Alpha-secretase dependent nuclear localization of the amyloid-ß precursor protein-binding protein Fe65 promotes DNA repair.

Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actin cytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65 is the transmembrane amyloid-ß precursor protein (APP), which can undergo regulated intramembrane proteolysis (RIP). Following ß- and γ-secretase-mediated RIP, the released APP intracellular domain (AICD) together with Fe65 can translocate to the nucleus and regulate transcription. In this study, we investigated if Fe65 nuclear localization can also be regulated by different α-secretases, also known to participate in RIP of APP and other transmembrane proteins. We found that in both Phorbol 12-myristate 13-acetate and all-trans retinoic acid differentiated neuroblastoma cells a strong negative impact on Fe65 nuclear localization, equal to the effect observed upon γ-secretase inhibition, could be detected following inhibition of all three (ADAM9, ADAM10 and ADAM17) α-secretases. Moreover, using the comet assay and analysis of Fe65 dependent DNA repair associated posttranslational modifications of histones, we could show that inhibition of α-secretase-mediated Fe65 nuclear translocation resulted in impaired capacity of the cells to repair DNA damage. Taken together this suggests that α-secretase processing of APP and/or other Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nuclear translocation and DNA repair.

Key Modulators of the Stress Granule Response TIA1, TDP-43, and G3BP1 Are Altered by Polyglutamine-Expanded ATXN7.

Spinocerebellar ataxia type 7 (SCA7) and other polyglutamine (polyQ) diseases are caused by expansions of polyQ repeats in disease-specific proteins. Aggregation of the polyQ proteins resulting in various forms of cellular stress, that could induce the stress granule (SG) response, is believed to be a common pathological mechanism in these disorders. SGs can contribute to cell survival but have also been suggested to exacerbate disease pathology by seeding protein aggregation. In this study, we show that two SG-related proteins, TDP-43 and TIA1, are sequestered into the aggregates formed by polyQ-expanded ATXN7 in SCA7 cells. Interestingly, mutant ATXN7 also localises to induced SGs, and this association altered the shape of the SGs. In spite of this, neither the ability to induce nor to disassemble SGs, in response to arsenite stress induction or relief, was affected in SCA7 cells. Moreover, we could not observe any change in the number of ATXN7 aggregates per cell following SG induction, although a small, non-significant, increase in total aggregated ATXN7 material could be detected using filter trap. However, mutant ATXN7 expression in itself increased the speckling of the SG-nucleating protein G3BP1 and the SG response. Taken together, our results indicate that the SG response is induced, and although some key modulators of SGs show altered behaviour, the dynamics of SGs appear normal in the presence of mutant ATXN7.

Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane, and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC).

The image analysis tool FRIC (Fluorescence Ratiometric Imaging of Chromatin) quantitatively monitors dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As a proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation as well as expression of the mutant lamin A protein "progerin," which causes Hutchinson-Gilford Progeria Syndrome. In summary FRIC is versatile, unbiased, robust, requires a minimum of experimental steps and is suitable for screening purposes.

Polyglutamine expanded Ataxin-7 induces DNA damage and alters FUS localization and function.

Polyglutamine (polyQ) diseases, such as Spinocerebellar ataxia type 7 (SCA7), are caused by expansions of polyQ repeats in disease specific proteins. The sequestration of vital proteins into aggregates formed by polyQ proteins is believed to be a common pathological mechanism in these disorders. The RNA-binding protein FUS has been observed in polyQ aggregates, though if disruption of this protein plays a role in the neuronal dysfunction in SCA7 or other polyQ diseases remains unclear. We therefore analysed FUS localisation and function in a stable inducible PC12 cell model expressing the SCA7 polyQ protein ATXN7. We found that there was a high degree of FUS sequestration, which was associated with a more cytoplasmic FUS localisation, as well as a decreased expression of FUS regulated mRNAs. In contrast, the role of FUS in the formation of γH2AX positive DNA damage foci was unaffected. In fact, a statistical increase in the number of γH2AX foci, as well as an increased trend of single and double strand DNA breaks, detected by comet assay, could be observed in mutant ATXN7 cells. These results were further corroborated by a clear trend towards increased DNA damage in SCA7 patient fibroblasts. Our findings suggest that both alterations in the RNA regulatory functions of FUS, and increased DNA damage, may contribute to the pathology of SCA7.

