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Shinji Hadano - One of the best experts on this subject based on the ideXlab platform.
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the n terminal intrinsically disordered region mediates intracellular localization and self oligomerization of ALS2
Biochemical and Biophysical Research Communications, 2021Co-Authors: Kento Shimakura, Asako Otomo, Kai Sato, Shun Mitsui, Suzuka Ono, Shinji HadanoAbstract:ALS2, a product of the causative gene for familial amyotrophic lateral sclerosis (ALS) type 2, plays a pivotal role in the regulation of endosome dynamics by activating small GTPase Rab5 via its intrinsic guanine nucleotide-exchange factor activity. Previously, we have reported that the N-terminal region of ALS2 has crucial roles in its endosomal localization and self-oligomerization, both of which are indispensable for the cellular function of ALS2. The N-terminus of ALS2 contains the regulator of chromosome condensation 1-like domain (RLD), which is predicted to form a seven-bladed β-propeller structure. Interestingly, the RLD is interrupted by the intrinsically disordered region (IDR), within which there are several amino acid residues which undergo phosphorylation. In this study, we sought to investigate as to whether and how the IDR as well as phosphorylation at either Ser483, Ser492 or Thr510 affect the intracellular localization and self-oligomerization of ALS2. All phospho- and dephospho-mimetic ALS2 mutants that were transiently expressed in HeLa cells were diffusely distributed throughout the cytosol with a partial localization to early endosomes. When expressed under Rac1-activating conditions, these mutants were localized to membrane ruffles as well as enlarged endosomes. Further, gel-filtration analysis revealed that these mutants primarily existed as a tetramer in cells. However, all these phenotypes were indistinguishable from those of wild-type ALS2. On the other hand, IDR-deleted ALS2 mutant was exclusively present in perinuclear aggregates colocalizing with the autophagy-related protein SQSTM1. Moreover, IDR-deleted ALS2 mutant formed an abnormally high molecular weight complex compared to wild-type ALS2. These results indicate that the IDR of ALS2 plays a crucial role not only in the regulation of intracellular localization but also in the self-oligomerization of ALS2 in cells, whereas phosphorylation of certain residues within the IDR exerts limited effects on such phenotypes.
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ALS2 the small gtpase rab17 interacting protein regulates maturation and sorting of rab17 associated endosomes
Biochemical and Biophysical Research Communications, 2020Co-Authors: Suzuka Ono, Asako Otomo, Mitsunori Fukuda, Kai Sato, Shuji Murakoshi, Shun Mitsui, Shinji HadanoAbstract:Small GTPase Rab17 has been shown to regulate a wide range of physiological processes including cell migration in tumor cells and dendrite morphogenesis in neurons. However, molecular mechanism underlying Rab17-mediated intracellular trafficking is still unclear. To address this issue, we focused on Rab17-interacting protein ALS2, which was also known as a guanine nucleotide exchange factor (GEF) for Rab5, and investigated how ALS2 contributed to Rab17-associated membrane trafficking in cells. Rab17 was primarily localized to endosomal compartments, particularly to recycling endosomes, which was dependent on Rab11 expression. Upon Rac1 activation, Rab17 along with ALS2 was recruited to membrane ruffles and early endosomes in a Rab5 activity-independent manner. While RABGEF1, another Rab17-interacting Rab5 GEF, functioned as a GEF for Rab17, ALS2 did not possess such catalytic activity but merely interacted with Rab17. Importantly, ALS2 acted downstream of RABGEF1, regulating the maturation of Rab17-residing nascent endosomes to early endosome antigen 1 (EEA1)-positive early endosomes. Further, these Rab17-residing nascent endosomes were arisen via clathrin-independent endocytosis (CIE). Collectively, ALS2 plays a crucial role in the regulation of Rab17-associated endosomal trafficking and maturation, probably through their physical interaction, in cells.
