Inhibition of catechol-O-methyl transferase (COMT) by tolcapone restores reductions in microtubule-associated protein 2 (MAP2) and synaptophysin (SYP) following exposure of neuronal cells to neurotropic HIV.

This investigation aimed to assess whether inhibition of cathecol-O-methyl transferase (COMT) by tolcapone could provide neuroprotection against HIV-associated neurodegenerative effects. This study was conducted based on a previous work, which showed that a single nucleotide polymorphism (SNP) at position 158 (val158met) in COMT, resulted in 40 % lower COMT activity. Importantly, this reduction confers a protective effect against HIV-associated neurocognitive disorders (HAND), which have been linked to HIV-associated brain changes. SH-SY5Y-differentiated neurons were exposed to macrophage-propagated HIV (neurotropic MACS2-Br strain) in the presence or absence of tolcapone for 6 days. RNA was extracted, and qPCR was performed using Qiagen RT2 custom array consisting of genes for neuronal and synaptic integrity, COMT and pro-inflammatory markers. Immunofluorescence was conducted to validate the gene expression changes at the protein level. Our findings demonstrated that HIV significantly increased the messenger RNA (mRNA) expression of COMT while reducing the expression of microtubule-associated protein 2 (MAP2) (p = 0.0015) and synaptophysin (SYP) (p = 0.012) compared to control. A concomitant exposure of tolcapone ameliorated the perturbed expression of MAP2 (p = 0.009) and COMT (p = 0.024) associated with HIV. Immunofluorescence revealed a trend reduction of SYP and MAP2 with exposure to HIV and that concomitant exposure of tolcapone increased SYP (p = 0.016) compared to HIV alone. Our findings demonstrated in vitro that inhibition of COMT can ameliorate HIV-associated neurodegenerative changes that resulted in the decreased expression of the structural and synaptic components MAP2 and SYP. As HIV-associated dendritic and synaptic damage are contributors to HAND, inhibition of COMT may represent a potential strategy for attenuating or preventing some of the symptoms of HAND.

Neural recovery after cortical injury: Effects of MSC derived extracellular vesicles on motor circuit remodeling in rhesus monkeys.

Reorganization of motor circuits in the cortex and corticospinal tract are thought to underlie functional recovery after cortical injury, but the mechanisms of neural plasticity that could be therapeutic targets remain unclear. Recent work from our group have shown that systemic treatment with mesenchymal stem cell derived (MSCd) extracellular vesicles (EVs) administered after cortical damage to the primary motor cortex (M1) of rhesus monkeys resulted in a robust recovery of fine motor function and reduced chronic inflammation. Here, we used immunohistochemistry for cfos, an activity-dependent intermediate early gene, to label task-related neurons in the surviving primary motor and premotor cortices, and markers of axonal and synaptic plasticity in the spinal cord. Compared to vehicle, EV treatment was associated with a greater density of cfos+ pyramidal neurons in the deep layers of M1, greater density of cfos+ inhibitory interneurons in premotor areas, and lower density of synapses on MAP2+ lower motor neurons in the cervical spinal cord. These data suggest that the anti-inflammatory effects of EVs may reduce injury-related upper motor neuron damage and hyperexcitability, as well as aberrant compensatory re-organization in the cervical spinal cord to improve motor function.

Downregulation of surface AMPA receptor expression in the striatum following prolonged social isolation, a role of mGlu5 receptors.