Phosphorylation of the amyloid precursor protein (APP) at Ser-675 promotes APP processing involving meprin ß.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of ß-amyloid (Aß) peptides. Aß is a cleavage product of the amyloid precursor protein (APP), and aberrant posttranslational modifications of APP can alter APP processing and increase Aß generation. In the AD brain, seven different residues, including Ser-675 (APP695 numbering) in the APP cytoplasmic domain has been found to be phosphorylated. Here, we show that expression of a phosphomimetic variant of Ser-675 in APP (APP-S675E), in human neuroblastoma SK-N-AS cells, reduces secretion of the soluble APP ectodomain (sAPPα), even though the total plasma membrane level of APP was unchanged compared with APP levels in cells expressing APPwt or APP-S675A. Moreover, the level of an alternative larger C-terminal fragment (CTF) increased in the APP-S675E cells, whereas the CTF form that was most abundant in cells expressing APPwt or APP-S675A decreased in the APP-S675E cells. Upon siRNA-mediated knockdown of the astacin metalloprotease meprin ß, the levels of the alternative CTF decreased and the CTF ratio was restored back to APPwt levels. Our findings suggest that APP-Ser-675 phosphorylation alters the balance of APP processing, increasing meprin ß-mediated and decreasing α-secretase-mediated processing of APP at the plasma membrane. As meprin ß cleavage of APP has been shown to result in formation of highly aggregation-prone, truncated Aß2-40/42 peptides, enhanced APP processing by this enzyme could contribute to AD pathology. We propose that it would be of interest to clarify in future studies how APP-Ser-675 phosphorylation promotes meprin ß-mediated APP cleavage.

Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1.

In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.

Altered p53 and NOX1 activity cause bioenergetic defects in a SCA7 polyglutamine disease model.

Spinocerebellar ataxia type 7 (SCA7) is one of the nine neurodegenerative disorders caused by expanded polyglutamine (polyQ) domains. Common pathogenic mechanisms, including bioenergetics defects, have been suggested for these so called polyQ diseases. However, the exact molecular mechanism(s) behind the metabolic dysfunction is still unclear. In this study we identified a previously unreported mechanism, involving disruption of p53 and NADPH oxidase 1 (NOX1) activity, by which the expanded SCA7 disease protein ATXN7 causes metabolic dysregulation. The NOX1 protein is known to promote glycolytic activity, whereas the transcription factor p53 inhibits this process and instead promotes mitochondrial respiration. In a stable inducible PC12 model of SCA7, p53 and mutant ATXN7 co-aggregated and the transcriptional activity of p53 was reduced, resulting in a 50% decrease of key p53 target proteins, like AIF and TIGAR. In contrast, the expression of NOX1 was increased approximately 2 times in SCA7 cells. Together these alterations resulted in a decreased respiratory capacity, an increased reliance on glycolysis for energy production and a subsequent 20% reduction of ATP in SCA7 cells. Restoring p53 function, or suppressing NOX1 activity, both reversed the metabolic dysfunction and ameliorated mutant ATXN7 toxicity. These results hence not only enhance the understanding of the mechanisms causing metabolic dysfunction in SCA7 disease, but also identify NOX1 as a novel potential therapeutic target in SCA7 and possibly other polyQ diseases.

Inhibition of autophagy via p53-mediated disruption of ULK1 in a SCA7 polyglutamine disease model.

Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine domains. These so-called polyglutamine (polyQ) diseases are all characterized by aggregation. Reducing the level of aggregating polyQ proteins via pharmacological activation of autophagy has been suggested as a therapeutic approach. However, recently, evidence implicating autophagic dysfunction in these disorders has also been reported. In this study, we show that the SCA7 polyglutamine protein ataxin-7 (ATXN7) reduces the autophagic activity via a previously unreported mechanism involving p53-mediated disruption of two key proteins involved in autophagy initiation. We show that in mutant ATXN7 cells, an increased p53-FIP200 interaction and co-aggregation of p53-FIP200 into ATXN7 aggregates result in decreased soluble FIP200 levels and subsequent destabilization of ULK1. Together, this leads to a decreased capacity for autophagy induction via the ULK1-FIP200-Atg13-Atg101 complex. We also show that treatment with a p53 inhibitor, or a blocker of ATXN7 aggregation, can restore the soluble levels of FIP200 and ULK1, as well as increase the autophagic activity and reduce ATXN7 toxicity. Understanding the mechanism behind polyQ-mediated inhibition of autophagy is of importance if therapeutic approaches based on autophagy stimulation should be developed for these disorders.