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functional links between sqstm1 and ALS2 in the pathogenesis of als cumulative impact on the protection against mutant sod1 mediated motor dysfunction in mice
Human Molecular Genetics, 2016Co-Authors: Shinji Hadano, Asako Otomo, Kai Sato, Shun Mitsui, Suzuka Ono, Lei Pan, Mizuki Kubo, Wakana Onodera, Koichiro Abe, Xue Ping ChenAbstract:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons in the brain and spinal cord. Multiple toxicity pathways, such as oxidative stress, misfolded protein accumulation, and dysfunctional autophagy, are implicated in the pathogenesis of ALS. However, the molecular basis of the interplay between such multiple factors in vivo remains unclear. Here, we report that two independent ALS-linked autophagy-associated gene products; SQSTM1/p62 and ALS2/alsin, but not antioxidant-related factor; NFE2L2/Nrf2, are implicated in the pathogenesis in mutant SOD1 transgenic ALS models. We generated SOD1H46R mice either on a Nfe2l2-null, Sqstm1-null, or Sqstm1/ALS2-double null background. Loss of SQSTM1 but not NFE2L2 exacerbated disease symptoms. A simultaneous inactivation of SQSTM1 and ALS2 further accelerated the onset of disease. Biochemical analyses revealed that loss of SQSTM1 increased the level of insoluble SOD1 at the intermediate stage of the disease, whereas no further elevation occurred at the end-stage. Notably, absence of SQSTM1 rather suppressed the mutant SOD1-dependent accumulation of insoluble polyubiquitinated proteins, while ALS2 loss enhanced it. Histopathological examinations demonstrated that loss of SQSTM1 accelerated motor neuron degeneration with accompanying the preferential accumulation of ubiquitin-positive aggregates in spinal neurons. Since SQSTM1 loss is more detrimental to SOD1H46R mice than lack of ALS2, the selective accumulation of such aggregates in neurons might be more insulting than the biochemically-detectable insoluble proteins. Collectively, two ALS-linked factors, SQSTM1 and ALS2, have distinct but additive protective roles against mutant SOD1-mediated toxicity by modulating neuronal proteostasis possibly through the autophagy-endolysosomal system.
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defective relocalization of ALS2 alsin missense mutants to rac1 induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation
FEBS Letters, 2011Co-Authors: Asako Otomo, Ryota Kunita, Johe Ikeda, Kyoko Suzukiutsunomiya, Shinji HadanoAbstract:Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.
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genetic background and gender effects on gross phenotypes in congenic lines of ALS2 alsin deficient mice
Neuroscience Research, 2010Co-Authors: Shinji Hadano, Asako Otomo, Ryota Kunita, Kyoko Suzukiutsunomiya, Shigeru Kakuta, Yasuhiro Yoshii, Lei Pan, Yasuo Iwasaki, Yoichiro Iwakura, Johe IkedaAbstract:Abstract 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.
Johe Ikeda - One of the best experts on this subject based on the ideXlab platform.
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defective relocalization of ALS2 alsin missense mutants to rac1 induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation
FEBS Letters, 2011Co-Authors: Asako Otomo, Ryota Kunita, Johe Ikeda, Kyoko Suzukiutsunomiya, Shinji HadanoAbstract:Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.
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genetic background and gender effects on gross phenotypes in congenic lines of ALS2 alsin deficient mice
Neuroscience Research, 2010Co-Authors: Shinji Hadano, Asako Otomo, Ryota Kunita, Kyoko Suzukiutsunomiya, Shigeru Kakuta, Yasuhiro Yoshii, Lei Pan, Yasuo Iwasaki, Yoichiro Iwakura, Johe IkedaAbstract:Abstract 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.
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loss of ALS2 alsin exacerbates motor dysfunction in a sod1h46r expressing mouse als model by disturbing endolysosomal trafficking
PLOS ONE, 2010Co-Authors: Shinji Hadano, Asako Otomo, Ryota Kunita, Yasuo Uchiyama, Kyoko Suzukiutsunomiya, Akira Akatsuka, Masato Koike, Masashi Aoki, Yasuto Itoyama, Johe IkedaAbstract: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.
