A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation.

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

Mechanical Loading Modulates AMPK and mTOR Signaling in Muscle Cells.

Skeletal muscle adaptation to exercise involves various phenotypic changes that enhance the metabolic and contractile functions. One key regulator of these adaptive responses is the activation of AMPK, which is influenced by exercise intensity. However, the mechanistic understanding of AMPK activation during exercise remains incomplete. In this study, we utilized an in vitro model to investigate the effects of mechanical loading on AMPK activation and its interaction with the mTOR signaling pathway. Proteomic analysis of muscle cells subjected to static loading (SL) revealed distinct quantitative protein alterations associated with RNA metabolism, with 10% SL inducing the most pronounced response compared to lower intensities of 5% and 2% as well as the control. Additionally, 10% SL suppressed RNA and protein synthesis while activating AMPK and inhibiting the mTOR pathway. We also found that SRSF2, necessary for pre-mRNA splicing, is regulated by AMPK and mTOR signaling, which, in turn, is regulated in an intensity-dependent manner by SL with the highest expression in 2% SL. Further examination showed that the ADP/ATP ratio was increased after 10% SL compared to the control and that SL induced changes in mitochondrial biogenesis. Furthermore, Seahorse assay results indicate that 10% SL enhances mitochondrial respiration. These findings provide novel insights into the cellular responses to mechanical loading and shed light on the intricate AMPK-mTOR regulatory network in muscle cells.

Genomic evidence for the suitability of Göttingen Minipigs with a rare seizure phenotype as a model for human epilepsy.

Epilepsy is a complex genetic disorder that affects about 2% of the global population. Although the frequency and severity of epileptic seizures can be reduced by a range of pharmacological interventions, there are no disease-modifying treatments for epilepsy. The development of new and more effective drugs is hindered by a lack of suitable animal models. Available rodent models may not recapitulate all key aspects of the disease. Spontaneous epileptic convulsions were observed in few Göttingen Minipigs (GMPs), which may provide a valuable alternative animal model for the characterisation of epilepsy-type diseases and for testing new treatments. We have characterised affected GMPs at the genome level and have taken advantage of primary fibroblast cultures to validate the functional impact of fixed genetic variants on the transcriptome level. We found numerous genes connected to calcium metabolism that have not been associated with epilepsy before, such as ADORA2B, CAMK1D, ITPKB, MCOLN2, MYLK, NFATC3, PDGFD, and PHKB. Our results have identified two transcription factor genes, EGR3 and HOXB6, as potential key regulators of CACNA1H, which was previously linked to epilepsy-type disorders in humans. Our findings provide the first set of conclusive results to support the use of affected subsets of GMPs as an alternative and more reliable model system to study human epilepsy. Further neurological and pharmacological validation of the suitability of GMPs as an epilepsy model is therefore warranted.

Resistance to chicken amyloid arthropathy is associated with a dysfunctional mutation in serum amyloid A.

Chicken amyloid arthropathy is a debilitating disease with a major impact on animal welfare. Since the disease is triggered by bacterial infection, preventative treatment also contributes to the widespread overuse of antibiotics. Bacterial infection initiates an acute phase response including increased serum amyloid A (SAA) production by the liver. SAA accumulates at sites of infection and in particular in large joints of affected birds. Interestingly, white egg-laying chickens (WL) are resistant to the disease whilst brown egg-laying chickens (BL) are most affected. Disease susceptibility has an immunological basis but the possible contribution of underlying genetic risk factors is not understood. Using a whole genome sequencing approach, we discovered a novel variant in the SAA gene in WL, which is predicted to result in an arginine to serine substitution at position 90 (SAA.R90S). Surprisingly, when overexpressed in chicken hepatocellular carcinoma cells, SAA.R90S was expressed at a higher rate and secreted to a greater degree than the wild-type SAA protein. Moreover, RNASeq analysis showed that the R90S mutant exerted a differential effect on the expression of core transcription factors linked to cell fate determination and cell differentiation. Comparative analysis of gene expression in murine CD4 T-cells stimulated with IL-6/SAA, suggests that SAA.R90S might block an induced cell fate change toward pro-inflammatory T helper 17 cells, which are required for immunological protection against pathogenic bacteria during an acute phase response. Our results provide first mechanistic insights into the genetic resistance of WL to amyloid arthropathy and could be applied to commercial layer breeding programs to improve animal welfare and reduce the negative effects of the overuse of antibiotics.

