Search

Voltage-gated sodium (NaV) and potassium (KV) channels are critical components of neuronal action potential generation and propagation. Here, we report that NaVβ1 encoded by SCN1b, an integral subunit of NaV channels, coassembles with and modulates the biophysical properties of KV1 and KV7 channels, but not KV3 channels, in an isoform-specific manner. Distinct domains of NaVβ1 are involved in modulation of the different KV channels. Studies with channel chimeras demonstrate that NaVβ1-mediated changes in activation kinetics and voltage dependence of activation require interaction of NaVβ1 with the channel's voltage-sensing domain, whereas changes in inactivation and deactivation require interaction with the channel's pore domain. A molecular model based on docking studies shows NaVβ1 lying in the crevice between the voltage-sensing and pore domains of KV channels, making significant contacts with the S1 and S5 segments. Cross-modulation of NaV and KV channels by NaVβ1 may promote diversity and flexibility in the overall control of cellular excitability and signaling. [ABSTRACT FROM AUTHOR]

Abstract: Huntington's disease is an autosomal dominant neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in the huntingtin protein, resulting in intracellular aggregate formation and neurodegeneration. How neuronal cells are affected by such a polyglutamine tract expansion remains obscure. To dissect the ways in which polyglutamine expansion can cause neural dysfunction, the authors generated Drosophila transgenic strains expressing either a nuclear targeted or cytoplasmic form of pathogenic (NHtt-152QNLS, NHtt-152Q), or nonpathogenic (NHtt-18QNLS, NHtt-18Q) N-terminal human huntingtin. These proteins were expressed in the dendritic arborization neurons of the larval peripheral nervous system and their effects on neuronal survival, morphology, and larval locomotion were examined. The authors found that NHtt-152QNLS larvae had altered dendrite morphology and larval locomotion, whereas NHtt-152Q, NHtt-18QNLS, and NHtt-18Q larvae did not. Furthermore, the authors examined the physiological defect underlying this disrupted larval locomotion in detail by recording spontaneous ongoing segmental nerve activity. NHtt-152QNLS larvae displayed uncoordinated activity between anterior and posterior segments. Moreover, anterior segments had shorter bursts and longer interburst intervals in NHtt-152QNLS larvae than in NHtt-18QNLS larvae, whereas posterior segments had longer bursts and shorter interburst intervals. These results suggest that the pathogenic protein disrupts neuron function without inducing cell death, and describe how this dysfunction leads to a locomotor defect. These results also suggest that sensory inputs are necessary for the coordination of anterior and posterior body parts during locomotion. From these analyses the authors show that examination of motor behaviors in the Drosophila larvae is a powerful new model to dissect non-cell-lethal mechanisms of mutant Htt toxicity. [ABSTRACT FROM AUTHOR]

Voltage-gated Na+ channel (VGSC) β1 subunifs regulate cell-cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na+ current (lNa) mediated by NaV1.6. In addition, β1 is required for highfrequency action potential firing. Transient INa is unchanged in Scn1b (β1) null CGNs; however, the resurgent INa, thought to underlie high-frequency firing in NaV1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing NaV1.6 is reduced in Scn1b null cerebellar neurons. In place of NaV1.6 at the AIS, we observed an increase in NaV1.1, whereas NaV1.2 was unchanged. This indicates that β1 is required for normal localization of NaV1.6 at the AlS during the postnatal developmental switch to NaV1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with a subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and NaV1.6 such that β1-mediated neurite outgrowth requires NaV1.6-mediated INa, and NaV1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development. [ABSTRACT FROM AUTHOR]

Expansion of CAG trinucleotide repeat within the coding region of several genes results in the production of proteins with expanded polyglutamine (PolyQ) stretch. The expression of these pathogenic proteins leads to PolyQ diseases, such as Huntington’s disease or several types of spinocerebellar ataxias. This family of neurodegenerative disorders is characterized by constant progression of the symptoms and molecularly, by the accumulation of mutant proteins inside neurons causing their dysfunction and eventually death. So far, no effective therapy actually preventing the physical and/or mental decline has been developed. Experimental therapeutic strategies either target the levels or processing of mutant proteins in an attempt to prevent cellular deterioration, or they are aimed at the downstream pathologic effects to reverse or ameliorate the caused damages. Certain pathomechanistic aspects of PolyQ disorders are discussed here. Relevance of disease models and recent knowledge of therapeutic possibilities is reviewed and updated. [ABSTRACT FROM AUTHOR]

