Ryota Kunita

Molecular and cellular function of ALS2/alsin: implication of membrane dynamics in neuronal development and degeneration.

ALS2 is a causative gene for a juvenile autosomal recessive form of motor neuron diseases (MNDs), including amyotrophic lateral sclerosis 2 (ALS2), juvenile primary lateral sclerosis, and infantile-onset ascending hereditary spastic paralysis. These disorders are characterized by ascending degeneration of the upper motor neurons with or without lower motor neuron involvement. Thus far, a total of 12 independent ALS2 mutations, which include a small deletion, non-sense mutation, or missense mutation spreading widely across the entire coding sequence, are reported. They are predicted to result in either premature termination of translation or substitution of an evolutionarily conserved amino acid. Thus, a loss of functions in the ALS2-coded protein accounts for motor dysfunction and/or degeneration in the ALS2-linked MNDs. The ALS2 gene encodes a novel 184kDa protein of 1657 amino acids, ALS2 or alsin, comprising three predicted guanine nucleotide exchange factor (GEF) domains: the N-terminal RCC1-like domain, the central Dbl homology and pleckstrin homology (DH/PH) domains, and the C-terminal vacuolar protein sorting 9 (VPS9) domain. In addition, eight consecutive membrane occupation and recognition nexus (MORN) motifs are noted in the region between DH/PH and VPS9 domains. ALS2 activates Rab5 small GTPase and involves in endosome/membrane trafficking and fusions in the cells, and also promotes neurite outgrowth in neuronal cultures. Further, a neuroprotective role for ALS2 against cytotoxicity; i.e., the mutant Cu/Zn-superoxide dismutase 1 (SOD1)-mediated toxicity, oxidative stress, and excitotoxicity, has recently been implied. This review outlines current understandings of the molecular and cellular functions of ALS2 and its related proteins on safeguarding the integrity of motor neurons, and sheds light on the molecular pathogenesis of MNDs as well as other conditions of neurodegenerative diseases.

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 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/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 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.

The Rab5 activator ALS2/alsin acts as a novel Rac1 effector through Rac1-activated endocytosis.

Mutations in the ALS2 gene cause a number of recessive motor neuron diseases, indicating that the ALS2 protein (ALS2/alsin) is vital for motor neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for Rab5 (Rab5GEF) and is involved in endosome dynamics. However, the spatiotemporal regulation of the ALS2-mediated Rab5 activation is unclear. Here we identified an upstream activator for ALS2 and showed a functional significance of the ALS2 activation in endosome dynamics. ALS2 preferentially interacts with activated Rac1. In the cells activated Rac1 recruits cytoplasmic ALS2 to membrane ruffles and subsequently to nascent macropinosomes via Rac1-activated macropinocytosis. At later endocytic stages macropinosomal ALS2 augments fusion of the ALS2-localized macropinosomes with the transferrin-positive endosomes, depending on the ALS2-associated Rab5GEF activity. These results indicate that Rac1 promotes the ALS2 membranous localization, thereby rendering ALS2 active via Rac1-activated endocytosis. Thus, ALS2 is a novel Rac1 effector and is involved in Rac1-activated macropinocytosis. All together, loss of ALS2 may perturb macropinocytosis and/or the following membrane trafficking, which gives rise to neuronal dysfunction in the ALS2-linked motor neuron diseases.

ALS2CL, the novel protein highly homologous to the carboxy-terminal half of ALS2, binds to Rab5 and modulates endosome dynamics.

ALS2, the causative gene product for juvenile recessive amyotrophic lateral sclerosis (ALS2), is a guanine‐nucleotide exchange factor for the small GTPase Rab5. Here, we report a novel ALS2 homologous gene, ALS2 C‐terminal like (ALS2CL), which encodes a 108‐kD ALS2CL protein. ALS2CL exhibited a specific but a relatively weak Rab5‐GEF activity with accompanying rather strong Rab5‐binding properties. In HeLa cells, co‐expression of ALS2CL and Rab5A resulted in a unique tubulation phenotype of endosome compartments with significant colocalization of ALS2CL and Rab5A. These results suggest that ALS2CL is a novel factor modulating the Rab5‐mediated endosome dynamics in the cells.

Defective relocalization of ALS2/alsin missense mutants to Rac1-induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation

ALS2 physically interacts with ALS2 by anti tag coimmunoprecipitation (View interaction)

ALS2/alsin deficiency in neurons leads to mild defects in macropinocytosis and axonal growth.

Loss of function mutations in the ALS2 gene account for a number of juvenile/infantile recessive motor neuron diseases, indicating that its gene product, ALS2/alsin, plays a crucial role in maintenance and survival for a subset of neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rab5 and is implicated in endosome dynamics in cells. However, the role of ALS2 in neurons remains unclear. To elucidate the neuronal ALS2 functions, we investigate cellular phenotypes of ALS2-deficient primary cultured neurons derived from Als2-knockout (KO) mice. Here, we show that ALS2 deficiency results not only in the delay of axon outgrowth in hippocampal neurons, but also in a decreased level of the fluid phase horseradish peroxidase (HRP) uptake, which represents the activity for macropinocytic endocytosis, in cortical neurons. Thus, ALS2 may act as a modulator in neuronal differentiation and/or development through regulation of membrane dynamics.

Genetic background and gender effects on gross phenotypes in congenic lines of ALS2/alsin-deficient mice

Loss-of-function mutations in human ALS2 account for several juvenile recessive motor neuron diseases (MNDs). To understand the molecular basis underlying motor dysfunction in ALS2-linked MNDs, several lines of Als2(-/-) mice with a mixed genetic background were thus far generated, and their phenotypes were thoroughly characterized. However, several phenotypic discrepancies among different Als2-deficient lines became evident. To investigate whether genetic backgrounds are associated with such discrepancies, we here generated congenic lines of Als2(-/-) mice on two different genetic backgrounds; C57BL/6 (B6) and FVB/N (FVB), and investigated their gross phenotypes. Both B6 and FVB congenic lines were viable and fertile with no evidences for obvious abnormalities. There were no differences in growth curves between wild-type and Als2(-/-) mice on each genetic background. Remarkably, Als2(-/-) mice on a FVB, but not a B6, background exhibited a shorter life span than wild-type litters. Further, B6 female, but not male, Als2(-/-) mice showed a significantly lower spontaneous rearing activity than wild-type litters. These genetic background- and/or gender-specific findings suggest the presence of modifiers for life span and motor activities in Als2(-/-) mice. These congenic mice should provide a useful means to understand the molecular and genetic basis for variable expression of pathological phenotypes in MNDs.