Expanded ataxin-7 cause toxicity by inducing ROS production from NADPH oxidase complexes in a stable inducible Spinocerebellar ataxia type 7 (SCA7) model.

BACKGROUND: Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common mechanisms of disease pathogenesis suggested for polyQ disorders include aggregation of the polyQ protein and induction of oxidative stress. However, the exact mechanism(s) of toxicity is still unclear. RESULTS: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity. The increase in ROS could be completely prevented by inhibition of NADPH oxidase (NOX) complexes suggesting that ATXN7 directly or indirectly causes oxidative stress by increasing superoxide anion production from these complexes. Moreover, we could observe that induction of mutant ATXN7 leads to a decrease in the levels of catalase, a key enzyme in detoxifying hydrogen peroxide produced from dismutation of superoxide anions. This could also contribute to the generation of oxidative stress. Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7. In contrast, ATXN7 aggregation was aggravated by treatments promoting oxidative stress. CONCLUSION: Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

Differential degradation of full-length and cleaved ataxin-7 fragments in a novel stable inducible SCA7 model.

Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine repeats, and a common toxic gain-of-function mechanism has been proposed. Proteolytic cleavage of several polyglutamine proteins has been identified and suggested to modulate the polyglutamine toxicity. In this study, we show that full-length and cleaved fragments of the SCA7 disease protein ataxin-7 (ATXN7) are differentially degraded. We found that the ubiquitin-proteosome system (UPS) was essential for the degradation of full-length endogenous ATXN7 or transgenic full-length ATXN7 with a normal or expanded glutamine repeat in both HEK 293T and stable PC12 cells. However, a similar contribution by UPS and autophagy was found for the degradation of proteolytically cleaved ATXN7 fragments. Furthermore, in our novel stable inducible PC12 model, induction of mutant ATXN7 expression resulted in toxicity and this toxicity was worsened by inhibition of either UPS or autophagy. In contrast, pharmacological activation of autophagy could ameliorate the ATXN7-induced toxicity. Based on our findings, we propose that both UPS and autophagy are important for the reduction of mutant ataxin-7-induced toxicity, and enhancing ATXN7 clearance through autophagy could be used as a potential therapeutic strategy in SCA7.

EMMPRIN regulates cytoskeleton reorganization and cell adhesion in prostate cancer.

BACKGROUND: Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment. METHODS: In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) versus malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization. RESULTS: EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression, or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins. CONCLUSIONS: Our results suggest that EMMPRIN regulates cell adhesion, invasion, and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis.

Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport.

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein-dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.

Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism.

The p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper-zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin-association domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N-terminal Phox and Bem1 (PB1) domain (residues 1-104) and a separate internal region (residues 178-224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187, and K189 residues are critical to the p62-mutant SOD1 interaction as substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin-independent fashion and targeted for the autophagy-lysosome degradation pathway.

Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord.

Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton.

Enzymatically inactive adenylate kinase 4 interacts with mitochondrial ADP/ATP translocase.

Adenylate kinase 4 (AK4) is a unique member with no enzymatic activity in vitro in the adenylate kinase (AK) family although it shares high sequence homology with other AKs. It remains unclear what physiological function AK4 might play or why it is enzymatically inactive. In this study, we showed increased AK4 protein levels in cultured cells exposed to hypoxia and in an animal model of the neurodegenerative disease amyotrophic lateral sclerosis. We also showed that short hairpin RNA (shRNA)-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and increased cell death. Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H(2)O(2) induced cell death. Proteomic studies revealed that the mitochondrial ADP/ATP translocases (ANTs) interacted with AK4 and higher amount of ANT was co-precipitated with AK4 when cells were exposed to H(2)O(2) treatment. In addition, structural analysis revealed that, while AK4 retains the capability of binding nucleotides, AK4 has a glutamine residue instead of a key arginine residue in the active site well conserved in other AKs. Mutation of the glutamine residue to arginine (Q159R) restored the adenylate kinase activity with GTP as substrate. Collectively, these results indicate that the enzymatically inactive AK4 is a stress responsive protein critical to cell survival and proliferation. It is likely that the interaction with the mitochondrial inner membrane protein ANT is important for AK4 to exert the protective benefits to cells under stress.

