High-Throughput Analysis of Bacterial Toxic Lipopolysaccharide in Water by Dual-Wavelength Monitoring Using a Ratiometric Fluorescent Chemosensor.

Lipopolysaccharide (LPS) is a bacterial toxin that causes fever in humans. Our small-molecule chemosensor named Zn-dpa-C2OPy shows rapid ratiometric fluorescence response to LPS in water with a detection limit of 11 pM, which is lower than that of our previously reported sensor. Spectroscopic measurements (fluorescence, absorbance, 1H NMR, and fluorescence lifetime), dynamic light scattering measurements, and transmission electron microscopy observations revealed that the fluorescence response was induced by the changes in the aggregation state via multi-point recognition of LPS through hydrophobic and electrostatic interactions, in addition to the coordination between the zinc(II)-dipicolylamine moiety of the chemosensor and the phosphate group of LPS. The proposed Zn-dpa-C2OPy chemosensor was applied to an original flow injection analysis (FIA) system with a self-developed dual-wavelength fluorophotometer, and a high throughput of 36 samples per hour was achieved. These results demonstrate the feasibility of this unique methodology combining a ratiometric fluorescent chemosensor and FIA for continuous online monitoring of LPS in water.

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.

Nanostructured Manganese Oxides within a Ring-Shaped Polyoxometalate Exhibiting Unusual Oxidation Catalysis.

Nanosized manganese oxides have recently received considerable attention for their synthesis, structures, and potential applications. Although various synthetic methods have been developed, precise synthesis of novel nanostructured manganese oxides are still challenging. In this study, using a structurally defined nanosized cavity inside a ring-shaped polyoxometalate, we succeeded in synthesizing two types of discrete 18 and 20 nuclear nanostructured manganese oxides, Mn18 and Mn20, respectively. In particular, Mn18 showed much higher catalytic activity than other manganese oxides for the oxygenation of alkylarenes including electron-deficient ones, and the reaction proceeded through a unique reaction mechanism due to its unusual manganese oxide structure.

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.

The N-terminal intrinsically disordered region mediates intracellular localization and self-oligomerization of ALS2.

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 ß-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.

ALS2, the small GTPase Rab17-interacting protein, regulates maturation and sorting of Rab17-associated endosomes.

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.

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.

Functional links between SQSTM1 and ALS2 in the pathogenesis of ALS: cumulative impact on the protection against mutant SOD1-mediated motor dysfunction in mice.

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.

Electrochemical detection of boric acid using gallacetophenonato-(ß-diketonato) ruthenium complex in water.

A simple and practical method for boron detection in water is desired in various fields such as seawater desalination, water conservation, and plant production. To develop a method for detecting boron as boric acid in water, we synthesized [Ru(acac)2(H2thap)] (acac = acetylacetonat ion, thap = 2',3',4'-trihydroxyacetophenonate (gallacetophenonate) ion) possessing a cis-diol moiety that interacts with boric acid. A comparison of UV-visible (UV-vis) absorption spectra measured in the presence and absence of boric acid at various pH values revealed that [Ru(acac)2(H2thap)] shows the highest response to boric acid at pH 8.5. Cyclic voltammograms (CVs) and differential pulse voltammograms (DPVs) of [Ru(acac)2(H2thap)] aqueous solution at pH 8.5 with varying boric acid concentrations showed a decrease in the peak current value at 0.032 V (vs. Ag|AgClaq.) and an increase in the peak current value at 0.444 V with increasing boric acid concentration. On the basis of the relationship between the ratio of current values (at 0.032 V and 0.444 V) and boric acid concentrations, the binding constant (assuming a 1:1 binding model) for the interaction between [Ru(acac)2(H2thap)] and boric acid was estimated to be 135.1 ± 9.1 mol-1 dm3, and the Limit of Detection (LOD) was calculated to be 1.03 mg B L-1.

Preparation of ultrasmall cyclodextrin nanogels by an inverse emulsion method using a cationic surfactant.

