Central nervous system specific high molecular weight ALS2/alsin homophilic complex is enriched in mouse brain synaptosomes.

ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function.

Altered oligomeric states in pathogenic ALS2 variants associated with juvenile motor neuron diseases cause loss of ALS2-mediated endosomal function.

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.

Persistent inflammation and T cell exhaustion in severe sepsis in the elderly.

INTRODUCTION: Sepsis is known as a complex immunological response with hyperinflammation in the acute phase followed by immunosuppression. Although aging is crucial in sepsis, the impact of aging on inflammation and immunosuppression is still unclear. The purpose of this study was to investigate the relationship between inflammation and immunosuppression in aged patients and mice after sepsis. METHODS: Fifty-five patients with severe sepsis and 30 healthy donors were prospectively enrolled, and 90-day survival was compared between elderly (≥ 65 years) and adult (18-64 years) septic patients with serial measurement of serum interleukin (IL)-6. Within 24 h after diagnosis of severe sepsis, peripheral blood mononuclear cells were stimulated ex vivo to measure expression of the activation maker CD25 in T cells, IL-2 levels in the supernatant, and proliferation. In the mouse study, young (6-8 weeks) and aged (20-22 months) C57/B6 mice were subjected to cecal ligation and puncture (CLP), and survival was compared after 7 days with serial measurement of serum IL-6. Expression of the negative co-stimulatory molecules, CD25, and IL-2 in CD4+ T cells was measured. RESULTS: The survival rate in elderly sepsis patients and aged septic mice was significantly lower than that in adult patients and young septic mice (60% vs. 93% in septic patients, 0% vs. 63% in septic mice, P < 0.05). Serum IL-6 levels in elderly sepsis patients and aged septic mice were persistently higher than those in adult patients and young septic mice. Expression of negative co-stimulatory molecules in CD4+ T cells in the spleen, lymph nodes, and peripheral blood was significantly higher in aged mice than in young mice (P < 0.01). Ex vivo stimulation decreased CD25 expression, IL-2 production, and proliferation to a greater extent in CD4+ T cells from elderly patients and aged septic mice than in those from adult patients and young septic mice. Elderly patients demonstrated increased detection of gram-negative bacteria at days 14-16 and 28-32 after sepsis (P < 0.05). CONCLUSIONS: Persistent inflammation and T cell exhaustion may be associated with decreased survival in elderly patients and mice after sepsis.

Fermentation of non-sterilized fish biomass with a mixed culture of film-forming yeasts and lactobacilli and its effect on innate and adaptive immunity in mice.

Non-sterilized fish waste containing fish bones was fermented using combined starter cultures of film-forming yeast (Candida ethanolica) and lactic acid bacteria (LAB; Lactobacillus casei and Lactobacillus rhamnosus) in order to obtain a liquefied fermented broth without spoiling. During the entire fermentation, the number of LAB cells was maintained at a high level (6 × 10(8)-5 × 10(7) cells/ml). Although the number of general bacteria was 10(6)cell/ml after adding non-sterilized fish biomass, its growth was suppressed to be 1-3 × 10(4) cells/ml. The entire biomass had completely liquefied and the fermented broth contained all 20 α-amino acids composed of protein and also various kinds of minerals in abundance. The weight of mice group fed the fermented broth content feed (sample feed) for 31 days significantly increased compared with that fed no broth feed (control feed) (21.37 g vs 20.76 g (p < 0.05). No abnormal behavior and appearance were observed. All internal organs (the heart, the liver, the lung, the intestines, and the spleen) of both groups were confirmed to be normal by visual observation. In peripheral blood, the percentages of NK cells and CD8+ T cells of the mice in the sample feed group increased significantly relative to those in the control feed group (NK cells: 19% vs 11%, CD8+ T cells: 9% vs 5%, p < 0.05). In the spleen, the percentage of NK cells in the sample feed group also increased significantly compared to that in the control feed group (p < 0.05). The fermented fish biomass is expected to be effective for innate and adaptive immunity and thus fit for animal feed.

Sepsis-induced hypercytokinemia and lymphocyte apoptosis in aging-accelerated Klotho knockout mice.