Major depressive disorder is a common and serious mood illness. The molecular mechanisms underlying the pathogenesis and symptomatology of depression are poorly understood at present. Multiple neurotransmitter systems are believed to be implicated in depression. Increasing evidence supports glutamatergic transmission as a critical element in depression and antidepressant activity. In this study, we investigated adaptive changes in expression of AMPA receptors in a key limbic reward structure, the striatum, in response to an anhedonic model of depression. Prolonged social isolation in adult rats caused anhedonic/depression- and anxiety-like behavior. In these depressed rats, surface levels of AMPA receptors, mainly GluA1 and GluA3 subunits, were reduced in the nucleus accumbens (NAc). Surface GluA1/A3 expression was also reduced in the caudate putamen (CPu) following chronic social isolation. No change was observed in expression of presynaptic synaptophysin, postsynaptic density-95, and dendritic microtubule-associated protein 2 in the striatum. Noticeably, chronic treatment with the metabotropic glutamate (mGlu) receptor 5 antagonist MTEP reversed the reduction of AMPA receptors in the NAc and CPu. MTEP also prevented depression- and anxiety-like behavior induced by social isolation. These data indicate that adulthood prolonged social isolation induces the adaptive downregulation of GluA1/A3-containing AMPA receptor expression in the limbic striatum. mGlu5 receptor activity is linked to this downregulation, and antagonism of mGlu5 receptors produces an antidepressant effect in this anhedonic model of depression.

Ginsenoside Rg1 attenuates cerebral ischemia-reperfusion injury due to inhibition of NOX2-mediated calcium homeostasis dysregulation in mice.

Background: The incidence of ischemic cerebrovascular disease is increasing in recent years and has been one of the leading causes of neurological dysfunction and death. Ginsenoside Rg1 has been found to protect against neuronal damage in many neurodegenerative diseases. However, the effect and mechanism by which Rg1 protects against cerebral ischemia-reperfusion injury (CIRI) are not fully understood. Here, we report the neuroprotective effects of Rg1 treatment on CIRI and its possible mechanisms in mice. Methods: A bilateral common carotid artery ligation was used to establish a chronic CIRI model in mice. HT22 cells were treated with Rg1 after OGD/R to study its effect on [Ca2+]i. The open-field test and pole-climbing experiment were used to detect behavioral injury. The laser speckle blood flowmeter was used to measure brain blood flow. The Nissl and H&E staining were used to examine the neuronal damage. The Western blotting was used to examine MAP2, PSD95, Tau, p-Tau, NOX2, PLC, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging was used to test the level of [Ca2+]i. Results: Rg1 treatment significantly improved cerebral blood flow, locomotion, and limb coordination, reduced ROS production, increased MAP2 and PSD95 expression, and decreased p-Tau, NOX2, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging results showed that Rg1 could inhibit calcium overload and resist the imbalance of calcium homeostasis after OGD/R in HT22 cells. Conclusion: Rg1 plays a neuroprotective role in attenuating CIRI by inhibiting oxidative stress, calcium overload, and neuroinflammation.

Antidepressant-like effect of ginsenoside Rb1 on potentiating synaptic plasticity via the miR-134-mediated BDNF signaling pathway in a mouse model of chronic stress-induced depression.

Background: Brain-derived neurotrophic factor (BDNF)-tropomyosin-related kinase B (TrkB) plays a critical role in the pathogenesis of depression by modulating synaptic structural remodeling and functional transmission. Previously, we have demonstrated that the ginsenoside Rb1 (Rb1) presents a novel antidepressant-like effect via BDNF-TrkB signaling in the hippocampus of chronic unpredictable mild stress (CUMS)-exposed mice. However, the underlying mechanism through which Rb1 counteracts stress-induced aberrant hippocampal synaptic plasticity via BDNF-TrkB signaling remains elusive. Methods: We focused on hippocampal microRNAs (miRNAs) that could directly bind to BDNF and are regulated by Rb1 to explore the possible synaptic plasticity-dependent mechanism of Rb1, which affords protection against CUMS-induced depression-like effects. Results: Herein, we observed that brain-specific miRNA-134 (miR-134) could directly bind to BDNF 3'UTR and was markedly downregulated by Rb1 in the hippocampus of CUMS-exposed mice. Furthermore, the hippocampus-targeted miR-134 overexpression substantially blocked the antidepressant-like effects of Rb1 during behavioral tests, attenuating the effects on neuronal nuclei-immunoreactive neurons, the density of dendritic spines, synaptic ultrastructure, long-term potentiation, and expression of synapse-associated proteins and BDNF-TrkB signaling proteins in the hippocampus of CUMS-exposed mice. Conclusion: These data provide strong evidence that Rb1 rescued CUMS-induced depression-like effects by modulating hippocampal synaptic plasticity via the miR-134-mediated BDNF signaling pathway.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Neuroprotective derivatives of tacrine that target NMDA receptor and acetyl cholinesterase - Design, synthesis and biological evaluation.

