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

Different Human Copper-Zinc Superoxide Dismutase Mutants, SOD1G93A and SOD1H46R, Exert Distinct Harmful Effects on Gross Phenotype in Mice

Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of fatal neurodegenerative diseases characterized by a selective loss of motor neurons in the brain and spinal cord. Creation of transgenic mice expressing mutant Cu/Zn superoxide dismutase (SOD1), as ALS models, has made an enormous impact on progress of the ALS studies. Recently, it has been recognized that genetic background and gender affect many physiological and pathological phenotypes. However, no systematic studies focusing on such effects using ALS models other than SOD1G93A mice have been conducted. To clarify the effects of genetic background and gender on gross phenotypes among different ALS models, we here conducted a comparative analysis of growth curves and lifespans using congenic lines of SOD1G93A and SOD1H46R mice on two different genetic backgrounds; C57BL/6N (B6) and FVB/N (FVB). Copy number of the transgene and their expression between SOD1G93A and SOD1H46R lines were comparable. B6 congenic mutant SOD1 transgenic lines irrespective of their mutation and gender differences lived longer than corresponding FVB lines. Notably, the G93A mutation caused severer disease phenotypes than did the H46R mutation, where SOD1G93A mice, particularly on a FVB background, showed more extensive body weight loss and earlier death. Gender effect on survival also solely emerged in FVB congenic SOD1G93A mice. Conversely, consistent with our previous study using B6 lines, lack of Als2, a murine homolog for the recessive juvenile ALS causative gene, in FVB congenic SOD1H46R, but not SOD1G93A, mice resulted in an earlier death, implying a genetic background-independent but mutation-dependent phenotypic modification. These results indicate that SOD1G93A- and SOD1H46R-mediated toxicity and their associated pathogenic pathways are not identical. Further, distinctive injurious effects resulted from different SOD1 mutations, which are associated with genetic background and/or gender, suggests the presence of several genetic modifiers of disease expression in the mouse genome

Aberration of miRNAs Expression in leukocytes from sporadic amyotrophic lateral sclerosis

Background: Accumulating evidence indicates that miRNAs play an important role in the development of amyotrophic lateral sclerosis (ALS). Most of previous studies on miRNA dysregulation in ALS focused on the alterative expression in ALS animal model or in limited samples from European patients with ALS. In the present study, the miRNA expression profiles were investigated in Chinese ALS patients to explore leukocytes miRNAs as a potential biomarker for the diagnosis of ALS.Methods: We analyzed the expression profiles of 1733 human mature miRNAs using microarray technology in leukocytes obtained from 5 patients with sporadic ALS (SALS) and 5 healthy controls. An independent group of 83 SALS patients, 24 Parkinson’s disease (PD) patients and 61 controls was used for validation by real-time polymerase chain reaction assay. Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. In addition, target genes and signaling information of validated differential expression miRNAs were predicted using Bioinformatics.Results: Eleven miRNAs, including four over-expressed and seven under-expressed miRNAs detected in SALS patients compared to healthy controls were selected for validation. Four under-expressed microRNAs, including hsa-miR-183, hsa-miR-193b, hsa-miR-451 and hsa-miR-3935, were confirmed in validation stage by comparison of 83 SALS patients and 61 HCs. Moreover, we identified a miRNA panel (hsa-miR-183, hsa-miR-193b, hsa-miR-451 and hsa-miR-3935) having a high diagnostic accuracy of SALS (AUC 0.857 for the validation group). However, only hsa-miR-183 was significantly lower in SALS patients than that in PD patients and in HCs, while no differences were found between PD patients and HCs. By bioinformatics analysis, we obtained a large number of target genes and signaling information that are linked to neurodegeneration. Conclusion: This study provided evidence of abnormal miRNA expression patterns in the peripheral blood leukocytes of SALS patients. Leukocytes miRNAs provide a promising opportunity for detection of SALS. The specificity of under-expression of hsa-miR-183 in SALS needs to be confirmed by further miRNA studies on other neurodegenerative diseases

Personalized Treatment for Infantile Ascending Hereditary Spastic Paralysis Based on In Silico Strategies

Infantile onset hereditary spastic paralysis (IAHSP) is a rare neurological disease diagnosed in less than 50 children worldwide. It is transmitted with a recessive pattern and originates from mutations of the ALS2 gene, encoding for the protein alsin and involved in differentiation and maintenance of the upper motoneuron. The exact pathogenic mechanisms of IAHSP and other neurodevelopmental diseases are still largely unknown. However, previous studies revealed that, in the cytosolic compartment, alsin is present as an active tetramer, first assembled from dimer pairs. The C-terminal VPS9 domain is a key interaction site for alsin dimerization. Here, we present an innovative drug discovery strategy, which identified a drug candidate to potentially treat a patient harboring two ALS2 mutations: one truncation at lysine 1457 (not considered) and the substitution of arginine 1611 with a tryptophan (R1611W) in the C-terminus VPS9. With a protein modeling approach, we obtained a R1611W mutant model and characterized the impact of the mutation on the stability and flexibility of VPS9. Furthermore, we showed how arginine 1611 is essential for alsin’s homo-dimerization and how, when mutated to tryptophan, it leads to an abnormal dimerization pattern, disrupting the formation of active tetramers. Finally, we performed a virtual screening, individuating an already therapy-approved compound (MK4) able to mask the mutant residue and re-establishing the alsin tetramers in HeLa cells. MK4 has now been approved for compassionate use

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

<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

Human WN1316-activating factors exert WN1316-mediated cytoprotective activity.

<p>Differentiated SH-SY5Y cells were treated with 8 μM WN1316 in the presence of purified GST-fusion proteins for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. Concentrations of the GST-fusion proteins used are as follows; GST; 4.4 μg/ml, GST-RBP4; 2.2 μg/ml, GST-AHSG; 1.9 μg/ml, GST- SERPINA1; 2.4 μg/ml, GST-ITIH4; 2.3 μg/ml, GST-A1BG; 1.5 μg/ml, GST-HPX; 1.7 μg/ml, GST-CFB; 3.3 μg/ml. 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 GST-RBP4 (**<i>p</i><0.001).</p

Protein factors in FBS exert WN1316-mediated cytoprotective activity.

<p>(A) Effect of FBS on WN1316-induced cytoprotection. Differentiated SH-SY5Y cells were treated with 8 μM WN1316 or DMSO for 3 h followed by 12 h of chase incubation without the compound, and then exposed to 40 μM menadione for 4 h. The cell viability was measured by AlamarBlue assay, 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). (B) Effect of heat-denatured FBS on the anti-oxidative stress activity of WN1316. Heat denaturation of FBS was performed at 65°C for 30 min and chilled ice-cold (denatured FBS), and heat denatured FBS was renatured by the incubation at 4°C for 16 h (renatured FBS). Differentiated SH-SY5Y cells were incubated with 8 μM WN1316 or DMSO for 3 h in DMEM supplemented with 10% FBS, 10% denatured FBS, or 10% renatured FBS, followed by 12 h of chase incubation without the compound, and then treated with 40 μM menadione for 4 h. The cell viability was calculated by AlamarBlue assay, 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