Amyotrophic lateral sclerosis (ALS) causes selective neurodegeneration in primary motor cortex, yet cell-type-specific molecular changes driving this vulnerability remain poorly understood. We present an integrated single-nucleus RNA- and ATAC-sequencing atlas of 778,330 nuclei from the primary motor cortex of 140 genetically characterised donors. ALS is associated with widespread transcriptional reprogramming driven by a common set of transcription factors (TFs) across multiple cell-types. Astrocytes harbour the most differentially expressed genes. Within astrocytes, a WDR49-expressing subpopulation is spatially associated with TDP-43 pathology, and genetic variants within WDR49 confer risk for both sporadic and monogenic autosomal dominant ALS. In patient-derived induced astrocytes, WDR49 protein abundance predicts the survival of co-cultured neurons. WDR49 localises to PML nuclear bodies, where it regulates astrocyte reactivity and secretion of EVs containing protein chaperones. Together, these in vivo and in vitro findings suggest that WDR49+ astrocytes mount a compensatory secretory response to extracellular protein aggregates, and that loss of this capacity lowers the threshold for ALS pathogenesis.

Charcot-Marie-Tooth disease (CMT) is a group of inherited progressive conditions affecting distal motor and sensory neurons, leading to muscle weakness, pain, and loss of sensation in limbs. CMT type 2A (CMT2A) is the most common form of axonal CMT and is associated with a more severe clinical manifestation. However, there are no treatments currently available. To investigate disease mechanisms and facilitate treatment discovery, we developed an in vitro model for CMT2A by introducing the patient-specific MFN2 R94Q/+ variant into human embryonic stem cells (hESCs). Isogenic variant and wild-type hESCs differentiated into spinal motor neurons with similar efficiency and gave rise to functional motor neurons in vitro. However, MFN2 R94Q/+ spinal motor neurons displayed impaired mitochondrial trafficking, resulting in altered distribution of mitochondria in axons. Unbiased quantitative proteomic profiling of the endogenous MFN2 interactome revealed dose-dependent remodelling by the R94Q variant across 412 proteins, highlighting candidate mechanisms in disease pathology. Importantly, we showed that mitochondrial trafficking defects could be alleviated by treatment with an HDAC6 inhibitor. Chemical inhibition of HDAC6 also rescued the motor phenotype in a zebrafish CMT2A model. Taken together, our study reveals a variant-specific insight into CMT2A disease mechanisms and confirms HDAC6 as a promising target for further therapeutic development.

ABSTRACT The disease gonorrhoea is caused by the sexually transmitted pathogen Neisseria gonorrhoeae. This bacterium is an obligate human pathogen that can survive intracellularly through the expression of specific pathogenicity determinants. Protein post‐translational modifications have been shown to be involved in the regulation of gene transcription and metabolism. Here, we studied the role of non‐enzymatic acetylation by acetyl‐phosphate in N. gonorrhoeae. This was achieved through the deletion of pta and ackA genes from the phosphotransacetylase‐acetate kinase pathway (PTA‐AKA) that modulate the level of acetyl‐phosphate in the cell. As predicted, more protein acetylation was observed in the ΔackA strain. Using immunoaffinity purification of acetylated peptides and LC‐MS/MS we demonstrated that 88% of the detectable N. gonorrhoeae proteome (1343 proteins) is acetylated. With many of the acetylated proteins involved in central metabolism especially in pyruvate utilisation. Growth studies showed that the ΔackA strain was unable to utilise pyruvate as a carbon source, whereas it could grow on glucose as well as the wild‐type. Furthermore, a deacetylase enzyme was identified and its gene mutated (Δhdac), this allowed the identification of a number of putative targets for HDAC, including phosphotransacetylase. We found that gonococcal pathogenicity was changed by acetyl‐phosphate concentration, with the ΔackA strain killing the wax moth larvae faster than the wild‐type, whereas the Δpta strain was non‐pathogenic in this model. The data obtained suggest that non‐enzymatic protein acetylation in N. gonorrhoeae plays an important role in the central metabolism, carbon source utilisation, and virulence of this bacterium.