Stable water-in-oil emulsion membranes can be prepared using [dilauryl(dimethyl)ammonium] bromide (DDAB), a cationic surfactant. We prepared ultrasmall cyclodextrin (γ-CyD) nanogels (γ-CyDngs) by forming ionic pairs between the secondary hydroxyl groups of γ-CyDs and DDAB. Fluorescence and NMR characterisation of the obtained γ-CyDngs revealed superior inclusion affinities compared with native γ-CyDs, beneficial for the solubilisation of hydrophobic compounds in water.

Simultaneous Reverse Shoulder Arthroplasty and Open Reduction With Internal Fixation for Bilateral Proximal Humerus Fractures in the Elderly: A Report of Two Cases.

Simultaneous bilateral proximal humerus fractures (PHFs) caused by a single trauma are considered rare. We herein report two patients with bilateral PHFs treated based on our treatment strategy: reverse shoulder arthroplasty (RSA) to obtain rigid stability for one shoulder and open reduction with internal fixation (ORIF) to regain the mobility of external and internal rotation for the other. By using Neer's four-segment fracture classification, patients underwent RSA for one shoulder with the more advanced fracture type and ORIF for the other. In Case 1, a 74-year-old female presented with bilaterally comminuted PHFs and underwent surgical treatment with RSA and ORIF. In Case 2, a 78-year-old female with the comorbidity of rheumatoid arthritis and a history of total elbow arthroplasty for both elbows had bilateral PHFs and underwent surgical treatment with RSA and ORIF. Postoperatively, both patients were instructed to perform exercises including passive range of motion (ROM) for a week, active ROM exercises for two weeks, and muscle strengthening for six weeks after the surgery. At the follow-up, both patients were satisfied with the pain relief and functional recovery. Especially, increased ROM for external and internal rotation was obtained in shoulders with ORIF. These case reports describe a viable treatment option comprising simultaneous RSA and ORIF, and this surgical approach may restore shoulder functions in cases of bilateral PHFs.

High-throughput quantitative analysis of axonal transport in cultured neurons from SOD1H46R ALS mice by using a microfluidic device.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons. We have previously shown that autophagosome-like vesicular structures are progressively accumulated in the spinal axons of an ALS mouse model, overexpressing human Cu/Zn superoxide dismutase (SOD1) mutant, prior to the onset of motor symptoms. This suggests that axonal transport perturbation can be an early sign of neuronal dysfunction. However, the exact causal relationship between axonal transport deficits and neurodegeneration is not fully understood. To clarify whether axonal transport of organelles even in neurons at early developmental stages was affected by overexpression of mutant SOD1, we conducted a microfluidic device-based high-throughput quantitative analysis of the axonal transport of acidic vesicles and mitochondria in primary cultured cortical neurons established from SOD1H46R transgenic mice. Compared to wild-type (WT), a significantly increased number of motile acidic vesicles, i.e., autophagosomes and/or late-endosomes, was observed in the axons of SOD1H46R neurons. By contrast, mitochondria moving along the axons were significantly decreased in SOD1H46R compared to WT. Since such phenotypes, where the axonal transport of these organelles is differently affected by mutant SOD1 expression, emerge before axonal degeneration, axonal transport deficits could dysregulate axon homeostasis, thereby ultimately accelerating neurodegeneration.