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ALS2 alsin deficiency in neurons leads to mild defects in macropinocytosis and axonal growth
Biochemical and Biophysical Research Communications, 2008Co-Authors: Asako Otomo, Shinji Hadano, Hikaru Mizumura, Ryota Kunita, Johe Ikeda, Kyoko Suzukiutsunomiya, Hitoshi Osuga, Kyuichiro OnoeAbstract: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.
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molecular and cellular function of ALS2 alsin implication of membrane dynamics in neuronal development and degeneration
Neurochemistry International, 2007Co-Authors: Shinji Hadano, Asako Otomo, Ryota Kunita, Johe Ikeda, Kyoko SuzukiutsunomiyaAbstract: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.
Asako Otomo - One of the best experts on this subject based on the ideXlab platform.
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the n terminal intrinsically disordered region mediates intracellular localization and self oligomerization of ALS2
Biochemical and Biophysical Research Communications, 2021Co-Authors: Kento Shimakura, Asako Otomo, Kai Sato, Shun Mitsui, Suzuka Ono, Shinji HadanoAbstract:ALS2, a product of the causative gene for familial amyotrophic lateral sclerosis (ALS) type 2, plays a pivotal role in the regulation of endosome dynamics by activating small GTPase Rab5 via its intrinsic guanine nucleotide-exchange factor activity. Previously, we have reported that the N-terminal region of ALS2 has crucial roles in its endosomal localization and self-oligomerization, both of which are indispensable for the cellular function of ALS2. The N-terminus of ALS2 contains the regulator of chromosome condensation 1-like domain (RLD), which is predicted to form a seven-bladed β-propeller structure. Interestingly, the RLD is interrupted by the intrinsically disordered region (IDR), within which there are several amino acid residues which undergo phosphorylation. In this study, we sought to investigate as to whether and how the IDR as well as phosphorylation at either Ser483, Ser492 or Thr510 affect the intracellular localization and self-oligomerization of ALS2. All phospho- and dephospho-mimetic ALS2 mutants that were transiently expressed in HeLa cells were diffusely distributed throughout the cytosol with a partial localization to early endosomes. When expressed under Rac1-activating conditions, these mutants were localized to membrane ruffles as well as enlarged endosomes. Further, gel-filtration analysis revealed that these mutants primarily existed as a tetramer in cells. However, all these phenotypes were indistinguishable from those of wild-type ALS2. On the other hand, IDR-deleted ALS2 mutant was exclusively present in perinuclear aggregates colocalizing with the autophagy-related protein SQSTM1. Moreover, IDR-deleted ALS2 mutant formed an abnormally high molecular weight complex compared to wild-type ALS2. These results indicate that the IDR of ALS2 plays a crucial role not only in the regulation of intracellular localization but also in the self-oligomerization of ALS2 in cells, whereas phosphorylation of certain residues within the IDR exerts limited effects on such phenotypes.
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altered oligomeric states in pathogenic ALS2 variants associated with juvenile motor neuron diseases cause loss of ALS2 mediated endosomal function
The FASEB Journal, 2020Co-Authors: Kai Sato, Asako Otomo, Kyoko Suzukiutsunomiya, Mahoko Takahashi Ueda, Yui Hiratsuka, Junya Sugiyama, Shuji Murakoshi, Shun Mitsui, Suzuka Ono, So NakagawaAbstract:Familial amyotrophic lateral sclerosis type 2 (ALS2) is a juvenile autosomal recessive motor neuron disease caused by the mutations in the ALS2 gene. The ALS2 gene product, ALS2/alsin, forms a homophilic oligomer and acts as a guanine nucleotide-exchange factor (GEF) for the small GTPase Rab5. This oligomerization is crucial for both Rab5 activation and ALS2-mediated endosome fusion and maturation in cells. Recently, we have shown that pathogenic missense ALS2 mutants retaining the Rab5 GEF activity fail to properly localize to endosomes via Rac1-stimulated macropinocytosis. However, the molecular mechanisms underlying dysregulated distribution of ALS2 variants remain poorly understood. Therefore, we sought to clarify the relationship between intracellular localization and oligomeric states of pathogenic ALS2 variants. Upon Rac family small GTPase 1 (Rac1) activation, all mutants tested moved from the cytosol to membrane ruffles but not to macropinosomes and/or endosomes. Furthermore, most WT ALS2 complexes were tetramers. Importantly, the sizes of an ALS2 complex carrying missense mutations in the N terminus of the regulator of chromosome condensation 1-like domain (RLD) or in-frame deletion in the pleckstrin homology domain were shifted toward higher molecular weight, whereas the C-terminal vacuolar protein sorting 9 (VPS9) domain missense mutant existed as a smaller dimeric or trimeric smaller form. Furthermore, in silico mutagenesis analyses using the RLD protein structure in conjunction with a cycloheximide chase assay in vitro disclosed that these missense mutations led to a decrease in protein stability. Collectively, disorganized higher structures of ALS2 variants might explain their impaired endosomal localization and the stability, leading to loss of the ALS2 function.
