Finally, it is possible that MTSCC may be one of renal neoplasms which frequently exhibit the neuroendocrine differentiation.     

胎儿胰腺发育中CgA和NSE的表达及其意义

目的检测人胎儿胰腺中CgA和NSE的表达特征,初步探讨胎儿胰腺发育过程弥散神经内分泌系统的形成及其生物学作用.方法用免疫组织化学SP法,检测嗜铬素A(chromograin A,CgA)和神经元特异性烯醇化酶(neuron specific enolase,NSE)在不同胎龄胎儿胰腺组织中的表达.结果CgA在16-38周的胰腺中均有表达,随胎儿发育,CgA阳性细胞分布状态和反应程度均有差异变化;NSE在22-38周胎儿胰腺中表达,集中定位于胰腺的内分泌部细胞.结论CgA和NSE在人胎儿胰腺中的阳性表达,反映出弥散神经内分泌系统在胎儿胰腺中的形成过程;表明胎儿胰岛的形成及其DNES细胞的分泌,有调节胰腺外分泌部发育分化的作用,也提示胰岛DNES细胞通过调节血糖可能对胎儿个体发育具有重要影响.     

Immunogold electron microscopic demonstration of distinct submembranous localization of the activated gammaPKC depending on the stimulation.

We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that gammaPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of gammaPKC-GFP on the plasma membrane, Ca(2+) ionophore and NMDA rapidly translocated gammaPKC-GFP to the plasma membrane and then restricted gammaPKC-GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca(2+) influx alone induced the association of gammaPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of gammaPKC in response to various stimuli suggests a novel mechanism for PKC activation.     

Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment

Bone morphogenetic proteins (BMPs) promote astrocytic differentiation of cultured subventricular zone stem cells. To determine whether BMPs regulate the astrocytic lineage in vivo, transgenic mice were constructed that overexpress BMP4 under control of the neuron-specific enolase (NSE) promoter. Overexpression of BMP4 was first detectable by Western analysis on embryonic day 16 and persisted into the adult. The overexpression of BMP4 resulted in a remarkable 40% increase in the density of astrocytes in multiple brain regions accompanied by a decrease in the density of oligodendrocytes ranging between 11 and 26%, depending on the brain region and the developmental stage. No changes in neuron numbers or the pattern of myelination were detected, and there were no gross structural abnormalities. Similar phenotypes were observed in three independently derived transgenic lines. Coculture of transgenic neurons with neural progenitor cells significantly enhanced astrocytic lineage commitment by the progenitors; this effect was blocked by the BMP inhibitor Noggin, indicating that the stimulation of astrogliogenesis was due to BMP4 release by the transgenic neurons. These observations suggest that BMP4 directs progenitor cells in vivo to commit to the astrocytic rather than the oligodendroglial lineage. Further, differentiation of radial glial cells into astrocytes was accelerated, suggesting that radial glia were a source of at least some of the supernumerary astrocytes. Therefore, BMPs are likely important mediators of astrocyte development in vivo.     

谷氨酸转运体GLAST mRNA在大鼠脑内表达的细胞定位

实验采用荧光双标技术研究谷氨酸转运体ＧＬＡＳＴ ｍ ＲＮＡ 在大鼠脑内表达的细胞定位, 研究表明, 在星形神经胶质细胞和神经元, ＧＬＡＳＴｍ ＲＮＡ 分别与神经胶质纤维蛋白（ＧＦＡＰ） 和神经元特异性烯醇化酶 （ＮＳＥ） 有表达共存, 提示ＧＬＡＳＴ ｍ ＲＮＡ在星形神经胶质细胞和神经元上都有表达。     

Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo.     

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats

Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially neuron specific enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague–Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100 µg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.     

Episodic activation of the rat GnRH promoter: role of the homeoprotein oct-1

Recent reports demonstrate that the rat GnRH promoter is activated in an episodic fashion in immortalized GnRH neurons, but little information is available on molecular processes that contribute to this phenomenon. In this study, we dissected the regions of the rat GnRH promoter that mediate these effects by testing a series of 5' deletion luciferase reporter constructs on the pattern of photonic emissions from single, living GT1-7 GnRH neuronal cells. Deletion analysis revealed that the region -2012/-1597 that contains the neuron-specific enhancer (NSE) was required for the elaboration of pulses of GnRH promoter activity. The importance of this region was supported by observations that episodic reporter activity could be transferred to a neutral nonpulsatile promoter (Rous sarcoma virus, RSV(180)). Immunoneutralization of Oct-1 as well as mutation of an octamer binding site located at -1787/-1783 (AT-a site) blocked the pulsatile GnRH promoter activity in GT1-7 neuronal cells. Taken together, our findings indicate that episodic GnRH gene expression is a promoter-dependent phenomenon, which is mediated by Oct-1 interaction with regulatory elements in the NSE region.     

A cell system with targeted disruption of the SMN gene: functional conservation of the SMN protein and dependence of Gemin2 on SMN

The motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenous SMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.     

Neuroprotection by α-Lipoic Acid in Streptozotocin-Induced Diabetes

Glial cells provide structural and metabolic support for neurons, and these cells become reactive to any insult to the central nervous system. The streptozotocin (STZ) rat model was used to study glial reactivity and the prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP), S100B protein, and neuron specific enolase (NSE) was determined as well as lipid peroxidation (LPO) and glutathione (GSH) levels in some brain tissues. Western blot analyses showed GFAP, S100B, and NSE levels significantly increased under STZ-induced diabetes in brain, and LPO level increased as well. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels were increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis.     

Calcium: A novel and efficient inducer of differentiation of adipose‐derived stem cells into neuron‐like cells

This study comparatively investigated the effectiveness of calcium and other well‐known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose‐derived stem cells (ADSCs) into neuronal‐like cells. ADSCs were immunophenotyped and differentiated into neuron‐like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron‐specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real‐time polymerase chain reaction (quantitative real‐time polymerase chain reaction (qRT‐PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite‐like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron‐like cells and instantaneous increase in the expression of neuronal markers.     

Neuroplasticity and neuroprotection in enteric neurons: Role of epithelial cells

Neurons of enteric nervous system (ENS) regulate intestinal epithelial cells (IEC) functions but whether IEC can impact upon the neurochemical coding and survival of enteric neurons remain unknown. Neuro-epithelial interactions were studied using a coculture model composed of IEC lines and primary culture of rat ENS or human neuroblastoma cells (SH-SY5Y). Neurochemical coding of enteric neurons was analysed by immunohistochemistry and quantitative PCR. Neuroprotective effects of IEC were tested by measuring neuron specific enolase (NSE) release or cell permeability to 7-amino-actinomycin D (7-AAD). Following coculture with IEC, the percentage of VIP-immunoreactive (IR) neurons but not NOS-IR and VIP mRNA expression were significantly increased. IEC significantly reduced dopamine-induced NSE release and 7-AAD permeability in culture of ENS and SH-SY5Y, respectively. Finally, we showed that NGF had neuroprotective effects but reduced VIP expression in enteric neurons. In conclusion, our study identified a novel role for IEC in the regulation of enteric neuronal properties.