The complex and multifactorial nature of neuropsychiatric diseases demands multi-target drugs that can intervene with various sub-pathologies underlying disease progression. Targeting the impairments in cholinergic and glutamatergic neurotransmissions with small molecules has been suggested as one of the potential disease-modifying approaches for Alzheimer's disease (AD). Tacrine, a potent inhibitor of acetylcholinesterase (AChE) is the first FDA approved drug for the treatment of AD. Tacrine is also a low affinity antagonist of N-methyl-D-aspartate receptor (NMDAR). However, tacrine was withdrawn from its clinical use later due to its hepatotoxicity. With an aim to develop novel high affinity multi-target directed ligands (MTDLs) against AChE and NMDAR, with reduced hepatotoxicity, we performed in silico structure-based modifications on tacrine, chemical synthesis of the derivatives and in vitro validation of their activities. Nineteen such derivatives showed inhibition with IC50 values in the range of 18.53 ± 2.09 - 184.09 ± 19.23 nM against AChE and 0.27 ± 0.05 - 38.84 ± 9.64 µM against NMDAR. Some of the selected compounds also protected rat primary cortical neurons from glutamate induced excitotoxicity. Two of the tacrine derived MTDLs, 201 and 208 exhibited in vivo efficacy in rats by protecting against behavioral impairment induced by administration of the excitotoxic agent, monosodium glutamate. Additionally, several of these synthesized compounds also exhibited promising inhibitory activitiy against butyrylcholinesterase. MTDL-201 was also devoid of hepatotoxicity in vivo. Given the therapeutic potential of MTDLs in disease-modifying therapy, our studies revealed several promising MTDLs among which 201 appears to be a potential candidate for immediate preclinical evaluations.

Umbilical artery tissue contains p75 neurotrophin receptor-positive pericyte-like cells that possess neurosphere formation capacity and neurogenic differentiation potential.

INTRODUCTION: The p75 neurotrophin receptor (p75NTR) is known as an efficient marker for the prospective isolation of mesenchymal stem cells (MSCs) and neural crest-derived stem cells (NCSCs). To date, there is quite limited information concerning p75NTR-expressing cells in umbilical cord (UC), although UC is known as a rich source of MSCs. We show for the first time the localization, phenotype, and functional properties of p75NTR+ cells in UC. METHODS: Human UC tissue sections were subjected to immunohistochemistry for MSC markers including p75NTR. Enzymatically isolated umbilical artery (UA) cells containing p75NTR+ cells were assessed for immunophenotype, clonogenic capacity, and differentiation potential. To identify the presence of neural crest-derived cells in the UA, P0-Cre/Floxed-EGFP reporter mouse embryos were used, and immunohistochemical analysis of UC tissue was performed. RESULTS: Immunohistochemical analysis revealed that p75NTR+ cells were specifically localized to the subendothelial area of the UA and umbilical vein. The p75NTR+ cells co-expressed PDGFRß, CD90, CD146, and NG2, phenotypic markers of MSCs and pericytes. Isolated UA cells possessed the potential to form neurospheres that further differentiated into neuronal and glial cell lineages. Genetic lineage tracing analysis showed that EGFP+ neural crest-derived cells were detected in the subendothelial area of UA with p75NTR immunoreactivity. CONCLUSIONS: These results show that UA tissue harbors p75NTR+ pericyte-like cells in the subendothelial area that have the capacity to form neurospheres and the potential for neurogenic differentiation. The lineage tracing data suggests the p75NTR+ cells are putatively derived from the neural crest.

Tea polyphenols attenuate staurosporine-induced cytotoxicity and apoptosis by modulating BDNF-TrkB/Akt and Erk1/2 signaling axis in hippocampal neurons.