Interaction of amyotrophic lateral sclerosis (ALS)-related mutant copper-zinc superoxide dismutase with the dynein-dynactin complex contributes to inclusion formation.

An important consequence of protein misfolding related to neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the formation of proteinaceous inclusions or aggregates within the central nervous system. We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interact and co-localize with the dynein-dynactin complex in cultured cells and affected tissues of ALS mice. In this study, we report that the interaction between mutant SOD1 and the dynein motor plays a critical role in the formation of large inclusions containing mutant SOD1. Disruption of the motor by overexpression of the p50 subunit of dynactin in neuronal and non-neuronal cell cultures abolished the association between aggregation-prone SOD1 mutants and the dynein-dynactin complex. The p50 overexpression also prevented mutant SOD1 inclusion formation and improved the survival of cells expressing A4V SOD1. Furthermore, we observed that two ALS-linked SOD1 mutants, H46R and H48Q, which showed a lower propensity to interact with the dynein motor, also produced less aggregation and fewer large inclusions. Overall, these data suggest that formation of large inclusions depends upon association of the abnormal SOD1s with the dynein motor. Whether the misfolded SOD1s directly perturb axonal transport or impair other functional properties of the dynein motor, this interaction could propagate a toxic effect that ultimately causes motor neuron death in ALS.

Retrograde axonal transport and motor neuron disease.

Transport of material between extensive neuronal processes and the cell body is crucial for neuronal function and survival. Growing evidence shows that deficits in axonal transport contribute to the pathogenesis of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we review recent data indicating that defects in dynein-mediated retrograde axonal transport are involved in ALS etiology. We discuss how mutant copper-zinc superoxide dismutase (SOD1) and an aberrant interaction between mutant SOD1 and dynein could perturb retrograde transport of neurotrophic factors and mitochondria. A possible contribution of axonal transport to the aggregation and degradation processes of mutant SOD1 is also reviewed. We further consider how the interference with axonal transport and protein turnover by mutant SOD1 could influence the function and viability of motor neurons in ALS.

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration.

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin-dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. Here, we report age-dependent constitutive activation of glycogen synthase kinase 3beta, protein kinase B, mitogen-activated protein kinase, and c-Jun-N-terminal kinase in adult p62(-/-) mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62(-/-) brain led to recovery of aggregated K63-ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain-derived neurotrophic factor levels. Our findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer-like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid-beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity.

Interaction between familial amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants and the dynein complex.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor neuron death. More than 90 mutations in the copper-zinc superoxide dismutase (SOD1) gene cause a subset of familial ALS. Toxic properties have been proposed for the ALS-linked SOD1 mutants, but the nature of the toxicity has not been clearly specified. Cytoplasmic inclusion bodies containing mutant SOD1 and a number of other proteins are a pathological hallmark of mutant SOD1-mediated familial ALS, but whether such aggregates are toxic to motor neurons remains unclear. In this study, we identified a dynein subunit as a component of the mutant SOD1-containing high molecular weight complexes using proteomic techniques. We further demonstrated interaction and colocalization between dynein and mutant SOD1, but not normal SOD1, in cultured cells and also in G93A and G85R transgenic rodent tissues. Moreover, the interaction occurred early, prior to the onset of symptoms in the ALS animal models and increased over the disease progression. Motor neurons with long axons are particularly susceptible to defects in axonal transport. Our results demonstrate a direct "gain-of-interaction" between mutant SOD1 and dynein, which may provide insights into the mechanism by which mutant SOD1 could contribute to a defect in retrograde axonal transport or other dynein functions. The aberrant interaction is potentially critical to the formation of mutant SOD1 aggregates as well as the toxic cascades leading to motor neuron degeneration in ALS.

p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.