SOD1-Related Cerebellar Ataxia and Motor Neuron Disease: Cp Variant as Functional Modifier?

We describe a novel superoxide dismutase (SOD1) mutation-associated clinical phenotype of cerebellar ataxia and motor neuron disease with a variant in the ceruloplasmin (Cp) gene, which may have possibly contributed to a multi-factorial phenotype, supported by genetic and protein structure analyses.

ApoE Isoforms Inhibit Amyloid Aggregation of Proinflammatory Protein S100A9.

Increasing evidence suggests that the calcium-binding and proinflammatory protein S100A9 is an important player in neuroinflammation-mediated Alzheimer's disease (AD). The amyloid co-aggregation of S100A9 with amyloid-ß (Aß) is an important hallmark of this pathology. Apolipoprotein E (ApoE) is also known to be one of the important genetic risk factors of AD. ApoE primarily exists in three isoforms, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). Even though the difference lies in just two amino acid residues, ApoE isoforms produce differential effects on the neuroinflammation and activation of the microglial state in AD. Here, we aim to understand the effect of the ApoE isoforms on the amyloid aggregation of S100A9. We found that both ApoE3 and ApoE4 suppress the aggregation of S100A9 in a concentration-dependent manner, even at sub-stoichiometric ratios compared to S100A9. These interactions lead to a reduction in the quantity and length of S100A9 fibrils. The inhibitory effect is more pronounced if ApoE isoforms are added in the lipid-free state versus lipidated ApoE. We found that, upon prolonged incubation, S100A9 and ApoE form low molecular weight complexes with stochiometric ratios of 1:1 and 2:1, which remain stable under SDS-gel conditions. These complexes self-assemble also under the native conditions; however, their interactions are transient, as revealed by glutaraldehyde cross-linking experiments and molecular dynamics (MD) simulation. MD simulation demonstrated that the lipid-binding C-terminal domain of ApoE and the second EF-hand calcium-binding motif of S100A9 are involved in these interactions. We found that amyloids of S100A9 are cytotoxic to neuroblastoma cells, and the presence of either ApoE isoforms does not change the level of their cytotoxicity. A significant inhibitory effect produced by both ApoE isoforms on S100A9 amyloid aggregation can modulate the amyloid-neuroinflammatory cascade in AD.

Reports of Batch-Dependent Suspected Adverse Events of the BNT162b2 mRNA COVID-19 Vaccine: Comparison of Results from Denmark and Sweden.

Background and Objective: An unexpected batch-dependent safety signal for the BNT162b2 mRNA COVID-19 vaccine was recently identified in a nationwide study from Denmark, but the generalizability of this finding is unknown. Therefore, we compared batch-dependent rates of suspected adverse events (SAEs) reported to national authorities in Denmark and Sweden. Materials and Methods: SAE and vaccine batch data were received from national authorities in Denmark and Sweden, and analyses of heterogeneity in the relationship between numbers of vaccine doses and SAEs per batch were performed, along with comparison of SAE rates and severities for batches that were shared between the two countries. Results: Significant batch-dependent heterogeneity was found in the number of SAEs per 1000 doses for both countries, with batches associated with high SAE rates detected in the early phase of the vaccination campaign and positive correlations observed between the two countries for the severity of SAEs from vaccine batches that they shared. Mild SAEs predominated in the batches used in the early part of the vaccination roll-out, where markedly higher SAE rates per 1000 doses in Denmark for the batches that were shared between the two countries suggested that a large proportion of these SAEs were under-reported in Sweden. Conclusions: The batch-dependent safety signal observed in Denmark and now confirmed in Sweden suggests that early commercial batches of BNT162b2 may have differed from those used later on, and these preliminary and hypothesis-generating results warrant further study.

Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming.

BACKGROUND: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.

Mitochondrial DNA in the tumour microenvironment activates neutrophils and is associated with worse outcomes in patients with advanced epithelial ovarian cancer.

BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.

The molecular pathogenesis of superoxide dismutase 1-linked ALS is promoted by low oxygen tension.