Disruption of autophagy has emerged as a common feature in many neurodegenerative diseases. Autophagy is a membrane-dependent pathway that requires many key regulators to quickly localize on and off membranes during induction, promoting membrane fusion. Previously, our bioinformatic approaches have shown that autophagy and Huntington disease (HD) are enriched in palmitoylated proteins. Palmitoylation involves the reversible addition of long-chain fatty acids to promote membrane binding. Herein, we show that inhibition of palmitoylation regulates the abundance of several key regulators of autophagy and leads to a partial block of autophagic flux. We confirm that the autophagy receptor SQSTM1/p62 (sequestosome 1) is palmitoylated and directed to the lysosome. Importantly, we report that SQSTM1 palmitoylation is significantly reduced in HD patient and mouse model brains. This finding reveals a novel mechanism contributing to the generation of empty autophagosomes previously seen in HD models and patient-derived cells.

Histone deacetylase 1 and 2 (HDAC1/2) are highly related enzymes that regulate histone acetylation levels in all cells, as catalytic and structural components of six unique multiprotein complexes: SIN3, NuRD, CoREST, MIDAC, MIER and RERE. Co-immunoprecipitation of HDAC1-Flag followed by mass spectrometry revealed that 92% of HDAC1 in mouse embryonic stem cells resides in 3 complexes, NuRD (49%), CoREST (28%) and SIN3 (15%). We compared the structures of MTA1:HDAC1 and MIDAC:HDAC1 to identify critical binding residues on the surface of HDAC1. Surprisingly, a single mutation, Y48E, disrupts binding to all complexes except SIN3. Rescue experiments performed with HDAC1-Y48E in HDAC1/2 double-knockout cells, showed that retention of SIN3 binding alone is sufficient for cell viability. Gene expression and histone acetylation patterns were perturbed in both Y48E and a second mutant cell line, HDAC1-E63R, indicating that cells require a full repertoire of the HDAC1/2 complexes to regulate their transcriptome appropriately. Comparative analysis of MTA1/HDAC1 and SIN3B/HDAC2 structures confirmed the differential modes of HDAC1 recruitment, such that Y48 interacts with ELM2/SANT domain-containing proteins, but not SIN3. The E63R mutation shows markedly reduced binding to NuRD and MiDAC complexes, but retains some CoREST binding. We provide novel molecular insights into the abundance, co-factors and assemblies of this crucial family of chromatin modifying machines. ### Competing Interest Statement The authors have declared no competing interest.

Background: Oxidative stress is a key feature of several neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). Identification of reliable biomarkers of oxidative stress would be beneficial for drug-target engagement studies. Methods: We performed unbiased quantitative mass spectrometry (MS)-based analysis to measure changes in protein abundance and oxidation in cerebrospinal fluid (CSF) from a cohort of ALS patients and healthy controls at two time points (approximately four months apart) to capture disease progression. In addition, we developed a sensitive and targeted quantitative MS method to measure glutathione oxidation state in the same sets of CSF samples. Results: Proteomic analysis of CSF revealed statistically significant changes in the abundance of several proteins, including CHIT1, CHI3L1, CHI3L2 and COL18A1 in ALS patients compared to healthy controls at both time points. Several sites of protein oxidation were significantly altered in ALS compared to healthy controls, and total levels of reversible protein oxidation were elevated in ALS patients. Given that glutathione oxidation could be a useful biomarker of oxidative stress, we also measured glutathione and its oxidation state in CSF in the same cohorts of samples. Total GSH (tGSH), GSSG levels and the GSSG/GSH ratio were significantly higher in the ALS than in the healthy control group for both time points. For the first visit, fold changes of tGSH, GSSG, and GSSG/GSH ratio in ALS compared to HC were 1.33 (p = 0.0215), 1.54 (p = 0.0041) and 1.80 (p = 0.0454), respectively. For the second visit, these values were 1.50 (p = 0.0143), 2.00 (p = 0.0018) and 2.14 (p = 0.0120), respectively. Furthermore, we found positive correlations between disease duration until the first visit and total glutathione (tGSH), GSSG and GSSG/GSH ratio. Finally, there was a strong positive correlation between the total intensity of reversibly oxidised proteins and the ratio of GSSG/GSH in ALS patients at both visits. Conclusion: We propose that measuring levels of glutathione oxidation in CSF could act as a stratification biomarker to select ALS patients for antioxidant therapy and an approach to monitor the treatment response to therapeutic agents targeting oxidative stress. ### Competing Interest Statement The authors have declared no competing interest.