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ALS2 the small gtpase rab17 interacting protein regulates maturation and sorting of rab17 associated endosomes
Biochemical and Biophysical Research Communications, 2020Co-Authors: Suzuka Ono, Asako Otomo, Mitsunori Fukuda, Kai Sato, Shuji Murakoshi, Shun Mitsui, Shinji HadanoAbstract:Small GTPase Rab17 has been shown to regulate a wide range of physiological processes including cell migration in tumor cells and dendrite morphogenesis in neurons. However, molecular mechanism underlying Rab17-mediated intracellular trafficking is still unclear. To address this issue, we focused on Rab17-interacting protein ALS2, which was also known as a guanine nucleotide exchange factor (GEF) for Rab5, and investigated how ALS2 contributed to Rab17-associated membrane trafficking in cells. Rab17 was primarily localized to endosomal compartments, particularly to recycling endosomes, which was dependent on Rab11 expression. Upon Rac1 activation, Rab17 along with ALS2 was recruited to membrane ruffles and early endosomes in a Rab5 activity-independent manner. While RABGEF1, another Rab17-interacting Rab5 GEF, functioned as a GEF for Rab17, ALS2 did not possess such catalytic activity but merely interacted with Rab17. Importantly, ALS2 acted downstream of RABGEF1, regulating the maturation of Rab17-residing nascent endosomes to early endosome antigen 1 (EEA1)-positive early endosomes. Further, these Rab17-residing nascent endosomes were arisen via clathrin-independent endocytosis (CIE). Collectively, ALS2 plays a crucial role in the regulation of Rab17-associated endosomal trafficking and maturation, probably through their physical interaction, in cells.
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functional links between sqstm1 and ALS2 in the pathogenesis of als cumulative impact on the protection against mutant sod1 mediated motor dysfunction in mice
Human Molecular Genetics, 2016Co-Authors: Shinji Hadano, Asako Otomo, Kai Sato, Shun Mitsui, Suzuka Ono, Lei Pan, Mizuki Kubo, Wakana Onodera, Koichiro Abe, Xue Ping ChenAbstract:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons in the brain and spinal cord. Multiple toxicity pathways, such as oxidative stress, misfolded protein accumulation, and dysfunctional autophagy, are implicated in the pathogenesis of ALS. However, the molecular basis of the interplay between such multiple factors in vivo remains unclear. Here, we report that two independent ALS-linked autophagy-associated gene products; SQSTM1/p62 and ALS2/alsin, but not antioxidant-related factor; NFE2L2/Nrf2, are implicated in the pathogenesis in mutant SOD1 transgenic ALS models. We generated SOD1H46R mice either on a Nfe2l2-null, Sqstm1-null, or Sqstm1/ALS2-double null background. Loss of SQSTM1 but not NFE2L2 exacerbated disease symptoms. A simultaneous inactivation of SQSTM1 and ALS2 further accelerated the onset of disease. Biochemical analyses revealed that loss of SQSTM1 increased the level of insoluble SOD1 at the intermediate stage of the disease, whereas no further elevation occurred at the end-stage. Notably, absence of SQSTM1 rather suppressed the mutant SOD1-dependent accumulation of insoluble polyubiquitinated proteins, while ALS2 loss enhanced it. Histopathological examinations demonstrated that loss of SQSTM1 accelerated motor neuron degeneration with accompanying the preferential accumulation of ubiquitin-positive aggregates in spinal neurons. Since SQSTM1 loss is more detrimental to SOD1H46R mice than lack of ALS2, the selective accumulation of such aggregates in neurons might be more insulting than the biochemically-detectable insoluble proteins. Collectively, two ALS-linked factors, SQSTM1 and ALS2, have distinct but additive protective roles against mutant SOD1-mediated toxicity by modulating neuronal proteostasis possibly through the autophagy-endolysosomal system.
