Abstract
BackgroundALS2/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 FindingsTo 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 SOD1H46R, but not SOD1G93A, 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 SOD1H46R 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 SOD1H46R 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/SignificanceBased 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 SOD1H46R-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.
Highlights
Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of progressive neurodegenerative disorders characterized by a selective loss of upper motor neurons (UMN) in the cerebral cortex and lower motor neurons (LMN) in the brainstem and spinal cord [1]
A copy number of the SOD1H46R transgene (,20 copies) [31], which affected the disease severity [33], remained unchanged in the course of these mating schemes (Figure S1). Both wild-type and Als22/2 mice showed a constant increase in their body weight (Figure S2A and S2B), while mice carrying the SOD1H46R transgene reached their maximum body weight at 12–14 weeks of age, and terminally decreased as disease symptoms progressed (Figure 1A and 1B)
These exacerbated phenotypes by amyotrophic lateral sclerosis 2 (ALS2) loss were restorable by crossing to transgenic mice expressing human ALS2 (ALS2-tg; line L34-1) (Als22/2; SOD1H46R;ALS2 mice), albeit ALS2 overexpression per se did not show any obvious beneficial effects on lifespan in SOD1H46R mice (Figure 1D and S3)
Summary
Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of progressive neurodegenerative disorders characterized by a selective loss of upper motor neurons (UMN) in the cerebral cortex and lower motor neurons (LMN) in the brainstem and spinal cord [1]. ALS2 is a causative gene for a juvenile autosomal recessive form of motor neuron diseases (MNDs) [4,5,6], including amyotrophic lateral sclerosis 2 (ALS2) [7] (OMIM 205100), juvenile primary lateral sclerosis (PLSJ) [8] (OMIM 606353), and infantile-onset ascending hereditary spastic paralysis (IAHSP) [9] (OMIM 607225) These disorders are characterized by ascending degeneration of UMN with or without LMN involvement. ALS2 is a guanine nucleotide exchange factor for the small GTPase Rab5 [15] and involves in macropinocytosis-associated endosome trafficking and fusion [16,17], and neurite outgrowth [17,18,19] Loss of these functions accounts for motor dysfunction and axonal degeneration in the ALS2-linked MNDs. Logically designed mouse studies could provide persuasive evidence that loss of ALS2 triggers motor neuron degeneration. Molecular mechanisms underlying the relationship between ALS2-associated cellular function and its neuroprotective role remain unclear
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