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Nav1.1α subunits. Sodium channels are modulated by β1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Nav1.1α subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of β1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b-/- versus Scn1b-/- mice. Scn1b-/- CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a-/- model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b-/- mice seize spontaneously, the seizure susceptibility of Scn1b-/- mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation. [ABSTRACT FROM AUTHOR]

A hallmark of polyglutamine diseases, including Huntington disease (HD), is the formation of β-sheet-rich aggregates, called amyloid, of causative proteins with expanded polyglutamines. However, it has remained unclear whether the polyglutamine amyloid is a direct cause or simply a secondary manifestation of the pathology. Here we show that huntingtin-exon1 (thtt) with expanded polyglutamines remarkably misfolds into distinct amyloid conformations under different temperatures, such as 4 °C and 37 °C. The 4 °C amyloid has loop/turn structures together with mostly β-sheets, including exposed polyglutamines, whereas the 37 °C amyloid has more extended and buried β-sheets. By developing a method to efficiently introduce amyloid into mammalian cells, we found that the formation of the 4 °C amyloid led to substantial toxicity, whereas the toxic effects of the 37 °C amyloid were very small. Importantly, thtt amyloids in different brain regions of HD mice also had distinct conformations. The thermo-labile thtt amyloid with loop/turn structures in the striatum showed higher toxicity, whereas the rigid thtt amyloid with more extended β-sheets in the hippocampus and cerebellum had only mild toxic effects. These studies show that the thtt protein with expanded polyglutamines can misfold into distinct amyloid conformations and, depending on the conformations, the amyloids can be either toxic or nontoxic. Thus, the amyloid conformation of thtt may be a critical determinant of cytotoxicity in HD. [ABSTRACT FROM AUTHOR]

A pathological hallmark of the Huntington's disease (HD) is intracellular inclusions containing a huntingtin (Htt) protein with an elongated polyglutamine tract. Aggregation of mutant Htt causes abnormal protein-protein interactions, and the functional dysregulation of aggregate-interacting proteins (AIPs) has been proposed as a pathomechanism of HD. Despite this, a molecular mechanism remains unknown how Htt aggregates sequester AIPs.Wenote an RNA-binding protein, TIA-1, as a model of AIPs containing a Q/N-rich sequence and suggest that in vitro and in vivo Htt fibrillar aggregates function as a structural template for inducing insoluble fibrillation of TIA-1. It is also plausible that such a cross-seeding activity of Htt aggregates represses the physiological function of TIA-1. We thus propose that Htt aggregates act as an intracellular hub for the cross-seeded fibrillation of Q/N-rich AIPs and that a cross-seeding reaction is a molecular origin to cause diverse pathologies in a polyglutamine disease. [ABSTRACT FROM AUTHOR]

Huntington’s disease is a hereditary neurodegenerative disorder caused by an aberrant polyglutamine expansion in the amino terminus of the huntingtin protein. The resultant mutant huntingtin form aggregates in neurons and causes neuronal dysfunction and degeneration in many ways including transcriptional dysregulation. Here, we report that the expression of mutant huntingtin in the mouse neuroblastoma cell results in massive transcriptional induction of several chemokines including monocyte chemoattractant protein-1 (MCP-1) and murine chemokine (KC). The mutant huntingtin expressing cells also exhibit proteasomal dysfunction and down-regulation of NF- κB activity in a time-dependent manner and both these phenomena regulate the expression of MCP-1 and KC. The expression of MCP-1 and KC are increased in the mutant huntingtin expressing cells in response to mild proteasome inhibition. However, the expression of MCP-1 and KC and proteasome activity are not altered and inflammation is rarely observed in the brain of 12-week-old Huntington’s disease transgenic mice in comparison with their age-matched controls. Our result suggests that the mutant huntingtin-induced proteasomal dysfunction can up-regulate the expression of MCP-1 and KC in the neuronal cells and therefore might trigger the inflammation process. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in ataxin-3 (ATX3; MJD1) protein. In biochemical experiments, we demonstrate that mutant ATX3exp specifically associated with the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1), an intracellular calcium (Ca2+) release channel. In electrophysiological and Ca2+ imaging experiments, we show that InsP3R1 was sensitized to activation by InsP3 in the presence of mutant ATX3exp. We found that feeding SCA3-YAC-84Q transgenic mice with dantrolene, a clinically relevant stabilizer of intracellular Ca2+ signaling, improved their motor performance and prevented neuronal cell loss in pontine nuclei and substantia nigra regions. Our results indicate that deranged Ca2+ signaling may play an important role in SCA3 pathology and that Ca2+ signaling stabilizers such as dantrolene may be considered as potential therapeutic drugs for treatment of SCA3 patients. [ABSTRACT FROM AUTHOR]