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defective relocalization of ALS2 alsin missense mutants to rac1 induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation
FEBS Letters, 2011Co-Authors: Asako Otomo, Ryota Kunita, Johe Ikeda, Kyoko Suzukiutsunomiya, Shinji HadanoAbstract:Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.
Ryota Kunita - One of the best experts on this subject based on the ideXlab platform.
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defective relocalization of ALS2 alsin missense mutants to rac1 induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation
FEBS Letters, 2011Co-Authors: Asako Otomo, Ryota Kunita, Johe Ikeda, Kyoko Suzukiutsunomiya, Shinji HadanoAbstract:Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.
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genetic background and gender effects on gross phenotypes in congenic lines of ALS2 alsin deficient mice
Neuroscience Research, 2010Co-Authors: Shinji Hadano, Asako Otomo, Ryota Kunita, Kyoko Suzukiutsunomiya, Shigeru Kakuta, Yasuhiro Yoshii, Lei Pan, Yasuo Iwasaki, Yoichiro Iwakura, Johe IkedaAbstract:Abstract 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.
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loss of ALS2 alsin exacerbates motor dysfunction in a sod1h46r expressing mouse als model by disturbing endolysosomal trafficking
PLOS ONE, 2010Co-Authors: Shinji Hadano, Asako Otomo, Ryota Kunita, Yasuo Uchiyama, Kyoko Suzukiutsunomiya, Akira Akatsuka, Masato Koike, Masashi Aoki, Yasuto Itoyama, Johe IkedaAbstract: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.
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ALS2 alsin deficiency in neurons leads to mild defects in macropinocytosis and axonal growth
Biochemical and Biophysical Research Communications, 2008Co-Authors: Asako Otomo, Shinji Hadano, Hikaru Mizumura, Ryota Kunita, Johe Ikeda, Kyoko Suzukiutsunomiya, Hitoshi Osuga, Kyuichiro OnoeAbstract: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.
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molecular and cellular function of ALS2 alsin implication of membrane dynamics in neuronal development and degeneration
Neurochemistry International, 2007Co-Authors: Shinji Hadano, Asako Otomo, Ryota Kunita, Johe Ikeda, Kyoko SuzukiutsunomiyaAbstract: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.
Rebecca S Devon - One of the best experts on this subject based on the ideXlab platform.
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ALS2 deficient mice exhibit disturbances in endosome trafficking associated with motor behavioral abnormalities
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Rebecca S Devon, J R Helm, Paul C Orban, K Gerrow, M A Barbieri, Claudia Schwab, L P Cao, Nagat Bissada, R Cruzaguado, T L DavidsonAbstract:ALS2 is an autosomal recessive form of spastic paraparesis (motor neuron disease) with juvenile onset and slow progression caused by loss of function of alsin, an activator of Rac1 and Rab5 small GTPases. To establish an animal model of ALS2 and derive insights into the pathogenesis of this illness, we have generated alsin-null mice. Cytosol from brains of ALS2−/− mice shows marked diminution of Rab5-dependent endosome fusion activity. Furthermore, primary neurons from ALS2−/− mice show a disturbance in endosomal transport of insulin-like growth factor 1 (IGF1) and BDNF receptors, whereas neuronal viability and endocytosis of transferrin and dextran seem unaltered. There is a significant decrease in the size of cortical motor neurons, and ALS2−/− mice are mildly hypoactive. Altered trophic receptor trafficking in neurons of ALS2−/− mice may underlie the histopathological and behavioral changes observed and the pathogenesis of ALS2.
