Lipopolysaccharide triggers exacerbated microglial activation, excessive cytokine release and behavioural disturbances in mice with truncated Fused-in-Sarcoma Protein (FUS).

CNS inflammation, including microglial activation, in response to peripheral infections are known to contribute to the pathology of both familial and sporadic neurodegenerative disease. The relationship between Fused-in-Sarcoma Protein (FUS)-mediated disease in the transgenic FUS[1-359] animals and the systemic inflammatory response have not been explored. Here, we investigated microglial activation, inflammatory gene expression and the behavioural responses to lipopolysaccharide-induced (LPS; 0.1 mg/kg) systemic inflammation in the FUS[1-359] transgenic mice. The pathology of these mice recapitulates the key features of mutant FUS-associated familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Here, pre-symptomatic 8-week-old mutant or wild type controls were challenged with LPS or with saline and sucrose intake, novel cage exploration, marble burying and swimming behaviours were analyzed. The level of pro-inflammatory gene expression was also determined, and microglial activation was evaluated. In chronic experiments, to discover whether the LPS challenge would affect the onset of ALS-like paralysis, animals were evaluated for clinical signs from 5 to 7 weeks post-injection. Compared to controls, acutely challenged FUS[1-359]-tg mice exhibited decreased sucrose intake and increased floating behaviours. The FUS[1-359]-tg mice exhibited an increase in immunoreactivity for Iba1-positive cells in the prefrontal cortex and ventral horn of the spinal cord, which was accompanied by increased expression of interleukin-1ß, tumour necrosis factor, cyclooxygenase-(COX)-1 and COX-2. However, the single LPS challenge did not alter the time to development of paralysis in the FUS[1-359]-tg mice. Thus, while the acute inflammatory response was enhanced in the FUS mutant animals, it did not have a lasting impact on disease progression.

Loss of PML nuclear bodies in familial amyotrophic lateral sclerosis-frontotemporal dementia.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders that share genetic causes and pathogenic mechanisms. The critical genetic players of ALS and FTD are the TARDBP, FUS and C9orf72 genes, whose protein products, TDP-43, FUS and the C9orf72-dipeptide repeat proteins, accumulate in form of cytoplasmic inclusions. The majority of the studies focus on the understanding of how cells control TDP-43 and FUS aggregation in the cytoplasm, overlooking how dysfunctions occurring at the nuclear level may influence the maintenance of protein solubility outside of the nucleus. However, protein quality control (PQC) systems that maintain protein homeostasis comprise a cytoplasmic and a nuclear arm that are interconnected and share key players. It is thus conceivable that impairment of the nuclear arm of the PQC may have a negative impact on the cytoplasmic arm of the PQC, contributing to the formation of the cytoplasmic pathological inclusions. Here we focused on two stress-inducible condensates that act as transient deposition sites for misfolding-prone proteins: Promyelocytic leukemia protein (PML) nuclear bodies (PML-NBs) and cytoplasmic stress granules (SGs). Upon stress, PML-NBs compartmentalize misfolded proteins, including defective ribosomal products (DRiPs), and recruit chaperones and proteasomes to promote their nuclear clearance. SGs transiently sequester aggregation-prone RNA-binding proteins linked to ALS-FTD and mRNAs to attenuate their translation. We report that PML assembly is impaired in the human brain and spinal cord of familial C9orf72 and FUS ALS-FTD cases. We also show that defective PML-NB assembly impairs the compartmentalization of DRiPs in the nucleus, leading to their accumulation inside cytoplasmic SGs, negatively influencing SG dynamics. Although it is currently unclear what causes the decrease of PML-NBs in ALS-FTD, our data highlight the existence of a cross-talk between the cytoplasmic and nuclear PQC systems, whose alteration can contribute to SG accumulation and cytoplasmic protein aggregation in ALS-FTD.

Early Alterations of RNA Binding Protein (RBP) Homeostasis and ER Stress-Mediated Autophagy Contributes to Progressive Retinal Degeneration in the rd10 Mouse Model of Retinitis Pigmentosa (RP).

