Κυριακή, 12 Μαΐου 2019

Molecular Neurobiology

Blockade of Rapid Influx of Extracellular Zn 2+ into Nigral Dopaminergic Neurons Overcomes Paraquat-Induced Parkinson's Disease in Rats


The herbicide paraquat (PQ) has been reported to enhance the risk of developing Parkinson's disease (PD) from epidemiological studies. PQ-induced reactive oxygen species (ROS) are linked with a selective loss of nigrostriatal dopaminergic neurons. Here, we first report a unique mechanism of nigrostriatal dopaminergic degeneration, in which rapid intracellular Zn2+ dysregulation via PQ-induced ROS production causes PD in rats. When the substantia nigra pars compacta (SNpc) of rats was perfused with PQ, extracellular concentrations of glutamate and Zn2+ were increased and decreased, respectively, in the SNpc. These changes were ameliorated by co-perfusion with Trolox, an antioxidative agent. In in vitro slice experiments, PQ rapidly increased extracellular Zn2+ influx via AMPA receptor activation. Both loss of nigrostriatal dopaminergic neurons and increase in turning behavior in response to apomorphine were markedly reduced by coinjection of PQ and intracellular Zn2+ chelator, i.e., ZnAF-2DA into the SNpc. Furthermore, loss of nigrostriatal dopaminergic neurons induced with a low dose of PQ, which did not induce any behavioral abnormality, was completely blocked by coinjection of ZnAF-2DA. The present study indicates that rapid influx of extracellular Zn2+ into dopaminergic neurons via AMPA receptor activation, which is initially induced by PQ-mediated ROS production in the SNpc, induces nigrostriatal dopaminergic degeneration, resulting in PQ-induced PD in rats. Intracellular Zn2+dysregulation in dopaminergic neurons is the cause of PQ-induced pathogenesis in the SNpc, and the block of intracellular Zn2+ toxicity leads to defending PQ-induced pathogenesis.

Absence of Mutation Enrichment for Genes Phylogenetically Conserved in the Olivocerebellar Motor Circuitry in a Cohort of Canadian Essential Tremor Cases


Essential Tremor is a prevalent neurological disorder of unknown etiology. Studies suggest that genetic factors contribute to this pathology. To date, no causative mutations in a gene have been reproducibly reported. All three structures of the olivocerebellar motor circuitry have been linked to Essential Tremor. We postulated that genes enriched for their expression in the olivocerebellar circuitry would be more susceptible to harbor mutations in Essential Tremor patients. A list of 11 candidate genes, enriched for their expression in the olivocerebellar circuitry, was assessed for their variation spectrum and frequency in a cohort of Canadian Essential Tremor cases. Our results from this list of 11 candidate genes do not support an association for Essential Tremor in our cohort of Canadian cases. The heterogenic nature of ET and modest size of the cohort used in this study are two confounding factors that could explain these results.

Sprouty2—a Novel Therapeutic Target in the Nervous System?


Clinical trials applying growth factors to alleviate symptoms of patients with neurological disorders have largely been unsuccessful in the past. As an alternative approach, growth factor receptors or components of their signal transduction machinery may be targeted directly. In recent years, the search for intracellular signaling integrator downstream of receptor tyrosine kinases provided valuable novel substrates. Among them are the Sprouty proteins which mainly act as inhibitors of growth factor-dependent neuronal and glial signaling pathways. In this review, we summarize the role of Sprouties in the lesioned central and peripheral nervous system with particular reference to Sprouty2 that is upregulated in various experimental models of neuronal degeneration and regeneration. Increased synthesis under pathological conditions makes Sprouty2 an attractive pharmacological target to enhance intracellular signaling activities, notably the ERK pathway, in affected neurons or activated astrocytes. Interestingly, high Sprouty2 levels are also found in malignant glioma cells. We recently demonstrated that abrogating Sprouty2 function strongly inhibits intracranial tumor growth and leads to significantly prolonged survival of glioblastoma bearing mice by induction of ERK-dependent DNA replication stress. On the contrary, knockdown of Sprouty proteins increases proliferation of activated astrocytes and, consequently, reduces secondary brain damage in neuronal lesion models such as kainic acid-induced epilepsy or endothelin-induced ischemia. Furthermore, downregulation of Sprouty2 improves nerve regeneration in the lesioned peripheral nervous system. Taken together, targeting Sprouties as intracellular inhibitors of the ERK pathway holds great promise for the treatment of various neurological disorders including gliomas. Since the protein lacks enzymatic activities, it will be difficult to develop chemical compounds capable to directly and specifically modulate Sprouty functions. However, interfering with Sprouty expression by gene therapy or siRNA treatment provides a realistic approach to evaluate the therapeutic potential of indirectly stimulating ERK activities in neurological disease.

