Apolipoprotein E Proteinopathy Is a Major Dementia-Associated Pathologic Biomarker in Individuals with or without the APOE Epsilon 4 Allele.

The amygdala is vulnerable to multiple or "mixed" mis-aggregated proteins associated with neurodegenerative conditions that can manifest clinically with amnestic dementia; the amygdala region is often affected even at earliest disease stages. With the original intent of identifying novel dementia-associated proteins, the detergent-insoluble proteome was characterized from the amygdalae of 40 participants from the University of Kentucky Alzheimer's Disease Center autopsy cohort. These individuals encompassed a spectrum of clinical conditions (cognitively normal to severe amnestic dementia). Polypeptides from the detergent-insoluble fraction were interrogated using liquid chromatography-electrospray ionization-tandem mass spectrometry. As anticipated, portions of peptides previously associated with neurologic diseases were enriched from subjects with dementia. Among all detected peptides, Apolipoprotein E (ApoE) stood out: even more than the expected Tau, APP/Aß, and α-Synuclein peptides, ApoE peptides were strongly enriched in dementia cases, including from individuals lacking the APOE ε4 genotype. The amount of ApoE protein detected in detergent-insoluble fractions was robustly associated with levels of complement proteins C3 and C4. Immunohistochemical staining of APOE ε3/ε3 subjects' amygdalae confirmed ApoE co-localization with C4 in amyloid plaques. Thus, analyses of human amygdala proteomics indicate that rather than being only an "upstream" genetic risk factor, ApoE is an aberrantly aggregated protein in its own right, and show that the ApoE protein may play active disease-driving mechanistic roles in persons lacking the APOE ε4 allele.

Lysine acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the preferential death of motor neurons. Approximately 10% of ALS cases are familial and 90% are sporadic. Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein implicated in familial ALS and frontotemporal dementia (FTD). The physiological function and pathological mechanism of FUS are not well understood, particularly whether post-translational modifications play a role in regulating FUS function. In this study, we discovered that FUS was acetylated at lysine-315/316 (K315/K316) and lysine-510 (K510) residues in two distinct domains. Located in the nuclear localization sequence, K510 acetylation disrupted the interaction between FUS and Transportin-1, resulting in the mislocalization of FUS in the cytoplasm and formation of stress granule-like inclusions. Located in the RNA recognition motif, K315/K316 acetylation reduced RNA binding to FUS and decreased the formation of cytoplasmic inclusions. Treatment with deacetylase inhibitors also significantly reduced the inclusion formation in cells expressing ALS mutation P525L. More interestingly, familial ALS patient fibroblasts showed higher levels of FUS K510 acetylation as compared with healthy controls. Lastly, CREB-binding protein/p300 acetylated FUS, whereas both sirtuins and histone deacetylases families of lysine deacetylases contributed to FUS deacetylation. These findings demonstrate that FUS acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS, implicating a potential role of acetylation in the pathophysiological process leading to FUS-mediated ALS/FTD.

Alpha adaptins show isoform-specific association with neurofibrillary tangles in Alzheimer's disease.

AIMS: The heterotetrameric assembly protein complex 2 (AP-2) is a central hub for clathrin-dependent endocytosis. The AP-2 α-adaptin subunit has two major isoforms, encoded by two separate genes: AP2A1 and AP2A2. Endocytosis has been implicated in the pathogenesis of neurodegenerative disease, and recent studies linked α-adaptins (gene variants, splicing defects and altered expression) with late-onset Alzheimer's disease (LOAD) risk. Here, we used multiple antibodies to investigate α-adaptin isoforms and their localization in human brains. METHODS: The specificities of 10 different α-adaptin antibodies were evaluated using immunoblots after human AP2A1 and AP2A2 plasmid transfection in cultured cells. Additional immunoblot analyses were then performed on protein homogenates from control and LOAD subjects. Formalin-fixed, paraffin-embedded brain sections from control and LOAD subjects were immunohistochemically stained, and immunofluorescence experiments were performed for quantitation of colocalisation with digital image analysis. RESULTS: Eight of the 10 evaluated antibodies recognised transfected α-adaptin proteins on immunoblots. The α-adaptin subspecies were relatively uniformly expressed in five different human brain regions. The α-adaptins were present in the detergent-insoluble fraction from cognitively impaired, but less so in control, brains. Immunohistochemical analyses showed colocalisation of AP2A1 with tau pathology in LOAD brains. By contrast, AP2A2 colocalised with microglial cells. CONCLUSIONS: These observations provide evidence of isoform-specific changes of α-adaptins in the brains of LOAD subjects. Antibodies that were verified to recognise AP2A1, but not AP2A2, labelled neurofibrillary tangles of LOAD patients. The findings extend our understanding of AP-2 proteins in the human brain in healthy and diseased states.

Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Mutations in the Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) gene cause a subset of familial ALS cases and are also implicated in sporadic ALS. FUS is typically localized to the nucleus. The ALS-related FUS mutations cause cytoplasmic mis-localization and the formation of stress granule-like structures. Abnormal cytoplasmic FUS localization was also found in a subset of frontotemporal dementia (FTLD) cases without FUS mutations. To better understand the function of FUS, we performed wild-type and mutant FUS pull-downs followed by proteomic identification of the interacting proteins. The FUS interacting partners we identified are involved in multiple pathways, including chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. FUS interacted with hnRNPA1 and Matrin-3, RNA binding proteins whose mutations were also reported to cause familial ALS, suggesting that hnRNPA1 and Matrin-3 may play common pathogenic roles with FUS. The FUS interactions displayed varied RNA dependence. Numerous FUS interacting partners that we identified are components of exosomes. We found that FUS itself was present in exosomes, suggesting that the secretion of FUS might contribute to the cell-to-cell spreading of FUS pathology. FUS interacting proteins were sequestered into the cytoplasmic mutant FUS inclusions that could lead to their mis-regulation or loss of function, contributing to ALS pathogenesis. Our results provide insights into the physiological functions of FUS as well as important pathways where mutant FUS can interfere with cellular processes and potentially contribute to the pathogenesis of ALS.

Subcellular localization and RNAs determine FUS architecture in different cellular compartments.

Mutations in Fused in sarcoma (FUS) gene cause a subset of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron degenerative disease. Wild-type FUS is largely localized in the nucleus, but mutant FUS accumulates in the cytoplasm and forms inclusions. It is unclear whether FUS depletion from the nucleus or FUS inclusions in the cytoplasm triggers motor neuron degeneration. In this study, we revealed that the nuclear and cytoplasmic FUS proteins form distinct local distribution patterns. The nuclear FUS forms oligomers and appears granular under confocal microscope. In contrast, the cytoplasmic FUS forms inclusions with no oligomers detected. These patterns are determined by the subcellular localization of FUS, regardless of wild-type or mutant protein. Moreover, mutant FUS remained or re-directed in the nucleus can oligomerize and behave similarly to the wild-type FUS protein. We further found that nuclear RNAs are critical to its oligomerization. Interestingly, the formation of cytoplasmic FUS inclusions is also dependent on RNA binding. Since the ALS mutations disrupt the nuclear localization sequence, mutant FUS is likely retained in the cytoplasm after translation and interacts with cytoplasmic RNAs. We therefore propose that local RNA molecules interacting with the FUS protein in different subcellular compartments play a fundamental role in determining FUS protein architecture and function.