The retinal degeneration 10 (rd10) mouse model is widely used to study retinitis pigmentosa (RP) pathomechanisms. It offers a rather unique opportunity to study trans-neuronal degeneration because the cell populations in question are separated anatomically and the mutated Pde6b gene is selectively expressed in rod photoreceptors. We hypothesized that RNA binding protein (RBP) aggregation and abnormal autophagy might serve as early pathogenic events, damaging non-photoreceptor retinal cell types that are not primarily targeted by the Pde6b gene defect. We used a combination of immunohistochemistry (DAB, immunofluorescence), electron microscopy (EM), subcellular fractionation, and Western blot analysis on the retinal preparations obtained from both rd10 and wild-type mice. We found early, robust increases in levels of the protective endoplasmic reticulum (ER) calcium (Ca2+) buffering chaperone Sigma receptor 1 (SigR1) together with other ER-Ca2+ buffering proteins in both photoreceptors and non-photoreceptor neuronal cells before any noticeable photoreceptor degeneration. In line with this, we found markedly altered expression of the autophagy proteins p62 and LC3, together with abnormal ER widening and large autophagic vacuoles as detected by EM. Interestingly, these changes were accompanied by early, prominent cytoplasmic and nuclear aggregation of the key RBPs including pTDP-43 and FET family RBPs and stress granule formation. We conclude that progressive neurodegeneration in the rd10 mouse retina is associated with early disturbances of proteostasis and autophagy, along with abnormal cytoplasmic RBP aggregation.

Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS.

Nuclear to cytoplasmic mislocalization and aggregation of multiple RNA-binding proteins (RBPs), including FUS, are the main neuropathological features of the majority of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobular degeneration (FTLD). In ALS-FUS, these aggregates arise from disease-associated mutations in FUS, whereas in FTLD-FUS, the cytoplasmic inclusions do not contain mutant FUS, suggesting different molecular mechanisms of FUS pathogenesis in FTLD that remain to be investigated. We have previously shown that phosphorylation of the C-terminal Tyr526 of FUS results in increased cytoplasmic retention of FUS due to impaired binding to the nuclear import receptor TNPO1. Inspired by the above notions, in the current study we developed a novel antibody against the C-terminally phosphorylated Tyr526 FUS (FUSp-Y526) that is specifically capable of recognizing phosphorylated cytoplasmic FUS, which is poorly recognized by other commercially available FUS antibodies. Using this FUSp-Y526 antibody, we demonstrated a FUS phosphorylation-specific effect on the cytoplasmic distribution of soluble and insoluble FUSp-Y526 in various cells and confirmed the involvement of the Src kinase family in Tyr526 FUS phosphorylation. In addition, we found that FUSp-Y526 expression pattern correlates with active pSrc/pAbl kinases in specific brain regions of mice, indicating preferential involvement of cAbl in the cytoplasmic mislocalization of FUSp-Y526 in cortical neurons. Finally, the pattern of immunoreactivity of active cAbl kinase and FUSp-Y526 revealed altered cytoplasmic distribution of FUSp-Y526 in cortical neurons of post-mortem frontal cortex tissue from FTLD patients compared with controls. The overlap of FUSp-Y526 and FUS signals was found preferentially in small diffuse inclusions and was absent in mature aggregates, suggesting possible involvement of FUSp-Y526 in the formation of early toxic FUS aggregates in the cytoplasm that are largely undetected by commercially available FUS antibodies. Given the overlapping patterns of cAbl activity and FUSp-Y526 distribution in cortical neurons, and cAbl induced sequestration of FUSp-Y526 into G3BP1 positive granules in stressed cells, we propose that cAbl kinase is actively involved in mediating cytoplasmic mislocalization and promoting toxic aggregation of wild-type FUS in the brains of FTLD patients, as a novel putative underlying mechanism of FTLD-FUS pathophysiology and progression.

PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD.

ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset.

Interactome screening of C9orf72 dipeptide repeats reveals VCP sequestration and functional impairment by polyGA.

Repeat expansions in the C9orf72 gene are a common cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, two devastating neurodegenerative disorders. One of the proposed mechanisms of GGGGCC repeat expansion is their translation into non-canonical dipeptide repeats, which can then accumulate as aggregates and contribute to these pathologies. There are five different dipeptide repeat proteins (polyGA, polyGR, polyPR, polyPA and polyGP), some of which are known to be neurotoxic. In the present study, we used BioID2 proximity labelling to identify the interactomes of all five dipeptide repeat proteins consisting of 125 repeats each. We identified 113 interacting partners for polyGR, 90 for polyGA, 106 for polyPR, 25 for polyPA and 27 for polyGP. Gene Ontology enrichment analysis of the proteomic data revealed that these target interaction partners are involved in a variety of functions, including protein translation, signal transduction pathways, protein catabolic processes, amide metabolic processes and RNA-binding. Using autopsy brain tissue from patients with C9orf72 expansion complemented with cell culture analysis, we evaluated the interactions between polyGA and valosin containing protein (VCP). Functional analysis of this interaction revealed sequestration of VCP with polyGA aggregates, altering levels of soluble valosin-containing protein. VCP also functions in autophagy processes, and consistent with this, we observed altered autophagy in cells expressing polyGA. We also observed altered co-localization of polyGA aggregates and p62 in cells depleted of VCP. All together, these data suggest that sequestration of VCP with polyGA aggregates contributes to the loss of VCP function, and consequently to alterations in autophagy processes in C9orf72 expansion disorders.

Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation.

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.

A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.

Hsp90-mediated regulation of DYRK3 couples stress granule disassembly and growth via mTORC1 signaling.

Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual-specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.

Aggregates of RNA Binding Proteins and ER Chaperones Linked to Exosomes in Granulovacuolar Degeneration of the Alzheimer's Disease Brain.

Granulovacuolar degeneration (GVD) occurs in Alzheimer's disease (AD) brain due to compromised autophagy. Endoplasmic reticulum (ER) function and RNA binding protein (RBP) homeostasis regulate autophagy. We observed that the ER chaperones Glucose - regulated protein, 78 KDa (GRP78/BiP), Sigma receptor 1 (SigR1), and Vesicle-associated membrane protein associated protein B (VAPB) were elevated in many AD patients' subicular neurons. However, those neurons which were affected by GVD showed lower chaperone levels, and there was only minor co-localization of chaperones with GVD bodies (GVBs), suggesting that neurons lacking sufficient chaperone-mediated proteostasis enter the GVD pathway. Consistent with this notion, granular, incipient pTau aggregates in human AD and pR5 tau transgenic mouse neurons were regularly co-localized with increased chaperone immunoreactivity, whereas neurons with mature neurofibrillary tangles lacked both the chaperone buildup and significant GVD. On the other hand, APP/PS1 (APPswe/PSEN1dE9) transgenic mouse hippocampal neurons that are devoid of pTau accumulation displayed only few GVBs-like vesicles, which were still accompanied by prominent chaperone buildup. Identifying a potential trigger for GVD, we found cytoplasmic accumulations of RBPs including Matrin 3 and FUS as well as stress granules in GVBs of AD patient and pR5 mouse neurons. Interestingly, we observed that GVBs containing aggregated pTau and pTDP-43 were consistently co-localized with the exosomal marker Flotillin 1 in both AD and pR5 mice. In contrast, intraneuronal 82E1-immunoreactive amyloid-ß in human AD and APP/PS1 mice only rarely co-localized with Flotillin 1-positive exosomal vesicles. We conclude that altered chaperone-mediated ER protein homeostasis and impaired autophagy manifesting in GVD are linked to both pTau and RBP accumulation and that some GVBs might be targeted to exocytosis.

Phenotypes and malignancy risk of different FUS mutations in genetic amyotrophic lateral sclerosis.

OBJECTIVE: Mutations in Fused in Sarcoma (FUS or TLS) are the fourth most prevalent in Western European familial amyotrophic lateral sclerosis (ALS) populations and have been associated with causing both early and very late disease onset. FUS aggregation, DNA repair deficiency, and genomic instability are contributors to the pathophysiology of FUS-ALS, but their clinical significance per se and their influence on the clinical variability have yet to be sufficiently investigated. The aim of this study was to analyze genotype-phenotype correlations and malignancy rates in a newly compiled FUS-ALS cohort. METHODS: We cross-sectionally reviewed FUS-ALS patient histories in a multicenter cohort with 36 novel cases and did a meta-analysis of published FUS-ALS cases reporting the largest genotype-phenotype correlation of FUS-ALS. RESULTS: The age of onset (median 39 years, range 11-80) was positively correlated with the disease duration. C-terminal domain mutations were found in 90%. Among all, P525L and truncating/ frameshift mutations most frequently caused juvenile onset, rapid disease progression, and atypical ALS often associated with negative family history while the R521 mutation site was associated with late disease onset and pure spinal phenotype. Malignancies were found in one of 40 patients. INTERPRETATION: We report the largest genotype-phenotype correlation of FUS-ALS, which enables a careful prediction of the clinical course in newly diagnosed patients. In this cohort, FUS-ALS patients did not have an increased risk for malignant diseases.

FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

Monitoring α-synuclein multimerization in vivo.