The Expression and Cellular Localisation of Neurotrophin and Neural Guidance Molecules in Peritoneal Ectopic Lesions


Endometriosis is a gynaecological disorder characterised by the presence of endometrial-like tissue outside the uterus. It affects 10–15% of women during their reproductive age. The existence of close and complex relationship between chronic pelvic pain and endometriosis are widely recognised. However, the mechanisms of pain generation in women with endometriosis remain poorly understood. Immunohistochemistry was used to assess the density of nerve fibres stained with protein gene product 9.5 (PGP9.5) and the expression of various neurotrophins including glial cell derived neurotrophic factor (GDNF), persephin, neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and neuronal guidance molecules semaphorin 3E and Slit-2 and their receptors Plexin-D1 and Robo4 in peritoneal ectopic lesions from women with endometriosis and uninvolved peritoneum samples. Neurotrophins and neuronal guidance molecules and their receptors are synthesised in situ within peritoneal ectopic lesion which suggest their role in facilitating and maintaining the growth of nerve fibres. These molecules were found to be overall most highly expressed in the glands of endometriotic peritoneal lesions. In addition, the presence of ectopic lesions within the peritoneal cavity may affect the environment; in turn, the peritoneum altered appeared to play a role in the growth of nerve fibres and their development and maintenance in peritoneal lesions. Through exploring different neuronally active factors in and around ectopic lesions which may be contributing to pain generation, this study provides an insight and better understanding of the pain mechanisms associated with peritoneal endometriosis.

SNAP25 Gene Polymorphisms Protect Against Parkinson's Disease and Modulate Disease Severity in Patients


Parkinson's disease (PD) is a α-synucleinopathy in which intracellular aggregates of α-synuclein (α-syn) result in neurodegeneration and in the impairment of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex-mediated release of neurotransmitters. SNAP25 is a SNARE complex component: its concentration is increased in the cerebrospinal fluid of PD patients and this is related to the severity of cognitive and motor symptoms. Five SNAP25 single-nucleotide polymorphisms (SNPs) that modulate gene expression and were described to play a role in neurologic conditions (rs363050, rs363039, rs363043, rs3746544, and rs1051312) were analyzed in a cohort of 412 sporadic Italian PD patients and 1103 healthy controls (HC) in order to identify possible correlation with the disease. The SNAP25 rs1051312 C allele and CC genotype confer protection against PD onset, in particular in males (p = 0.003, OR(95%CI) = 0.67(0.51–0.88)) (pc = 0.008, OR(95%CI) = 0.28(0.10–0.70)). Co-segregation analyses revealed that the rs1051312 effect was reinforced when present within the rs363043 C-rs3746544 T-rs1051312 C haplotype (p = 3.3 × 10−4, OR = 0.47, 95%CI = 0.31–0.72), once again in males. Finally, rs363039 influenced age at onset (p = 0.02) and MMSE (Mini-Mental State Examination) scores (p = 0.01). The SNAP25 SNPs analyzed herein modulate gene expression at different levels as they are involved in binding miRNA and transcription factors; this suggests a possible synergistic effect of SNAP25 SNPs in the pathogenesis of PD. A replication in a larger and independent sample will help to further explore this hypothesis.