Parkin, a ubiquitin ligase, is responsible for autosomal recessive juvenile parkinsonism (AR-JP). We identified parkin-associated endothelin receptor-like receptor (Pael-R) as a substrate of parkin, whose accumulation is thought to induce unfolded protein response (UPR) -mediated cell death, leading to dopaminergic neurodegeneration. To create an animal model of AR-JP, we generated parkin knockout/Pael-R transgenic ( parkin-ko/ Pael-R-tg) mice. parkin-ko/ Pael-R-tg mice exhibited early and progressive loss of dopaminergic as well as noradrenergic neurons without formation of inclusion bodies, recapitulating the pathological features of AR-JP. Evidence of activation of UPR and up-regulation of dopamine and its metabolites were observed throughout the lifetime. Moreover, complex I activity of mitochondria isolated from parkin-ko/ Pael-R-tg mice was significantly reduced later in life. These findings suggest that persistent induction of unfolded protein stress underlies chronic progressive catecholaminergic neuronal death, and that dysfunction of mitochondrial complex I and oxidative stress might be involved in the progression of Parkinson’s disease. parkin-ko/ Pael-R-tg mice represents an AR-JP mouse model displaying chronic and selective loss of catecholaminergic neurons. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a polyglutamine [poly(Q)] disease with an expanded poly(Q) stretch in the N terminus of the huntingtin protein (htt). A major pathological feature of HD neurons is inclusion bodies, detergent-insoluble aggregates composed of poly(Q)-expanded mutant htt (mhtt). Misfolding of mhtt is thought to confer a toxic property via formation of aggregates. Although toxic molecular species are still debated, it is important to clarify the aggregation mechanism to understand the pathogenesis of mhtt. We show Cdk5/p35 suppresses the formation of mhtt inclusion bodies in cell lines and primary neurons. Although we expressed the N-terminal exon 1 fragment of htt lacking phosphorylation sites for Cdk5 in COS-7 cells, the kinase activity of Cdk5 was required for the suppression. Furthermore, Cdk5/p35 suppressed inclusion formation of atrophin-1, another poly(Q) protein, raising the possibility that Cdk5/p35 generally suppresses inclusion formation of poly(Q) proteins. Microtubules (MTs) were a downstream component of Cdk5/p35 in the suppression of inclusion formation; Cdk5/p35 disrupted MTs, which were required for the formation of inclusions. Moreover, stabilization of MTs by Taxol induced inclusions even with overexpression of Cdk5/p35. The formation of inclusions was also regulated by manipulating the Cdk5/p35 activity in primary rat or mouse cortical neuron cultures. These results indicate that Cdk5-dependent regulation of MT organization is involved in the development of aggregate formation and subsequent pathogenesis of poly(Q) diseases. This Cdk5 inhibition of htt aggregates is a novel mechanism different from htt phosphorylation and interaction with Cdk5 reported previously (Luo et al., 2005; Anne et al., 2007). [ABSTRACT FROM AUTHOR]

In Huntington's disease (HD), mutant Huntingtin, which contains expanded polyglutamine stretches, forms nuclear aggregates in neurons. The interactions of several transcriptional factors with mutant Huntingtin, as well as altered expression of many genes in HD models, imply the involvement of transcriptional dysregulation in the HD pathological process. The precise mechanism remains obscure, however. Here, we show that mutant Huntingtin aggregates interact with the components of the NF-Y transcriptional factor in vitro and in HD model mouse brain. An electrophoretic mobility shift assay using HD model mouse brain lysates showed reduction in NF-Y binding to the promoter region of HSP70, one of the NF-Y targets. RT–PCR analysis revealed reduced HSP70 expression in these brains. We further clarified the importance of NF-Y for HSP70 transcription in cultured neurons. These data indicate that mutant Huntingtin sequesters NF-Y, leading to the reduction of HSP70 gene expression in HD model mice brain. Because suppressive roles of HSP70 on the HD pathological process have been shown in several HD models, NF-Y could be an important target of mutant Huntingtin. [ABSTRACT FROM AUTHOR]