Self-assembled FUS binds active chromatin and regulates gene transcription.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Fused in sarcoma (FUS) is a DNA/RNA binding protein and mutations in FUS cause a subset of familial ALS. Most ALS mutations are clustered in the C-terminal nuclear localization sequence of FUS and consequently lead to the accumulation of protein inclusions in the cytoplasm. It remains debatable whether loss of FUS normal function in the nucleus or gain of toxic function in the cytoplasm plays a more critical role in the ALS etiology. Moreover, the physiological function of FUS in the nucleus remains to be fully understood. In this study, we found that a significant portion of nuclear FUS was bound to active chromatin and that the ALS mutations dramatically decreased FUS chromatin binding ability. Functionally, the chromatin binding is required for FUS transcription activation, but not for alternative splicing regulation. The N-terminal QGSY (glutamine-glycine-serine-tyrosine)-rich region (amino acids 1-164) mediates FUS self-assembly in the nucleus of mammalian cells and the self-assembly is essential for its chromatin binding and transcription activation. In addition, RNA binding is also required for FUS self-assembly and chromatin binding. Together, our results suggest a functional assembly of FUS in the nucleus under physiological conditions, which is different from the cytoplasmic inclusions. The ALS mutations can cause loss of function in the nucleus by disrupting this assembly and chromatin binding.

ALS mutant SOD1 interacts with G3BP1 and affects stress granule dynamics.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS.

HDAC6 regulates mutant SOD1 aggregation through two SMIR motifs and tubulin acetylation.

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.

Exploratory Mass Spectrometry of Cerebrospinal Fluid from Persons with Autopsy-Confirmed LATE-NC.

Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Participants (n = 29) had been autopsied and had antemortem CSF draws in a longitudinal cohort of older adults at the University of Kentucky AD Research Center. Cases were designated as LATE-NC + if they had LATE-NC stage > 1 (n = 9); the remaining 20 cases were designated LATE-NC-. This convenience sample of CSF specimens was analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry. Relative quantification was performed using Sciex software. Peptides referent to a total of 949 proteins were identified in the samples depleted of abundant proteins, and 820 different proteins were identified in the non-depleted samples. When the Bonferroni/false-discovery statistical correction was applied to account for having made multiple comparison tests, only 4 proteins showed differential expression (LATE-NC + vs LATE-NC-) in the non-depleted samples (RBP4, MIF, IGHG3, and ITM2B). Post hoc western blots confirmed that RBP4 expression was higher in the LATE-NC + cases at the group level. In summary, an exploratory assessment of proteomes of autopsy-confirmed LATE-NC and non-LATE-NC CSF did not demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was, however, an increase in RBP4 protein levels in CSF from LATE-NC cases.

Impaired activity and membrane association of most calpain-5 mutants causal for neovascular inflammatory vitreoretinopathy.

Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on ß-strands in the protease core 2 domain. Conformational changes induced by Ca2+binding result in these ß-strands forming a ß-sheet and a hydrophobic pocket which docks W286 side chain away from the catalytic cleft, enabling calpain activation based on comparison with the Ca2+-bound CAPN1 protease core. The pathologic variants R243L, L244P, K250N, and R289W are predicted to disrupt the ß-strands, ß-sheet, and hydrophobic pocket, impairing calpain activation. The mechanism by which these variants impair membrane association is unclear. G376S impacts a conserved residue in the CBSW domain and is predicted to disrupt a loop containing acidic residues which may contribute to membrane binding. G267S did not impair membrane association and resulted in a slight but significant increase in autoproteolytic and proteolytic activity. However, G267S is also identified in individuals without NIV. Combined with the autosomal dominant pattern of NIV inheritance and evidence that CAPN5 may dimerize, the results are consistent with a dominant negative mechanism for the five pathogenic variants which resulted in impaired CAPN5 activity and membrane association and a gain-of-function for the G267S variant.

Exploratory mass spectrometry of cerebrospinal fluid from persons with autopsy-confirmed LATE-NC.