Contribution of Serum Lipid Profiles to Outcome After Endovascular Thrombectomy for Anterior Circulation Ischemic Stroke


The contribution of lipids, including low- and high-density lipoprotein cholesterol (LDL-C and HDL-C, respectively) and triglycerides (TG), to stroke outcomes is still debated. We sought to determine the impact of LDL-C concentrations on the outcome of patients with ischemic stroke in the anterior circulation who received treatment with endovascular thrombectomy (EVT). We performed a retrospective analysis of consecutive patients with acute ischemic stroke treated at a tertiary center between 2012 and 2016. Patients treated with EVT for large artery occlusion in the anterior circulation were selected. The primary endpoint was functional outcome at 3 months as measured with the modified Rankin Scale (mRS). Secondary outcome measures included hospital death and final infarct volume (FIV). Blood lipid levels were determined in a fasting state, 1 day after admission. We studied a total of 174 patients (44.8% men) with a median age of 74 years (interquartile range [IQR] 61–82) and median National Institutes of Health Stroke Scale at admission of 18 (14–22). Bridging therapy with intravenous tissue-plasminogen activator (t-PA) was administered in 122 (70.5%). The median LDL-C was 90 mg/dl (72–115). LDL-C demonstrated a U-type relationship with FIV (p = 0.036). Eighty-three (50.0%) patients had an mRS of 0–2 at 3 months. This favorable outcome was independently associated with younger age (OR 0.944, 95% CI 0.90–0.99, p = 0.012), thrombolysis in cerebral infarction 2b-3 reperfusion (OR 5.12, 95% CI 1.01–25.80, p = 0.015), smaller FIV (0.97 per cm3, 95% CI 0.97–0.99, p < 0.001), good leptomeningeal collaterals (OR 5.29, 95% CI 1.48–18.9, p = 0.011), and LDL-C more than 77 mg/dl (OR 0.179, 95% CI 0.04–0.74, p = 0.018). A higher LDL-C concentration early in the course of a stroke caused by large artery occlusion in the anterior circulation is independently associated with a favorable clinical outcome at 3 months. Further studies into the pathophysiological mechanisms underlying this observation are warranted.

Diltiazem Promotes Regenerative Axon Growth


Axotomy results in permanent loss of function after brain and spinal cord injuries due to the minimal regenerative propensity of the adult central nervous system (CNS). To identify pharmacological enhancers of axon regeneration, 960 compounds were screened for cortical neuron axonal regrowth using an in vitro cortical scrape assay. Diltiazem, verapamil, and bromopride were discovered to facilitate axon regeneration in rat cortical cultures, in the presence of chondroitin sulfate proteoglycans (CSPGs). Diltiazem, an L-type calcium channel blocker (L-CCB), also promotes axon outgrowth in adult primary mouse dorsal root ganglion (DRG) and induced human sensory (iSensory) neurons.

Modeling Alzheimer's Disease by Induced Pluripotent Stem Cells Carrying APP D678H Mutation


Alzheimer's disease (AD), probably caused by abnormal accumulation of β-amyloid (Aβ) and aberrant phosphorylation of tau, is the most common cause of dementia among older people. Generation of patient-specific neurons by induced pluripotent stem cell (iPSC) technology facilitates exploration of the disease features in live human neurons from AD patients. In this study, we generated iPSCs from two familial AD patients carrying a heterozygous D678H mutation in the APP gene (AD-iPSCs). The neurons derived from our AD-iPSCs demonstrated aberrant accumulation of intracellular and secreted Aβ42 and Aβ40, reduction of serine 9 phosphorylation in glycogen synthase kinase 3β (GSK3β) hyperphosphorylation of threonine 181 and serine 396 in tau protein, impaired neurite outgrowth, downregulation of synaptophysin, and increased caspase 1 activity. The comparison between neurons derived from a sibling pair of wild-type and mutated iPSCs successfully recapitulated these AD phenotypes. Treatment with indole compound NC009-1 (3-((1H-Indole-3-yl)methyl)-4-(2-nitrophenyl)but-3-en-2-one), a potential Aβ aggregation reducer, normalized the Aβ levels and GSK3β and tau phosphorylation, attenuated caspase 1 activity, and improved neurite outgrowth in AD-iPSC-derived neurons. Thus, APP D678H iPSCs-derived neurons recapitulate the cellular characteristics relevant to AD and enable exploration of the underlying pathogenesis and therapeutic strategies for AD.