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cross species characterization of the ALS2 gene and analysis of its pattern of expression in development and adulthood
Neurobiology of Disease, 2005Co-Authors: Rebecca S Devon, J R Helm, Justin D Topp, Paul C Orban, Claudia Schwab, Yu Zhou Yang, Terry D Pape, Tara Lynne Davidson, Daniel A Rogers, Francois GroslouisAbstract:Mutations in the ALS2 gene, which encodes alsin, cause autosomal recessive juvenile-onset amyotrophic lateral sclerosis (ALS2) and related conditions. Using both a novel monoclonal antibody and LacZ knock-in mice, we demonstrate that alsin is widely expressed in neurons of the CNS, including the cortex, brain stem and motor neurons of the spinal cord. Interestingly, the highest levels of alsin are found in the molecular layer of the cerebellum, a brain region not previously implicated in ALS2. During development, alsin is expressed by day E9.5, but CNS expression does not become predominant until early postnatal life. At the subcellular level, alsin is tightly associated with endosomal membranes and is likely to be part of a large protein complex that may include the actin cytoskeleton. ALS2 is present in primates, rodents, fish and flies, but not in the nematode worm or yeast, and is more highly conserved than expected among mammals. Additionally, the product of a second, widely expressed gene, ALS2 C-terminal like (ALS2CL), may subserve or modulate some of the functions of alsin as an activator of Rab and Rho GTPases.
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mutation screening of the ALS2 gene in sporadic and familial amyotrophic lateral sclerosis
JAMA Neurology, 2003Co-Authors: Collette K Hand, Rebecca S Devon, Francois Groslouis, Daniel Rochefort, Jawad Khoris, Vincent Meininger, Jeanpierre Bouchard, William Camu, Michael R Hayden, Guy A RouleauAbstract:Background Mutations in the ALS2 gene cause juvenile-onset autosomal recessive amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia. Objective To assess the role of ALS2 among more common forms of ALS. Methods DNA from 95 unrelated familial, 95 unrelated sporadic, and 11 early-onset ALS patients was screened for mutations in ALS2 by denaturing high-performance liquid chromatography and direct sequencing of polymerase chain reaction–amplified fragments. Each variant identified was also analyzed among control subjects. All 34 exons of ALS2 plus the 5′ and 3′ untranslated region were screened. Results We detected 23 novel sequence variants; however, none is disease-associated. Conclusion Mutations of ALS2 are not a common cause of ALS.
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the first nonsense mutation in alsin results in a homogeneous phenotype of infantile onset ascending spastic paralysis with bulbar involvement in two siblings
Clinical Genetics, 2003Co-Authors: Rebecca S Devon, J R Helm, Y Leitner, Guy A Rouleau, Tally LermansagieAbstract:: Eight mutations in the ALS2 gene have been described as causing autosomal-recessive juvenile-onset forms of the motor neuron diseases amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. All mutations are small deletions that are predicted to result in a frameshift and premature truncation of the alsin protein. Here we describe a ninth ALS2 mutation, in two siblings affected by infantile-onset ascending spastic paraplegia with bulbar involvement. This mutation is predicted to result in the substitution of an amino acid by a stop codon, and thus is the first nonsense mutation detected in this gene. It is probable that full-length alsin is required for the proper development and/or functioning of upper motor neurons.
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a gene encoding a putative gtpase regulator is mutated in familial amyotrophic lateral sclerosis 2
Nature Genetics, 2001Co-Authors: Shinji Hadano, Asako Otomo, Junko Showguchimiyata, Yoshiko Yanagisawa, Rebecca S Devon, Hitoshi Osuga, Collette K Hand, Natsuki Miyamoto, Yoshinori Okada, Roshni R SingarajaAbstract:Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.