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α-synuclein (α-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α-Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α-Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α-Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α-Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α-Syn at serine 129 enhances the accumulation and toxicity of α-Syn. Altogether, our study establishes a novel model to study α-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.-Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α-synuclein multimerization in vivo.

Cuprizone-induced graded oligodendrocyte vulnerability is regulated by the transcription factor DNA damage-inducible transcript 3.

Oligodendrocytes are integral to efficient neuronal signaling. Loss of myelinating oligodendrocytes is a central feature of many neurological diseases, including multiple sclerosis (MS). The results of neuropathological studies suggest that oligodendrocytes react with differing sensitivity to toxic insults, with some cells dying early during lesion development and some cells being resistant for weeks. This proposed graded vulnerability has never been demonstrated but provides an attractive window for therapeutic interventions. Furthermore, the biochemical pathways associated with graded oligodendrocyte vulnerability have not been well explored. We used immunohistochemistry and serial block-face scanning electron microscopy (3D-SEM) to show that cuprizone-induced metabolic stress results in an "out of phase" degeneration of oligodendrocytes. Although expression induction of stress response transcription factors in oligodendrocytes occurs within days, subsequent oligodendrocyte apoptosis continues for weeks. In line with the idea of an out of phase degeneration of oligodendrocytes, detailed ultrastructural reconstructions of the axon-myelin unit demonstrate demyelination of single internodes. In parallel, genome wide array analyses revealed an active unfolded protein response early after initiation of the cuprizone intoxication. In addition to the cytoprotective pathways, the pro-apoptotic transcription factor DNA damage-inducible transcript 3 (DDIT3) was induced early in oligodendrocytes. In advanced lesions, DDIT3 was as well expressed by activated astrocytes. Toxin-induced oligodendrocyte apoptosis, demyelination, microgliosis, astrocytosis, and acute axonal damage were less intense in the Ddit3-null mutants. This study identifies DDIT3 as an important regulator of graded oligodendrocyte vulnerability in a MS animal model. Interference with this stress cascade might offer a promising therapeutic approach for demyelinating disorders.

Golgin A4 in CSF and granulovacuolar degenerations of patients with Alzheimer disease.

OBJECTIVE: To isolate and identify a new, as yet unknown molecule in CSF that could serve as marker for Alzheimer disease. METHODS: We immunized mice with human CSF and fused hybridomas for monoclonal antibodies and screened these antibodies for their capacity to discriminate CSF of patients with Alzheimer disease from CSF of controls. We then chromatographically isolated the antigen to the best discriminating antibody and identified the antigen using mass spectrometric methods. Thereafter, we quantified the CSF concentration of the antigen in a new cohort of patients with Alzheimer disease and controls and performed immunohistochemistry of postmortem brain tissue derived from patients with Alzheimer disease and controls. RESULTS: We generated >200 hybridomas and selected 1 antibody that discriminated CSF from patients with Alzheimer disease from that of controls. We identified golgin A4 as the antigen detected by this antibody. Golgin A4 concentration was significantly higher in CSF from patients with Alzheimer disease than in CSF of controls (145 [interquartile range 125-155] vs 115 [ 99-128] pg/mL, p < 0.001) and demonstrated a substantial discriminative power (area under the receiver operating characteristic curve 0.80, 95% confidence interval 0.67-0.94). Immunohistochemistry of postmortem brain sections from patients with Alzheimer disease revealed a significant accumulation of golgin A4 in granulovacuolar degeneration bodies (GVBs). CONCLUSIONS: These results support the notion that golgin A4 could serve as a diagnostic marker in Alzheimer disease. For validation of this notion, prospective multicenter diagnostic studies will evaluate golgin A4 as diagnostic marker for Alzheimer disease. Furthermore, it has to be determined whether the association of golgin A4 with GVBs is an epiphenomenon or whether golgin A4 plays a more direct role in Alzheimer disease, allowing it to serve as a target in therapeutic treatment strategies. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that elevated CSF golgin A4 levels identify patients with Alzheimer disease.

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation.

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.

The ALS-linked E102Q mutation in Sigma receptor-1 leads to ER stress-mediated defects in protein homeostasis and dysregulation of RNA-binding proteins.