Background: Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Methods: Participants were recruited from a longitudinal cohort of older adults at the University of Kentucky AD Research Center. A convenience sample of clinically obtained lumbar puncture cerebrospinal fluid (CSF) samples was analyzed from 29 older adults that had autopsy confirmation of the presence or absence of LATE-NC. Nine of the participants had autopsy-confirmed LATE-NC. Antemortem CSF specimens were analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry using an Eksigent Ekspert nanoLC 400 system in line with a Sciex 6600+ mass spectrometer. Protein identification was performed using Protein Pilot (Sciex, ver. 5) software, and relative quantification was performed using the SWATH processing microApp in PeakView and MarkerView software (Sciex), respectively. Following data analyses, additional studies were performed using western blots. Results: A total of 830 proteins were identified in the samples depleted of abundant proteins, and 730 proteins were identified in the non-depleted samples. Whereas some dementia-related proteins were detected (Aß peptide and α-synuclein protein), others were not (TDP-43, TMEM106B, and tau proteins). When the Bonferroni correction was applied to correct for multiple comparisons, only 4 proteins showed differential expression (LATE-NC vs non-LATE-NC) in the nondepleted samples (RBP4, MIF, IGHG3 and ITM2B), whereas none showed statistically different changes in the depleted samples. Post-hoc western blots confirmed that RBP4 expression was higher in the LATE-NC cases at the group level, but there was overlap between the levels of RBP4 in LATE-NC and non-LATE-NC cases. Conclusions: An exploratory assessment of CSF proteomes of autopsy-confirmed LATE-NC and non-LATE-NC cases from a community-based cohort failed to demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was intriguing increase in RBP4 protein levels in CSF from LATE-NC cases. This may provide clues about pathogenetic mechanisms in LATE-NC.

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.

S-acylation regulates the membrane association and activity of Calpain-5.

Calpain-5 (CAPN5) is a member of the calpain family of calcium-activated neutral thiol proteases. CAPN5 is partly membrane associated, despite its lack of a transmembrane domain. Unlike classical calpains, CAPN5 contains a C-terminal C2 domain. C2 domains often have affinity to lipids, mediating membrane association. We recently reported that the C2 domain of CAPN5 was essential for its membrane association and the activation of its autolytic activity. However, despite the removal of the C2 domain by autolysis, the N-terminal fragment of CAPN5 remained membrane associated. S-acylation, also referred to as S-palmitoylation, is a reversible post-translational lipid modification of cysteine residues that promotes membrane association of soluble proteins. In the present study several S-acylated cysteine residues were identified in CAPN5 with the acyl-PEG exchange method. Data reported here demonstrate that CAPN5 is S-acylated on up to three cysteine residues including Cys-4 and Cys-512, and likely Cys-507. The D589N mutation in a potential calcium binding loop within the C2 domain interfered with the S-acylation of CAPN5, likely preventing initial membrane association. Mutating specific cysteine residues of CAPN5 interfered with both its membrane association and the activation of CAPN5 autolysis. Taken together, our results suggest that the S-acylation of CAPN5 is critical for its membrane localization which appears to favor its enzymatic activity.

Mitochondrial dysfunction in amyotrophic lateral sclerosis.

The etiology of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains to be better understood. Based on the studies from ALS patients and transgenic animal models, it is believed that ALS is likely to be a multifactorial and multisystem disease. Many mechanisms have been postulated to be involved in the pathology of ALS, such as oxidative stress, glutamate excitotoxicity, mitochondrial damage, defective axonal transport, glia cell pathology and aberrant RNA metabolism. Mitochondria, which play crucial roles in excitotoxicity, apoptosis and cell survival, have shown to be an early target in ALS pathogenesis and contribute to the disease progression. Morphological and functional defects in mitochondria were found in both human patients and ALS mice overexpressing mutant SOD1. Mutant SOD1 was found to be preferentially associated with mitochondria and subsequently impair mitochondrial function. Recent studies suggest that axonal transport of mitochondria along microtubules and mitochondrial dynamics may also be disrupted in ALS. These results also illustrate the critical importance of maintaining proper mitochondrial function in axons and neuromuscular junctions, supporting the emerging "dying-back" axonopathy model of ALS. In this review, we will discuss how mitochondrial dysfunction has been linked to the ALS variants of SOD1 and the mechanisms by which mitochondrial damage contributes to the disease etiology.

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.

The C2 domain of calpain 5 contributes to enzyme activation and membrane localization.