SQSTM1, a protective factor of SOD1-linked motor neuron disease, regulates the accumulation and distribution of ubiquitinated protein aggregates in neuron.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motor neurons in the brain and spinal cord. Recent studies have shown that mutations in SQSTM1 are linked to ALS. It has also been demonstrated that a systemic loss of SQSTM1 exacerbates disease phenotypes in an ALS mouse model. However, it is still unclear whether and how SQSTM1 in the central nervous system (CNS) specifically regulates ALS-associated disease phenotypes. To address this issue, we generated CNS-specific Sqstm1 deficient SOD1H46R transgenic mice, and conducted gross phenotype analyses as well as the immunohistochemical and biochemical examinations of spinal cord tissues using these mice. CNS-specific SQSTM1 deficiency accelerated the disease onset and shortened the lifespan of SOD1H46R mice. The CNS-specific SQSTM1 ablation also resulted in increased number of ubiquitin-positive aggregates, while their size rather became much smaller. Remarkably, ubiquitin-positive aggregates, which were usually present in extracellular space and/or neuropil in SOD1H46R mice, were preferentially localized to soma and neurites of spinal neurons in CNS-specific SQSTM1 deficient SOD1H46R mice. Next, to further clarify the function of SQSTM1 in neurons, we investigated the contribution of SQSTM1 to the accumulation of polyubiquitinated proteins in relation to the ubiquitin proteasome system (UPS) and the autophagy-endolysosomal system (APELS) in primary cultured motor neurons (PMNs). Loss of SQSTM1 in PMNs resulted in decreased accumulation of insoluble polyubiquitinated proteins, which was induced by simultaneous treatment with proteasome and lysosome inhibitors, suggesting a pivotal role of SQSTM1 in the formation of insoluble protein aggregates. However, SQSTM1 silencing had a limited impact on the susceptibility to proteasome and/or lysosome inhibitor-induced apoptosis in PMNs. Taken together, neuronal SQSTM1, whose functions are associated with both the UPS and APELS, might primarily regulate the distribution and accumulation of misfolded protein aggregates in the CNS, thereby protecting neurons from degeneration in mice.

Development of microsatellite markers for the geographically parthenogenetic stick insect Phraortes elongatus (Insecta: Phasmatodea).

Many plant and animal species exhibit geographic parthenogenesis, wherein unisexual (= parthenogenetic) lineages are more common in their marginal habitats such as high latitude or altitudes than their closely related bisexual counterparts. The Japanese stick insect, Phraortes elongatus (Thunberg) (Insecta: Phasmatodea), is known as a geographically parthenogenetic species due to the existence of both bisexual and unisexual populations. Here, we developed microsatellite markers to infer the genetic variation among populations of P. elongatus. Totally, 13 primer pairs were developed for the species, and they were tested on 47 samples collected from both a bisexual population and a unisexual population. All 13 loci were polymorphic in the bisexual population, whereas no loci were polymorphic in the unisexual population. The loss of variation in the unisexual population implies automixis with terminal fusion or gamete duplication as the mode of parthenogenesis. The markers developed in this study will be helpful for further comprehensive analysis of the genetic diversity and gene flow between bisexual and parthenogenetic lineages of P. elongatus.

SQSTM1L341V variant that is linked to sporadic ALS exhibits impaired association with MAP1LC3 in cultured cells.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are genetically, pathologically and clinically-related progressive neurodegenerative diseases. Thus far, several SQSTM1 variations have been identified in patients with ALS and FTD. However, it remains unclear how SQSTM1 variations lead to neurodegeneration. To address this issue, we investigated the effects of ectopic expression of SQSTM1 variants, which were originally identified in Japanese and Chinese sporadic ALS patients, on the cellular viability, their intracellular distributions and the autophagic activity in cultured cells. Expression of SQSTM1 variants in PC12 cells exerted no observable effects on viabilities under both normal and oxidative-stressed conditions. Further, although expression of SQSTM1 variants in PC12 cells and Sqstm1-deficient mouse embryonic fibroblasts resulted in the formation of numerous granular SQSTM1-positive structures, called SQSTM1-bodies, their intracellular distributions were indistinguishable from those of wild-type SQSTM1. Nonetheless, quantitative colocalization analysis of SQSTM1-bodies with MAP1LC3 demonstrated that among ALS-linked SQSTM1 variants, L341V variant showed the significantly lower level of colocalization. However, there were no consistent effects on the autophagic activities among the variants examined. These results suggest that although some ALS-linked SQSTM1 variations have a discernible effect on the intracellular distribution of SQSTM1-bodies, the impacts of other variations on the cellular homeostasis are rather limited at least under transiently-expressed conditions.

Hemophagocytosis induced by Leishmania donovani infection is beneficial to parasite survival within macrophages.