Tea polyphenols (TP) are the major ingredients in tea beverages that display health-benefits including anti-oxidation, anti-inflammation, anti-aging, attenuating blood pressure and deflating. In this study, we investigated the neuroprotective effects of TP to attenuate staurosporine (STS)-induced cytotoxicity. Rat hippocampal neurons were isolated, cultured and incubated with STS to induce neurite collapse and apoptosis, however, the medication of TP eliminated these adverse effects and maintained the morphology of neurons. STS decreased the expression of pro-BDNF, downregulated the TrkB/Akt/Bcl-2 signaling axis and promoted the activation of Erk1/2 and caspase-3. In contrast, TP rescued the expression of pro-BDNF and antagonistically restored the biochemistry of aforementioned signaling effectors. Consistently, the activity of TP can be attenuated by the inhibition of TrkB or Akt by small chemicals K252a and LY294002. Therefore, BDNF-TrkB and Akt signaling axis is essential for TP-mediated neuroprotective effects. In summary, TP showed beneficial effects to protect neurons from exogenous insults such as STS-induced neural cytotoxicity and cell death.

Mesial temporal extraventricular neurocytoma (mtEVN): A case report and literature review.

AIM: We describe a case of mesial temporal extraventricular neurocytoma (mtEVN) in a 23-year-old male presenting with drug-resistant seizures and review the literature on this rare tumor. METHODS: A PubMed search was queried using the MeSH term "neurocytoma" and key search terms "extraventricular", "temporal", and "epilepsy". Titles and abstracts were screened for temporal neurocytomas. References were reviewed to identify further studies. RESULTS: Twenty case reports were selected comparing the presentation, radiological, histopathological, and surgical outcomes of neocortex temporal EVNs (ntEVN) and mtEVNs. CONCLUSION: Gross total resection of mtEVNs under intraoperative electrocorticography monitoring typically affords an excellent prognosis and successful seizure control.

Morphological alterations induced by the exposure to TiO2 nanoparticles in primary cortical neuron cultures and in the brain of rats.

Nowadays, nanoparticles (NPs) of titanium dioxide (TiO2) are abundantly produced. TiO2 NPs are present in various food products, in paints, cosmetics, sunscreens and toothpastes. However, the toxicity of TiO2 NPs on the central nervous system has been poorly investigated until now. The aim of this study was to evaluate the toxicity of TiO2 NPs on the central nervous system in vitro and in vivo. In cell cultures derived from embryonic cortical brain of rats, a significant decrease in neuroblasts was observed after 24 to 96 h of incubation with TiO2 NPs (5 to 20 µg/ml). This phenomenon resulted from an inhibition of neuroblast proliferation and a concomitant increase in apoptosis. In the same time, a gliosis, characterized by an increase in proliferation of astrocytes and the hypertrophy of microglial cells, occurred. The phagocytosis of TiO2 NPs by microgliocytes was also observed. In vivo, after intraperitoneal injection, the TiO2 NPs reached the brain through the blood brain barrier and the nanoparticles promoted various histological injuries such as cellular lysis, neuronal apoptosis, and inflammation. A reduction of astrocyte population was observed in some brain area such as plexiform zone, cerebellum and subependymal area. An oxidative stress was also detected by immunohistochemistry in neurons of hippocampus, cerebellum and in subependymal area. In conclusion, our study demonstrated clearly the toxic impact of TiO2 NPs on rat brain and neuronal cells and pointed about not yet referenced toxicity impacts of TiO2 such as the reduction of neuroblast proliferation both in vitro and in vivo.

Exercise improves recognition memory and synaptic plasticity in the prefrontal cortex for rats modelling vascular dementia.