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of motor neurons (MNs) and their target muscles. Misfolded proteins which often form intracellular aggregates are a pathological hallmark of ALS. Disruption of the functional interplay between protein degradation (ubiquitin proteasome system and autophagy) and RNA-binding protein homeostasis has recently been suggested as an integrated model that merges several ALS-associated proteins into a common pathophysiological pathway. The E102Q mutation in one such candidate gene, the endoplasmic reticulum (ER) chaperone Sigma receptor-1 (SigR1), has been reported to cause juvenile ALS. Although loss of SigR1 protein contributes to neurodegeneration in several ways, the molecular mechanisms underlying E102Q-SigR1-mediated neurodegeneration are still unclear. In the present study, we showed that the E102Q-SigR1 protein rapidly aggregates and accumulates in the ER and associated compartments in transfected cells, leading to structural alterations of the ER, nuclear envelope and mitochondria and to subsequent defects in proteasomal degradation and calcium homeostasis. ER defects and proteotoxic stress generated by E102Q-SigR1 aggregates further induce autophagy impairment, accumulation of stress granules and cytoplasmic aggregation of the ALS-linked RNA-binding proteins (RBPs) matrin-3, FUS, and TDP-43. Similar ultrastructural abnormalities as well as altered protein degradation and misregulated RBP homeostasis were observed in primary lymphoblastoid cells (PLCs) derived from E102Q-SigR1 fALS patients. Consistent with these findings, lumbar α-MNs of both sALS as well as fALS patients showed cytoplasmic matrin-3 aggregates which were not co-localized with pTDP-43 aggregates. Taken together, our results support the notion that E102Q-SigR1-mediated ALS pathogenesis comprises a synergistic mechanism of both toxic gain and loss of function involving a vicious circle of altered ER function, impaired protein homeostasis and defective RBPs.

Autosomal dominant spinal muscular atrophy with lower extremity predominance: A recognizable phenotype of BICD2 mutations.

INTRODUCTION: Heterozygous BICD2 gene mutations cause a form of autosomal dominant spinal muscular atrophy with lower extremity predominance (SMALED). METHODS: We analyzed the BICD2 gene in a selected group of 25 index patients with neurogenic muscle atrophy. RESULTS: We identified 2 new BICD2 missense mutations, c.2515G>A, p.Gly839Arg, in a family with autosomal dominant inheritance, and c.2202G>T, p.Lys734Asn, as a de novo mutation in an isolated patient with similar phenotype. The patients had congenital foot contractures, muscle atrophy of the legs, and slowly progressive weakness of the shoulder girdle. There was no apparent sensory or brain dysfunction. One patient died of unrelated reasons at age 52 years. Autopsy revealed no upper motor neuron and only moderate lower motor neuron loss, but there was distal corticospinal tract degeneration and marked neurogenic muscular atrophy. CONCLUSION: These findings give further insight into the clinical and pathoanatomical consequences of BICD2 mutations. Muscle Nerve 54: 496-500, 2016.

Aberrant association of misfolded SOD1 with Na(+)/K(+)ATPase-α3 impairs its activity and contributes to motor neuron vulnerability in ALS.

Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron excitability and spinal circuits are pathological hallmarks of ALS, but the underlying molecular mechanisms remain unresolved. Here, we sought to understand whether the expression of mutant SOD1 protein could contribute to altering processes governing motor neuron excitability. We used the conformation specific antibody B8H10 which recognizes a misfolded state of SOD1 (misfSOD1) to longitudinally identify its interactome during early disease stage in SOD1G93A mice. This strategy identified a direct isozyme-specific association of misfSOD1 with Na(+)/K(+)ATPase-α3 leading to the premature impairment of its ATPase activity. Pharmacological inhibition of Na(+)/K(+)ATPase-α3 altered glutamate receptor 2 expression, modified cholinergic inputs and accelerated disease pathology. After mapping the site of direct association of misfSOD1 with Na(+)/K(+)ATPase-α3 onto a 10 amino acid stretch that is unique to Na(+)/K(+)ATPase-α3 but not found in the closely related Na(+)/K(+)ATPase-α1 isozyme, we generated a misfSOD1 binding deficient, but fully functional Na(+)/K(+)ATPase-α3 pump. Adeno associated virus (AAV)-mediated expression of this chimeric Na(+)/K(+)ATPase-α3 restored Na(+)/K(+)ATPase-α3 activity in the spinal cord, delayed pathological alterations and prolonged survival of SOD1G93A mice. Additionally, altered Na(+)/K(+)ATPase-α3 expression was observed in the spinal cord of individuals with sporadic and familial ALS. A fraction of sporadic ALS cases also presented B8H10 positive misfSOD1 immunoreactivity, suggesting that similar mechanism might contribute to the pathology.