Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

BACKGROUND: ALS2/alsin is a guanine nucleotide exchange factor for the small GTPase Rab5 and involved in macropinocytosis-associated endosome fusion and trafficking, and neurite outgrowth. ALS2 deficiency accounts for a number of juvenile recessive motor neuron diseases (MNDs). Recently, it has been shown that ALS2 plays a role in neuroprotection against MND-associated pathological insults, such as toxicity induced by mutant Cu/Zn superoxide dismutase (SOD1). However, molecular mechanisms underlying the relationship between ALS2-associated cellular function and its neuroprotective role remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: To address this issue, we investigated the molecular and pathological basis for the phenotypic modification of mutant SOD1-expressing mice by ALS2 loss. Genetic ablation of Als2 in SOD1(H46R), but not SOD1(G93A), transgenic mice aggravated the mutant SOD1-associated disease symptoms such as body weight loss and motor dysfunction, leading to the earlier death. Light and electron microscopic examinations revealed the presence of degenerating and/or swollen spinal axons accumulating granular aggregates and autophagosome-like vesicles in early- and even pre-symptomatic SOD1(H46R) mice. Further, enhanced accumulation of insoluble high molecular weight SOD1, poly-ubiquitinated proteins, and macroautophagy-associated proteins such as polyubiquitin-binding protein p62/SQSTM1 and a lipidated form of light chain 3 (LC3-II), emerged in ALS2-deficient SOD1(H46R) mice. Intriguingly, ALS2 was colocalized with LC3 and p62, and partly with SOD1 on autophagosome/endosome hybrid compartments, and loss of ALS2 significantly lowered the lysosome-dependent clearance of LC3 and p62 in cultured cells. CONCLUSIONS/SIGNIFICANCE: Based on these observations, although molecular basis for the distinctive susceptibilities to ALS2 loss in different mutant SOD1-expressing ALS models is still elusive, disturbance of the endolysosomal system by ALS2 loss may exacerbate the SOD1(H46R)-mediated neurotoxicity by accelerating the accumulation of immature vesicles and misfolded proteins in the spinal cord. We propose that ALS2 is implicated in endolysosomal trafficking through the fusion between endosomes and autophagosomes, thereby regulating endolysosomal protein degradation in vivo

小児期の近位型脊髄性筋萎縮症の臨床像と分子遺伝学的診断

脊髄性筋萎縮症(SMA)は脊髄の前角細胞の変性,脱落により,筋萎縮や筋力低下を呈する常染色体性劣性の遺伝病である.本疾患は発症年齢,最高到達運動能力,経過により3つの型に分類される.分子遺伝学的には,1990年に3型ともに5q13に存在することが証明され,さらに1995年SMAの候補遺伝子としてSMN(survival motor neuron)遺伝子とNAIP(neuronal apoptosis inhibitory protein)遺伝子が報告された.本研究ではSMA患者を臨床的に分類し,SMN遺伝子,NAIP遺伝子について解析した.また,臨床型と遺伝子学的解析結果の関係を検討した.対象はSMA患者39名と精神遅滞,心肥大を各々伴つたatypical SMA2名である.1990年のInternational SMA Collaboration Workshop Reportの臨床型分類をもとに39名を分類した.この分類における発症年齢,最高到達運動能力,経過の3つの基準のすべてを満たし,臨床型に分類できたのは39例中20例であった.発症年齢,運動能力,経過のそれぞれについて分類した結果,発症年齢,経過では幅拡い臨床像を示し,臨床的に分類するのには最高到達運動能力および経過が有用であった.また,遺伝子解析の結果,全体の85%(33/39:I型90%,II型100%,III型54%)においてSMN遺伝子のexon 7のみ,またはexon 7および8のホモ接合性の欠失を認めた.また,I型の2名(5% 2/39)においてSMN遺伝子の欠失と同時にNAIP遺伝子のexon 5および欠失が認められた.両遺伝子の認められた2例ともに1型の重症例であったが,その他のSMN遺伝子のみの欠失した症例は臨床症状にかなりの幅が認めれていた.いずれの欠失も認められなかつた7例のうち5例は皿型であった.以上より臨床的重症度と遺伝子の欠失サイズの相関が示唆された.atypical SMAのうち1例にSMN遺伝子の欠失が認められた.また,精神遅滞をともなつた1例は欠失が認められず,異なる疾患の可能性も考えられた.Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degenration of anterior horn cells in the spinal cord, leading to symmetrical proximal muscle weakness and atrophy. Affected individuals are classified into three types depending on the onset age, highest motor functional ability and clinical course. In 1990, the gene for SMA was mapped to chromosome region 5g13 for all three types of SMA. In 1995, the SMN (survival motor neuron) and NAIP (neuronal apoptosis inhibitory protein) genes were reported as candidate genes for SMA. In this study, we analyzed clinical features of SMA patients and deletions of these two candidate genes. The subjects included 39 SMA patients and two atypical SMA cases, one with mental retardation and the other with cardiomegaly. We classified 39 SMA cases based on the classification devised by the International SMA Consortium Meeting Report. Twenty cases satisfied all three criteria of this classification, and could clearly be classified as one of the three types. Our results of classifying cases according to each criterion suggested that clinical features of SMA constitute a broad spectrum encompassing the three types. Thus, it is most useful to classify patients according to their highest motor functional ability. Thirty-three cases of typical SMA (85% of all, 90% of type I, 100% of type II and 54% of type III) showed deletions of exons 7 and 8 or only exon 7 of the SMN gene. Two of type I (5% of all) showed deletions of exon 5 and 6 of the NAIP gene with deletions of the SMA genes. The other cases (2 of type I and 5 of type III) had no deletions of these genes. Involvement of the SMN and NAIP genes appears to be related to the severity of SMA. The one atypical case showed deletion of the SMN gene. The other one with mental retardation may be heterogeneous