Genetic Knockdown of mGluR5 in Striatal D1R-Containing Neurons Attenuates l -DOPA-Induced Dyskinesia in Aphakia Mice


l-DOPA is the main pharmacological therapy for Parkinson's disease. However, long-term exposure to l-DOPA induces involuntary movements termed dyskinesia. Clinical trials show that dyskinesia is attenuated by metabotropic glutamate receptor type 5 (mGluR5) antagonists. Further, the onset of dyskinesia is delayed by nicotine and mGluR5 expression is lower in smokers than in non-smokers. However, the mechanisms by which mGluR5 modulates dyskinesia and how mGluR5 and nicotine interact have not been established. To address these issues, we studied the role of mGluR5 in D1R-containing neurons in dyskinesia and examined whether nicotine reduces dyskinesia via mGluR5. In the aphakia mouse model of Parkinson's disease, we selectively knocked down mGluR5 in D1R-containing neurons (aphakia-mGluR5KD-D1). We found that genetic downregulation of mGluR5 decreased dyskinesia in aphakia mice. Although chronic nicotine increased the therapeutic effect of l-DOPA in both aphakia and aphakia-mGluR5KD-D1 mice, it caused a robust reduction in dyskinesia only in aphakia, and not in aphakia-mGluR5KD-D1 mice. Downregulating mGluR5 or nicotine treatment after l-DOPA decreased ERK and histone 3 activation, and FosB expression. Combining nicotine and mGluR5 knockdown did not have an added antidyskinetic effect, indicating that the effect of nicotine might be mediated by downregulation of mGluR5 expression. Treatment of aphakia-mGluR5KD-D1 mice with a negative allosteric modulator did not further modify dyskinesia, suggesting that mGluR5 in non-D1R-containing neurons does not play a role in its development. In conclusion, this work suggests that mGluR5 antagonists reduce dyskinesia by mainly affecting D1R-containing neurons and that the effect of nicotine on dyskinetic signs in aphakia mice is likely via mGluR5.

Early Manifestations of Brain Aging in Mice Due to Low Dietary Folate and Mild MTHFR Deficiency


Folate is an important B vitamin required for methylation reactions, nucleotide and neurotransmitter synthesis, and maintenance of homocysteine at nontoxic levels. Its metabolism is tightly linked to that of choline, a precursor to acetylcholine and membrane phospholipids. Low folate intake and genetic variants in folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, have been suggested to impact brain function and increase the risk for cognitive decline and late-onset Alzheimer's disease. Our study aimed to assess the impact of genetic and nutritional disturbances in folate metabolism, and their potential interaction, on features of cognitive decline and brain biochemistry in a mouse model. Wild-type and Mthfr+/− mice, a model for the MTHFR 677 C>T polymorphism, were fed control or folate-deficient diets from weaning until 8 and 10 months of age. We observed short-term memory impairment measured by the novel object paradigm, altered transcriptional levels of synaptic markers and epigenetic enzymes, as well as impaired choline metabolism due to the Mthfr+/− genotype in cortex or hippocampus. We also detected changes in mRNA levels of Presenillin-1, neurotrophic factors, one-carbon metabolic and epigenetic enzymes, as well as reduced levels of S-adenosylmethionine and acetylcholine, due to the folate-deficient diet. These findings shed further insights into the mechanisms by which genetic and dietary folate metabolic disturbances increase the risk for cognitive decline and suggest that these mechanisms are distinct.

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