Machado–Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine tract near the C-terminus of the MJD-1 gene product, ataxin-3. Ataxin-3 is degraded by the proteasome. However, the precise mechanism of ataxin-3 degradation remains to be elucidated. In this study, we show direct links between ataxin-3 and the proteasome. p45, an ATPase subunit of the 19S proteasome, interacts with ataxin-3 in vitro and stimulates the degradation of ataxin-3 in an in vitro reconstituted degradation assay system. The effect of p45 on ataxin-3 degradation is blocked by MG132, a proteasome inhibitor. In N2a or 293 cells, overexpression of p45 strikingly enhances the clearance of both normal and expanded ataxin-3, but not alpha synuclein or SOD1, implying a functional specificity of p45 in this proteolytic process. The N-terminus of ataxin-3, which serves as a recognition site by p45, is necessary for the proteolytic process of ataxin-3. We also show that other three ATPases of the 19S proteasome, MSS1, p48, and p56 have no effect on ataxin-3 degradation. These data provide evidence that p45 plays an important role in regulating ataxin-3 degradation by the proteasome. [ABSTRACT FROM AUTHOR]

Apolipoprotein E4 (apoE4) encoded by ε4 allele is a strong genetic risk factor for Alzheimer’s disease (AD). ApoE4 carriers have accelerated amyloid β-protein (Aβ) deposition in their brains, which may account for their unusual susceptibility to AD. We hypothesized that the accelerated Aβ deposition in the brain of apoE4 carriers is mediated through cholesterol-enriched low-density membrane (LDM) domains. Thus, the concentrations of Aβ and various lipids in LDM domains were quantified in the brains of homozygous apoE3 and apoE4 knock-in (KI) mice, and in the brains of those mice bred with β-amyloid precursor protein (APP) transgenic mice (Tg2576). The Aβ40 and Aβ42 concentrations and the Aβ42 proportions in LDM domains did not differ between apoE3 and apoE4 KI mice up to 18 months of age. The Aβ40 concentration in the LDM domains was slightly, but significantly higher in apoE3/APP mice than in apoE4/APP mice. The lipid composition of LDM domains was modulated in an apoE isoform-specific manner, but its significance for Aβ deposition remains unknown. These data show that the apoE isoform-specific effects on the Aβ concentration in LDM domains do not occur in KI mouse models. [ABSTRACT FROM AUTHOR]

Sodium channel β4 is a very recently identified auxiliary subunit of the voltage-gated sodium channels. To find the primarily affected gene in Huntington's disease (HD) pathogenesis, we profiled HD transgenic mice using a high-density oligonucleotide array and identified β4 as an expressed sequence tag (EST) that was significantly down-regulated in the striatum of HD model mice and patients. Reduction in β4 started at a presymptomatic stage in HD mice, whereas other voltage-gated ion channel subunits were decreased later. In contrast, spinal cord neurons, which generate only negligible levels of expanded polyglutamine aggregates, maintained normal levels of β4 expression even at the symptomatic stage. Overexpression of β4 induced neurite outgrowth in Neuro2a cells, and caused a thickening of dendrites and increased density of dendritic spines in hippocampal primary neurons, indicating that β4 modulates neurite outgrowth activities. These results suggest that down-regulation of β4 may lead to abnormalities of sodium channel and neurite degeneration in the striatum of HD transgenic mice and patients with HD. [ABSTRACT FROM AUTHOR]

Polyglutamine (polyQ) expansion in many proteins, including huntingtin and ataxin-3, is pathogenic and responsible for neuronal dysfunction and degeneration. Although at least nine neurodegenerative diseases are caused by expanded polyQ, the pathogenesis of these diseases is still not well understood. In the present study, we used Caenorhabditis elegans to study the molecular mechanism of polyQ-mediated toxicity. We expressed full-length and truncated ataxin-3 with different lengths of polyQ in the nervous system of C. elegans. We show that expanded polyQ interrupts synaptic transmission, and induces swelling and aberrant branching of neuronal processes. Using an ubiquitinated fluorescence reporter construct, we also showed that polyQ aggregates impair the ubiquitin–proteasome system in C. elegans. These results may provide information for further understanding the pathogenesis of polyQ diseases. [ABSTRACT FROM AUTHOR]