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS). Disease pathogenesis is linked to destabilization, disorder and aggregation of the SOD1 protein. However, the non-genetic factors that promote disorder and the subsequent aggregation of SOD1 have not been studied. Mainly located to the reducing cytosol, mature SOD1 contains an oxidized disulfide bond that is important for its stability. Since O2 is required for formation of the bond, we reasoned that low O2 tension might be a risk factor for the pathological changes associated with ALS development. By combining biochemical approaches in an extensive range of genetically distinct patient-derived cell lines, we show that the disulfide bond is an Achilles heel of the SOD1 protein. Culture of patient-derived fibroblasts, astrocytes, and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under low O2 tensions caused reductive bond cleavage and increases in disordered SOD1. The effects were greatest in cells derived from patients carrying ALS-linked mutations in SOD1. However, significant increases also occurred in wild-type SOD1 in cultures derived from non-disease controls, and patients carrying mutations in other common ALS-linked genes. Compared to fibroblasts, MNACs showed far greater increases in SOD1 disorder and even aggregation of mutant SOD1s, in line with the vulnerability of the motor system to SOD1-mediated neurotoxicity. Our results show for the first time that O2 tension is a principal determinant of SOD1 stability in human patient-derived cells. Furthermore, we provide a mechanism by which non-genetic risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could promote disease initiation and progression.

Cell-type- and region-specific restriction of neurotropic flavivirus infection by viperin.

BACKGROUND: Flaviviruses are a group of diverse and emerging arboviruses and an immense global health problem. A number of flaviviruses are neurotropic, causing severe encephalitis and even death. Type I interferons (IFNs) are the first line of defense of the innate immune system against flavivirus infection. IFNs elicit the concerted action of numerous interferon-stimulated genes (ISGs) to restrict both virus infection and replication. Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an ISG with broad-spectrum antiviral activity against multiple flaviviruses in vitro. Its activity in vivo restricts neurotropic infections to specific regions of the central nervous system (CNS). However, the cell types in which viperin activity is required are unknown. Here we have examined both the regional and cell-type specificity of viperin in the defense against infection by several model neurotropic flaviviruses. METHODS: Viral burden and IFN induction were analyzed in vivo in wild-type and viperin-/- mice infected with Langat virus (LGTV). The effects of IFN pretreatment were tested in vitro in primary neural cultures from different brain regions in response to infection with tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Zika virus (ZIKV). RESULTS: Viperin activity restricted nonlethal LGTV infection in the spleen and the olfactory bulb following infection via a peripheral route. Viperin activity was also necessary to restrict LGTV replication in the olfactory bulb and the cerebrum following CNS infection, but not in the cerebellum. In vitro, viperin could restrict TBEV replication in primary cortical neurons, but not in the cerebellar granule cell neurons. Interferon-induced viperin was also very important in primary cortical neurons to control TBEV, WNV, and ZIKV. CONCLUSIONS: Our findings show that viperin restricts replication of neurotropic flaviviruses in the CNS in a region- and cell-type-specific manner. The most important sites of activity are the olfactory bulb and cerebrum. Activity within the cerebrum is required in the cortical neurons in order to restrict spread. This study exemplifies cell type and regional diversity of the IFN response within the CNS and shows the importance of a potent broad-spectrum antiviral ISG.

Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects.

BACKGROUND: Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. METHODS: Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR-/-) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. RESULTS: Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. CONCLUSIONS: These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.

The roof plate boundary is a bi-directional organiser of dorsal neural tube and choroid plexus development.

The roof plate is a signalling centre positioned at the dorsal midline of the central nervous system and generates dorsalising morphogenic signals along the length of the neuraxis. Within cranial ventricles, the roof plate gives rise to choroid plexus, which regulates the internal environment of the developing and adult brain and spinal cord via the secretion of cerebrospinal fluid. Using the fourth ventricle as our model, we show that the organiser properties of the roof plate are determined by its boundaries with the adjacent neuroepithelium. Through a combination of in ovo transplantation, co-culture and electroporation techniques in chick embryos between embryonic days 3 and 6, we demonstrate that organiser properties are maintained by interactions between the non-neural roof plate and the neural rhombic lip. At the molecular level, this interaction is mediated by Delta-Notch signalling and upregulation of the chick homologue of Hes1: chairy2. Gain- and loss-of-function approaches reveal that cdelta1 is both necessary and sufficient for organiser function. Our results also demonstrate that while chairy2 is specifically required for the maintenance of the organiser, its ectopic expression is not sufficient to recapitulate organiser properties. Expression of atonal1 in the rhombic lip adjacent at the roof plate boundary is acutely dependent on both boundary cell interactions and Delta-Notch signalling. Correspondingly, the roof plate boundary organiser also signals to the roof plate itself to specify the expression of early choroid plexus markers. Thus, the roof plate boundary organiser signals bi-directionally to acutely coordinate the development of adjacent neural and non-neural tissues.

Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

Herpes simplex infection and the risk of Alzheimer's disease: A nested case-control study.

BACKGROUND: Herpes simplex virus (HSV) is thought to play an etiological role in the development of Alzheimer's disease (AD). METHODS: Plasma samples from 360 AD cases (75.3% women, mean age 61.2 years) and 360 age- and sex-matched dementia-free controls, taken on average 9.6 years before AD diagnosis, were analyzed for anti-HSV antibodies (immunoglobulin G, IgG, and immunoglobulin M, IgM) by enzyme-linked immunosorbent assays. RESULTS: In the complete sample group, the presence of anti-HSV IgG and IgM antibodies did not increase the risk of AD significantly (odds ratio (OR) 1.636, P = .069 and OR 1.368, P = .299, respectively). In cases with 6.6 years or more between plasma sampling and AD diagnosis (n = 270), there was a significant association between presence of anti-HSV IgG antibodies and AD (OR 2.250, P = .019). CONCLUSION: Among persons with a follow-up time of 6.6 years or more, HSV infection was significantly associated with AD.

Reactivated herpes simplex infection increases the risk of Alzheimer's disease.

BACKGROUND: Previous studies have suggested a link between herpes simplex virus (HSV) type 1 and the development of Alzheimer's disease (AD). METHODS: The present analysis included 3432 persons (53.9% women, mean age at inclusion 62.7 ± 14.4 years) with a mean follow-up time of 11.3 years. The number of incident AD cases was 245. Serum samples were analyzed for anti-HSV antibodies (immunoglobulin (Ig)G and IgM) by enzyme-linked immunosorbent assays. RESULTS: The presence of anti-HSV IgG antibodies was not associated with an increased risk for AD, controlled for age and sex (hazard ratio, HR, 0.993, P = .979). However, the presence of anti-HSV IgM at baseline was associated with an increased risk of developing AD (HR 1.959, P = .012). CONCLUSION: Positivity for anti-HSV IgM, a sign of reactivated infection, was found to almost double the risk for AD, whereas the presence of anti-HSV IgG antibodies did not affect the risk.

The scaffolding function of LSD1 controls DNA methylation in mouse ESCs.

Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Dissecting the genetic landscape of GPCR signaling through phenotypic profiling in C. elegans.

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans. These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts.

Lrrn1 is required for formation of the midbrain-hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick.

The midbrain-hindbrain boundary (MHB) acts as an organiser/signalling centre to pattern tectal and cerebellar compartments. Cells in adjacent compartments must be distinct from each other for boundary formation to occur at the interface. Here we have identified the leucine-rich repeat (LRR) neuronal 1 (Lrrn1) protein as a key regulator of this process in chick. The Lrrn family is orthologous to the Drosophila tartan/capricious (trn/caps) family. Differential expression of trn/caps promotes an affinity difference and boundary formation between adjacent compartments in a number of contexts; for example, in the wing, leg and eye imaginal discs. Here we show that Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. Lrrn1 is down-regulated in the anterior hindbrain by the organiser signalling molecule FGF8, thereby creating a differential affinity between these two compartments. Lrrn1 is required for the formation of MHB--loss of function leads to a loss of the morphological constriction and loss of Fgf8. Cells overexpressing Lrrn1 violate the boundary and result in a loss of cell restriction between midbrain and hindbrain compartments. Lrrn1 also regulates the glycosyltransferase Lunatic Fringe, a modulator of Notch signalling, maintaining its expression in midbrain cells which is instrumental in MHB boundary formation. Thus, Lrrn1 provides a link between cell affinity/compartment segregation, and cell signalling to specify boundary cell fate.