OBJECTIVES: Functional electrical stimulation (FES) may induce involuntary exercise and make beneficial effects on vascular dementia (VD) by strengthening the BDNF-pCREB-mediated pathway and hippocampal plasticity. Whether FES improves recognition memory and synaptic plasticity in the prefrontal cortex (PFC) was investigated by establishing a VD model. METHODS: The VD rats were administered with two weeks of voluntary exercise, forced exercise, or involuntary exercise induced with FES. Sham-operated and control groups were also included. The behavioral changes were assessed with the novel object recognition test and novel object location test. The expression levels of key proteins related to synaptic plasticity in the PFC were also detected. RESULTS: All types of exercise improved the rats' novel object recognition index, but only voluntary exercise and involuntary exercise induced with FES improved the novel object location index. Any sort of exercise enhanced the expression of key proteins in the PFC. CONCLUSION: Involuntary exercise induced with FES can improve recognition memory in VD better than forced exercise. The mechanism is associated with increased synaptic plasticity in the PFC. FES may be a useful alternative tool for cognitive rehabilitation.

Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability.

In vitro estimating strategies for potential neurotoxicity are required to screen multiple substances. In a previous study, we showed that exposure to low-concentrations of some chemicals, such as organotin, decreased the expression of GluR2 protein, which is a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors, and led to neuronal vulnerability. This result suggested that GluR2 decreases as an index of neuronal cell sensitivity and vulnerability to various toxic insults. Accordingly, we developed a versatile method that is a large scale determination of GluR2 protein expression in the presence of environmental chemicals by means of AlphaLISA technology. Various analytical conditions were optimized, and then GluR2 protein amount was measured by the method using AlphaLISA. The GluR2 amounts were strongly correlated with that of measured by western blotting, which is currently used to determine GluR2 expression. An ideal standard curve could be written with the authentic GluR2 protein from 0 ng to 100 ng. Subsequently, twenty environmental chemicals were screened and nitenpyram was identified as a chemical which lead to decrease in GluR2 protein expression. This assay may provide a tool for detecting neurotoxic chemicals according to decreases in GluR2 protein expression.

Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors.

Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.

Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment.

SNCA/α-synuclein aggregation plays a crucial role in synucleinopathies such as Parkinson disease and dementia with Lewy bodies. Aggregating and nonaggregating SNCA species are degraded by the autophagy-lysosomal pathway (ALP). Previously, we have shown that the ALP is not only responsible for SNCA degradation but is also involved in the intracellular aggregation process of SNCA. An additional role of extracellular SNCA in the pathology of synucleinopathies substantiating a prion-like propagation hypothesis has been suggested since released SNCA species and spreading of SNCA pathology throughout neural cells have been observed. However, the molecular interplay between intracellular pathways, SNCA aggregation, release, and response of the local microenvironment remains unknown. Here, we attributed SNCA-induced toxicity mainly to secreted species in a cell culture model of SNCA aggregation and in SNCA transgenic mice: We showed that ALP inhibition by bafilomycinA1 reduced intracellular SNCA aggregation but increased secretion of smaller oligomers that exacerbated microenvironmental response including uptake, inflammation, and cellular damage. Low-aggregated SNCA was predominantly released by exosomes and RAB11A-associated pathways whereas high-aggregated SNCA was secreted by membrane shedding. In summary, our study revealed a novel role of the ALP by linking protein degradation to nonclassical secretion for toxic SNCA species. Thus, impaired ALP in the diseased brain not only limits intracellular degradation of misfolded proteins, but also leads to a detrimental microenvironmental response due to enhanced SNCA secretion. These findings suggest that the major toxic role of SNCA is related to its extracellular species and further supports a protective role of intracellular SNCA aggregation.

Maturation of embryonic tissues in a lymph node: a new approach for bioengineering complex organs.

Given our recent finding that the lymph node (LN) can serve as an in vivo factory to generate complex structures like liver, pancreas, and thymus, we investigated whether LN could also support early development and maturation from several mid-embryonic (E14.5/15.5) mouse tissues including brain, thymus, lung, stomach, and intestine. Here we observed brain maturation in LN by showing the emergence of astrocytes with well-developed branching processes. Thymus maturation in LN was monitored by changes in host immune cells. Finally, newly terminally differentiated mucus-producing cells were identified in ectopic tissues generated by transplantation of lung, stomach and intestine in LN. Thus, we speculate the LN offers a unique approach to study the intrinsic and extrinsic differentiation potential of cells and tissues during early development, and provides a new site for bioengineering complex body parts.