小児期の近位型脊髄性筋萎縮症の臨床像と分子遺伝学的診断

脊髄性筋萎縮症(SMA)は脊髄の前角細胞の変性,脱落により,筋萎縮や筋力低下を呈する常染色体性劣性の遺伝病である.本疾患は発症年齢,最高到達運動能力,経過により3つの型に分類される.分子遺伝学的には,1990年に3型ともに5q13に存在することが証明され,さらに1995年SMAの候補遺伝子としてSMN(survival motor neuron)遺伝子とNAIP(neuronal apoptosis inhibitory protein)遺伝子が報告された.本研究ではSMA患者を臨床的に分類し,SMN遺伝子,NAIP遺伝子について解析した.また,臨床型と遺伝子学的解析結果の関係を検討した.対象はSMA患者39名と精神遅滞,心肥大を各々伴つたatypical SMA2名である.1990年のInternational SMA Collaboration Workshop Reportの臨床型分類をもとに39名を分類した.この分類における発症年齢,最高到達運動能力,経過の3つの基準のすべてを満たし,臨床型に分類できたのは39例中20例であった.発症年齢,運動能力,経過のそれぞれについて分類した結果,発症年齢,経過では幅拡い臨床像を示し,臨床的に分類するのには最高到達運動能力および経過が有用であった.また,遺伝子解析の結果,全体の85%(33/39:I型90%,II型100%,III型54%)においてSMN遺伝子のexon 7のみ,またはexon 7および8のホモ接合性の欠失を認めた.また,I型の2名(5% 2/39)においてSMN遺伝子の欠失と同時にNAIP遺伝子のexon 5および欠失が認められた.両遺伝子の認められた2例ともに1型の重症例であったが,その他のSMN遺伝子のみの欠失した症例は臨床症状にかなりの幅が認めれていた.いずれの欠失も認められなかつた7例のうち5例は皿型であった.以上より臨床的重症度と遺伝子の欠失サイズの相関が示唆された.atypical SMAのうち1例にSMN遺伝子の欠失が認められた.また,精神遅滞をともなつた1例は欠失が認められず,異なる疾患の可能性も考えられた.Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degenration of anterior horn cells in the spinal cord, leading to symmetrical proximal muscle weakness and atrophy. Affected individuals are classified into three types depending on the onset age, highest motor functional ability and clinical course. In 1990, the gene for SMA was mapped to chromosome region 5g13 for all three types of SMA. In 1995, the SMN (survival motor neuron) and NAIP (neuronal apoptosis inhibitory protein) genes were reported as candidate genes for SMA. In this study, we analyzed clinical features of SMA patients and deletions of these two candidate genes. The subjects included 39 SMA patients and two atypical SMA cases, one with mental retardation and the other with cardiomegaly. We classified 39 SMA cases based on the classification devised by the International SMA Consortium Meeting Report. Twenty cases satisfied all three criteria of this classification, and could clearly be classified as one of the three types. Our results of classifying cases according to each criterion suggested that clinical features of SMA constitute a broad spectrum encompassing the three types. Thus, it is most useful to classify patients according to their highest motor functional ability. Thirty-three cases of typical SMA (85% of all, 90% of type I, 100% of type II and 54% of type III) showed deletions of exons 7 and 8 or only exon 7 of the SMN gene. Two of type I (5% of all) showed deletions of exon 5 and 6 of the NAIP gene with deletions of the SMA genes. The other cases (2 of type I and 5 of type III) had no deletions of these genes. Involvement of the SMN and NAIP genes appears to be related to the severity of SMA. The one atypical case showed deletion of the SMN gene. The other one with mental retardation may be heterogeneous

<i>N</i>-linked glycoproteins exert WN1316-mediated cytoprotective activity.