Sepsis is primarily a disease of the aged, with 65% of sepsis cases reported in patients older than 65 years and 80% of deaths due to sepsis occurring in this age group. Klotho knockout mice (Klotho mice) are a mouse model of accelerated aging and shortened life span. The purpose of the study was to elucidate the immunological changes occurring in Klotho mice during sepsis. Five-week-old homozygous female Klotho knockout (Klotho) and wild-type (WT) mice were subjected to 1 × 27-gauge cecal ligation and puncture (CLP), and survival was compared after 4 days. Another set of mice was killed at 8 h after CLP or sham surgery, and the spleen, thymus, and serum were harvested. Apoptosis was measured by flow cytometry by using caspase 3. Serum cytokines and bacterial colony count in peritoneal lavage were also analyzed. Klotho septic mice started to die at 8 to 12 h after CLP, and the final survival of Klotho mice was significantly lower than that of WT mice (0% vs. 100%, P < 0.01). Increased bacterial count in the peritoneal cavity and decreased recruitment of neutrophils and macrophages to the peripheral cavity were observed in Klotho-CLP mice. Both flow cytometric and immunohistological analyses showed a dramatic increase in caspase 3-positive cells in the thymus and spleen of Klotho-CLP mice (P < 0.01). Serum concentrations of interleukin 6, tumor necrosis factor α, and interleukin 10 were higher in Klotho-CLP mice than in WT-CLP mice. Hypercytokinemia with impaired bacterial clearance and increased apoptosis of lymphocytes may be related to poor survival in Klotho-septic mice.

Reduction of immunocompetent T cells followed by prolonged lymphopenia in severe sepsis in the elderly.

OBJECTIVE: To investigate the immunological changes caused by severe sepsis in elderly patients. DESIGN: One-year, prospective observational study. SETTING: Emergency department and intensive care unit of a single university hospital. PATIENTS: Seventy-three patients with severe sepsis and 72 healthy donors. MEASUREMENTS AND MAIN RESULTS: In elderly septic patients (aged 65 yr and over), 3-month survival was significantly reduced compared with that for adult patients (18-64 yr) (60% vs. 89%, p < 0.01). We found that lymphopenia was prolonged for at least 21 days in elderly nonsurvivors of sepsis, while the number of lymphocytes recovered in both adult and elderly survivors of sepsis. In order to examine the immunological status of septic patients, blood samples were collected within 48 hrs of diagnosis of severe sepsis, and peripheral blood mononuclear cells were purified for flow cytometric analysis. T cell levels were significantly reduced in both adult and elderly septic patients, compared with those in healthy donors (56% and 57% reduction, respectively). Interestingly, the immunocompetent CD28+ subset of CD4+ T cells decreased, whereas the immunosuppressive PD-1+ T cells and the percentage of regulatory T cells (CD4+ T cells that are both Foxp3+ and CD25+) increased in elderly patients, especially nonsurvivors, presumably reflecting the initial signs of immunosuppression. CONCLUSION: Reduction of immunocompetent T cells followed by prolonged lymphopenia may be associated with poor prognosis in elderly septic patients.

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

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.

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.

Loss of ALS2/Alsin exacerbates motor dysfunction in a SOD1-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.

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.

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.

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, a novel ALS2-interactor, modulates ALS2-mediated endosome dynamics.

ALS2, the causative gene product for a number of recessive motor neuron diseases, is a guanine-nucleotide exchange factor for Rab5, and acts as a modulator for endosome dynamics. Recently, we have identified a novel ALS2 homolog, ALS2CL, which is highly homologous to the C-terminal half of ALS2. In this study, we investigate the molecular features of ALS2CL and its functional relationship with ALS2. A majority of ALS2CL is present as a homo-dimeric form, which can interact with the ALS2-oligomer, resulting in the formation of the large ALS2/ALS2CL heteromeric complex. In cultured cells, overexpressed ALS2CL is colocalized with ALS2 onto membranous compartments. Further, ALS2CL dominantly suppresses the endosome enlargement induced by a constitutively active form of ALS2, and results in an extensive perinuclear tubulo-membranous phenotype, which are dependent upon the ALS2CL-ALS2 interaction. Collectively, ALS2CL is a novel ALS2-interacting protein and is implicated in ALS2-mediated endosome dynamics.

Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin exhibit age-dependent neurological deficits and altered endosome trafficking.

ALS2/alsin is a member of guanine nucleotide exchange factors for the small GTPase Rab5 (Rab5GEFs), which act as modulators in endocytic pathway. Loss-of-function mutations in human ALS2 account for a number of juvenile recessive motor neuron diseases (MNDs). However, the normal physiological role of ALS2 in vivo and the molecular mechanisms underlying motor dysfunction are still unknown. To address these issues, we have generated mice homozygous for disruption of the Als2 gene. The Als2-null mice observed through 21 months of age demonstrated no obvious developmental, reproductive or motor abnormalities. However, immunohistochemical and electrophysiological analyses identified an age-dependent, slowly progressive loss of cerebellar Purkinje cells and disturbance of spinal motor neurons associated with astrocytosis and microglial cell activation, indicating a subclinical dysfunction of motor system in Als2-null mice. Further, quantitative epidermal growth factor (EGF)-uptake analysis identified significantly smaller-sized EGF-positive endosomes in Als2-null fibroblasts, suggesting an alteration of endosome/vesicle trafficking in the cells. Collectively, while loss of ALS2 does not produce a severe disease phenotype in mice, these Als2-null animals should provide a useful model with which to understand the interplay between endosomal dynamics and the long-term viability of large neurons such as Purkinje cells and spinal motor neurons.

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.