Adaptive responses induced by 24S-hydroxycholesterol through liver X receptor pathway reduce 7-ketocholesterol-caused neuronal cell death.

Lipid peroxidation products have been known to induce cellular adaptive responses and enhance tolerance against subsequent oxidative stress through up-regulation of antioxidant compounds and enzymes. 24S-hydroxycholesterol (24SOHC) which is endogenously produced oxysterol in the brain plays an important role in maintaining brain cholesterol homeostasis. In this study, we evaluated adaptive responses induced by brain-specific oxysterol 24SOHC in human neuroblastoma SH-SY5Y cells. Cells treated with 24SOHC at sub-lethal concentrations showed significant reduction in cell death induced by subsequent treatment with 7-ketocholesterol (7KC) in both undifferentiated and retinoic acid-differentiated SH-SY5Y cells. These adaptive responses were also induced by other oxysterols such as 25-hydroxycholesterol and 27-hydroxycholesterol which are known to be ligands of liver X receptor (LXR). Co-treatment of 24SOHC with 9-cis retinoic acid, a retinoid X receptor ligand, enhanced the adaptive responses. Knockdown of LXRß by siRNA diminished the adaptive responses induced by 24SOHC almost completely. The treatment with 24SOHC induced the expression of LXR target genes, such as ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1). The 24SOHC-induced adaptive responses were significantly attenuated by siRNA for ABCG1 but not by siRNA for ABCA1. Taken together, these results strongly suggest that 24SOHC at sub-lethal concentrations induces adaptive responses via transcriptional activation of LXR signaling pathway, thereby protecting neuronal cells from subsequent 7KC-induced cytotoxicity.

Human endometrial stem cells as a new source for programming to neural cells.

Human EnSC (endometrial-derived stem cell) is an abundant and easily available source for cell replacement therapy. Many investigations have shown the potency of the cells to differentiate into several mesoderm-derived cell lineages, including osteocytes and adipocytes. Here, the potency of EnSC in neural differentiation has been investigated. Flow cytometric analysis showed that they were positive for CD90, CD105, OCT4, CD44 and negative for CD31, CD34, CD133. The characterized cells were induced into neural differentiation by bFGF (basic fibroblast growth factor), PDGF (platelet-derived growth factor) and EGF (epidermal growth factor) signalling molecules, respectively in a sequential protocol, and differentiated cells were analysed for expression of neuronal markers by RT-PCR (reverse transcription-PCR) and immunocytochemistry, including Nestin, GABA (γ-aminobutyric acid), MAP2 (microtubule-associated protein 2), ß3-tub (class III ß-tubulin) and NF-L (neurofilament-light) at the level of their mRNAs. The expression of MAP2, ß3-tub and NF-L proteins in EnSC was confirmed 28 days PT (post-treatment) by immunocytochemistry. In conclusion, EnSC can respond to signalling molecules that are usually used as standards in neural differentiation and can programme neuronal cells, making these cells worth considering as a unique source for cell therapy in neurodegenerative disease.

Cytotoxic effects of G(M1) ganglioside and amyloid ß-peptide on mouse embryonic neural stem cells.

AD (Alzheimer's disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aßs (amyloid ß-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between G(M1) ganglioside and Aßs. Several reports have documented the bifunctional roles of Aßs in NSCs (neural stem cells), but the precise effects of G(M1) and Aß on NSCs have not yet been clarified. We evaluated the effect of G(M1) and Aß-(1-40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either G(M1) or Aß-(1-40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of G(M1) and Aß-(1-40). The exogenously added G(M1) and Aß-(1-40) were confirmed to incorporate into NECs. The Ras-MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with G(M1) and Aß-(1-40), but caspase 3 was activated. NECs treated with G(M1) and Aß-(1-40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that G(M1) and Aß-(1-40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aß-(1-40) and G(M1) co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.