The enzymatic characteristics of the ubiquitous calpain 5 (CAPN5) remain undescribed despite its high expression in the central nervous system and links to eye development and disease. CAPN5 contains the typical protease core domains but lacks the C terminal penta-EF hand domain of classical calpains, and instead contains a putative C2 domain. This study used the SH-SY5Y neuroblastoma cell line stably transfected with CAPN5-3xFLAG variants to assess the potential roles of the CAPN5 C2 domain in Ca2+ regulated enzyme activity and intracellular localization. Calcium dependent autoproteolysis of CAPN5 was documented and characterized. Mutation of the catalytic Cys81 to Ala or addition of EGTA prevented autolysis. Eighty µM Ca2+ was sufficient to stimulate half-maximal CAPN5 autolysis in cellular lysates. CAPN5 autolysis was inhibited by tri-leucine peptidyl aldehydes, but less effectively by di-Leu aldehydes, consistent with a more open conformation of the protease core relative to classical calpains. In silico modeling revealed a type II topology C2 domain including loops with the potential to bind calcium. Mutation of the acidic amino acid residues predicted to participate in Ca2+ binding, particularly Asp531 and Asp589, resulted in a decrease of CAPN5 membrane association. These residues were also found to be invariant in several genomes. The autolytic fragment of CAPN5 was prevalent in membrane-enriched fractions, but not in cytosolic fractions, suggesting that membrane association facilitates the autoproteolytic activity of CAPN5. Together, these results demonstrate that CAPN5 undergoes Ca2+-activated autoproteolytic processing and suggest that CAPN5 association with membranes enhances CAPN5 autolysis.

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.

The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics.

Stress granules (SGs) are ribonucleoprotein aggregates that form in response to stress conditions. The regulation of SG dynamics is not fully understood. Permanent pathological SG-like structures were reported in neurodegenerative diseases such as amyotrophic lateral sclerosis. The Ras GTPase-activating protein-binding protein G3BP1 is a central regulator of SG dynamics. We found that the lysine 376 residue (K376) of G3BP1, which is in the RRM RNA binding domain, was acetylated. Consequently, G3BP1 RNA binding was impaired by K376 acetylation. In addition, the acetylation-mimicking mutation K376Q impaired the RNA-dependent interaction of G3BP1 with poly(A)-binding protein 1 (PABP1), but its RNA-independent interactions with caprin-1 and USP10 were little affected. The formation of G3BP1 SGs depended on G3BP1 RNA binding; thus, replacement of endogenous G3BP1 with the K376Q mutant or the RNA binding-deficient F380L/F382L mutant interfered with SG formation. Significant G3BP1 K376 acetylation was detected during SG resolution, and K376-acetylated G3BP1 was seen outside SGs. G3BP1 acetylation is regulated by histone deacetylase 6 (HDAC6) and CBP/p300. Our data suggest that the acetylation of G3BP1 facilitates the disassembly of SGs, offering a potential avenue to mitigate hyperactive stress responses under pathological conditions.

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.

Polarized light and oviposition site selection in the yellow fever mosquito: no evidence for positive polarotaxis in Aedes aegypti.

Aquatic insects and insects associated with water use horizontally polarized light (i.e., positive polarotaxis) to detect potential aquatic or moist oviposition sites. Mosquitoes lay their eggs onto wet substrata, in water, water-filled tree/rock holes, or man-made small containers/bottles/old tyres containing water. Until now it has remained unknown whether mosquitoes are polarotactic or not. The knowledge how mosquitoes locate water would be important to develop new control measures against them. Thus, we studied in dual-choice laboratory experiments the role of horizontally polarized light in the selection of oviposition sites in blood-fed, gravid females of the yellow fever mosquito, Aedes aegypti. On the basis of our results we propose that Ae. aegypti is not polarotactic. Thus the yellow fever mosquito is the first known water-associated insect species that does not detect water by means of the horizontally polarized water-reflected light. This can be explained by the reflection-polarization characteristics of small-volume water-filled cavities/containers preferred by Ae. aegypti as oviposition sites.