<p>Differentiated SH-SY5Y cells were pretreated with 8 μM WN1316 in DMEM containing 0.2 mg/ml FBS, Con A pass, or Con A elute for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (<i>p</i><0.0001) followed by Dunnett’s <i>post hoc</i> test compared with DMSO-treated control (**<i>p</i><0.001, *<i>p</i><0.01).</p

Deglycosylation does not affect WN1316-mediated cytoprotective activity.

<p>Differentiated SH-SY5Y cells were pretreated with 8 μM WN1316 in DMEM containing 0.28 mg/ml Blue pass treated with or without EnzMix and PNGaseF for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. As control experiments, differentiated SH-SY5Y cells were treated with 8 μM WN1316 (as a positive control) or DMSO (as a vehicle control) in DMEM supplemented with 2% FBS (corresponds to a protein concentration of approximately 0.5 mg/ml). The cell viability was calculated by AlamarBlue, and was expressed as a relative value (relative cytoprotective activity; -fold) of the WN1316-treated samples for vehicle control (DMSO) set as 1. Data are expressed as mean ± SD (n = 4). Statistical significance was evaluated by one-way ANOVA (<i>p</i><0.0001) followed by Dunnett’s <i>post hoc</i> test compared with DMSO-treated control (**<i>p</i><0.001).</p

Silver staining and Western blot analysis of Blue pass fraction treated with or without glycosidase.

<p>Blue pass fraction was treated with or without EnzMix (removal <i>N</i>- and <i>O</i>-glycans) and PNGaseF (removal of <i>N</i>-glycans) at 37°C for 16 h. The molecular masses and homogeneities of the proteins were analyzed by SDS-PAGE (A) and Western blotting with anti-AHSG antibody as a glycoprotein standard (B). Proteins were visualized by silver staining using a Silver Stain Kit Wako (Wako).</p

Bromocriptine Mesylate Attenuates Amyotrophic Lateral Sclerosis: A Phase 2a, Randomized, Double-Blind, Placebo-Controlled Research in Japanese Patients.

OBJECTIVE:Bromocriptine mesylate (BRC), a dopamine D2 receptor agonist has been shown to confer neuroprotection, sustained motor function and slowed disease progression in mouse models of amyotrophic lateral sclerosis (ALS) Here we report a first in human trial in ALS. DESIGN:A multicenter, Riluzole add-on, randomized, double-blind, placebo controlled 102-week extension BRC clinical trial. METHODS:The trial was conducted between January 2009 and March 2012 on 36 Japanese ALS patients. A 12-week treatment with Riluzole observational period was followed by combined treatment (Riluzole + BRC; n = 29 or Riluzole + placebo; n = 7). The dosing commenced at 1.25 mg/day increasing in steps at two weeks intervals to a maximum of 15 mg/day. The efficacy of BRC was evaluated by comparing BRC and placebo groups upon completion of stepwise dosing at 14 weeks 2 points (1st endpoint) and upon completion or discontinuation of the study (2nd endpoint) of the dosing. RESULTS:Statistics analyses revealed a marginal BRC treatment efficacy with P≦20%to placebo by 1st and 2nd endpoint analysis. In the 1st endpoint analysis, BRC group was significantly effective on the scores of ALSAQ40-communicaton (P = 1.2%), eating and drinking (P = 2.2%), ALSFRS-R total (P = 17.6%), grip strength (P = 19.8%) compared to the placebo group. In the 2nd endpoint analysis, differences between the scores of Limb Norris Scale (P = 18.3%), ALSAQ40-communication (P = 11.9%), eating and drinking (P = 13.6%), and neck forward-bent test (P = 15.4%) of BRC group were detected between the two groups. There was no significant difference between the treatment groups for adverse events or serious drug reactions incidence. CONCLUSIONS:BRC sustains motoneuronal function at least in part through BRC treatment. Further analysis involving a Phase 2b or 3 clinical trial is required but BRC currently shows promise for ALS treatment. TRIAL REGISTRATION:UMIN Clinical Trials UMIN000008527