Visceral leishmaniasis (VL) is caused by parasitic protozoa of the genus Leishmania and is characterized by clinical manifestations such as fever, hepatosplenomegaly and anemia. Hemophagocytosis, the phenomenon of phagocytosis of blood cells by macrophages, is found in VL patients. In a previous study we established an experimental model of VL, reproducing anemia in mice for the first time, and identified hemophagocytosis by heavily infected macrophages in the spleen as a possible cause of anemia. However, the mechanism for parasite-induced hemophagocytosis or its role in parasite survival remained unclear. Here, we established an in vitro model of Leishmania-induced hemophagocytosis to explore the molecules involved in this process. In contrast to naïve RAW264.7 cells (mouse macrophage cell line) which did not uptake freshly isolated erythrocytes, RAW264.7 cells infected with L. donovani showed enhanced phagocytosis of erythrocytes. Additionally, for hemophagocytes found both in vitro and in vivo, the expression of signal regulatory protein α (SIRPα), one of the receptors responsible for the 'don't-eat-me' signal was suppressed by post-transcriptional control. Furthermore, the overlapped phagocytosis of erythrocytes and Leishmania parasites within a given macrophage appeared to be beneficial to the parasites; the in vitro experiments showed a higher number of parasites within macrophages that had been induced to engulf erythrocytes. Together, these results suggest that Leishmania parasites may actively induce hemophagocytosis by manipulating the expression of SIRPα in macrophages/hemophagocytes, in order to secure their parasitism.

Oral activity of the antimalarial endoperoxide 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against Leishmania donovani complex.

Visceral leishmaniasis (VL) is a major problem worldwide and causes significant morbidity and mortality. Existing drugs against VL have limitations, including their invasive means of administration long duration of treatment regimens. There are also concerns regarding increasing treatment relapses as well as the identification of resistant clinical strains with the use of miltefosine, the sole oral drug for VL. There is, therefore, an urgent need for new alternative oral drugs for VL. In the present study, we show the leishmanicidal effect of a novel, oral antimalarial endoperoxide N-251. In our In vitro studies, N-251 selectively and specifically killed Leishmania donovani D10 amastigotes with no accompanying toxicity toward the host cells. In addition, N-251 exhibited comparable activities against promastigotes of L. donovani D10, as well as other L. donovani complex parasites, suggesting a wide spectrum of activity. Furthermore, even after a progressive infection was established in mice, N-251 significantly eliminated amastigotes when administered orally. Finally, N-251 suppressed granuloma formation in mice liver through parasite death. These findings indicate the therapeutic effect of N-251 as an oral drug, hence suggest N-251 to be a promising lead compound for the development of a new oral chemotherapy against VL.

Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1H46R-expressing ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective loss of upper and lower motor neurons. Recent studies have shown that mutations in SQSTM1 are linked to ALS. SQSTM1 encodes SQSTM1/p62 that regulates not only autophagy via the association with MAP1LC3/LC3 and ubiquitinated proteins but also the KEAP1-NFE2L2/Nrf2 anti-oxidative stress pathway by interacting with KEAP1. Previously, we have demonstrated that loss of SQSTM1 exacerbates disease phenotypes in a SOD1H46R-expressing ALS mouse model. To clarify the effects of SQSTM1 overexpression in this model, we generated SQSTM1 and SOD1 H46R double-transgenic (SQSTM1;SOD1 H46R ) mice. SQSTM1;SOD1 H46R mice exhibited earlier disease onset and shorter lifespan than did SOD1 H46R mice. Conversely, disease progression after the onset rather slightly but significantly slowed in SQSTM1;SOD1 H46R mice. However, there were observable differences neither in the number of Nissl positive neurons nor in the distribution of ubiquitin-positive and/or SQSTM1-positive aggregates between SOD1 H46R and SQSTM1;SOD1 H46R mice. It was noted that these protein aggregates were mainly observed in neuropil, and partly localized to astrocytes and/or microglia, but not to MAP2-positive neuronal cell bodies and dendrites at the end-stage of disease. Nonetheless, the biochemically-detectable insoluble SQSTM1 and poly-ubiquitinated proteins were significantly and progressively increased in the spinal cord of SQSTM1;SOD1 H46R mice compared to SOD1 H46R mice. These results suggest that overexpression of SQSTM1 in SOD1 H46R mice accelerates disease onset by compromising the protein degradation pathways.