Bacterial species utilise different receptors at the cell membrane to adhere to cells. Previously, we demonstrated that interference with CD9, a human tetraspanin, reduces adherence of multiple species of bacteria to cells. CD9 is not a receptor but organises numerous commandeered host proteins at the cell membrane; however, the full interactome has not yet been delineated. Using a CD9 proximity labelling model, a first for CD9, we observed a diverse interactome, with 710 enriched proteins in uninfected cells. Proximal proteins were associated with various cellular processes, including extracellular matrix (ECM)-receptor interactions and tight junctions. Several known bacterial receptors were also detected, including CD44, CD46 and CD147. The interactome was dynamic during infection with two distinct bacterial species, Neisseria meningitidis and Staphylococcus aureus. In total, 12 human proteins were enriched during meningococcal infection, compared to one during staphylococcal infection, demonstrating different host factor requirements during CD9-mediated bacterial adherence. CD44 or CD147 knockdown reduced staphylococcal and meningococcal adherence, respectively, but not vice versa. However, in combination with CD9 interference, no additive effects were observed, demonstrating association of these proteins during infection. We have developed a tool that measures changes within the CD9 interactome, demonstrated CD9 as a universal organiser of bacterial 'adhesion platforms', and shown efficacy of a disrupting CD9-derived peptide.

Histone deacetylase 1 and 2 (HDAC1/2) regulate histone acetylation as catalytic and structural components of six unique multiprotein complex families: SIN3, NuRD, CoREST, MIDAC, MIER, and RERE. Co-immunoprecipitation of HDAC1-Flag followed by mass spectrometry revealed that 92% of HDAC1 in mouse embryonic stem cells resides in three complexes, NuRD (49%), CoREST (28%), and SIN3 (15%). We compared the structures of MTA1:HDAC1 and MIDEAS:HDAC1 to identify critical binding residues on the surface of HDAC1. Surprisingly, a single mutation, Y48E, disrupts binding to all complexes except SIN3. Rescue experiments performed with HDAC1-Y48E in HDAC1/2 double-knockout cells showed that retention of SIN3 binding alone is sufficient for cell viability. Gene expression and histone acetylation patterns were perturbed in both Y48E and a second mutant cell line, HDAC1-E63R, indicating that cells require a full repertoire of the HDAC1/2 complexes to regulate their transcriptome appropriately. Comparative analysis of MTA1/HDAC1 and SIN3B/HDAC2 structures confirmed the differential modes of HDAC1 recruitment, with Y48 interacting with ELM2/SANT domain-containing proteins, but not SIN3. The E63R mutation shows markedly reduced binding to NuRD and MiDAC complexes but retains some CoREST binding. We provide novel molecular insights into the abundance, co-factors and assemblies of this crucial family of chromatin modifying machines.

Salmonella Typhi secretes typhoid toxin that activates cellular DNA damage responses (DDR) during acute typhoid fever. Human infection challenge studies revealed that the toxin suppresses bacteraemia via unknown mechanisms. Using quantitative proteomic analysis on the plasma of bacteraemic participants, we demonstrate that wild-type toxigenic Salmonella induced secretion of lysozyme (LYZ) and apolipoprotein C3 (APOC3). Recombinant typhoid toxin or Salmonella infection recapitulated LYZ and APOC3 secretion in cultured cells, which involved ATM/ATR-dependent DDRs and confirmed observations in typhoid fever. LYZ caused spheroplast formation, inhibited the Salmonella type 3 secretion system, and intracellular infections. LYZ expression was regulated by p53 in a cell type-specific manner and driven by mitochondrial oxidative stress that caused nuclear DDRs and p53-mediated senescence responses. Addition of LYZ inhibited oxidative DNA damage and resulting senescence responses caused by typhoid toxin. Our findings may indicate that toxin-induced DDRs elicit antimicrobial responses, which suppress Salmonella bacteraemia during typhoid fever.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterised by the selective loss of motor neurons in the motor cortex, brainstem and spinal cord. In 1993, the first ALS-linked gene mutations were identified in the Cu,Zn superoxide dismutase (SOD1) gene, which account for approximately 20 % of familial ALS cases. The mechanism of toxicity in this subset of patients is thought to arise from a gain-of-toxic function from the protein's propensity to misfold and aggregate into cytoplasmic inclusions. Immunohistochemical studies have shown that misfolded wildtype SOD1 (wtSOD1) is also detected in the motor neurons and glial cells of ALS patients without SOD1 mutations. It is proposed that disrupted, or aberrant, post-translational modifications cause wtSOD1 to adopt a toxic conformation similar to that of the mutant protein. Subsequent mechanistic studies have shown that this misfolded wtSOD1 can disrupt cellular function and lead to motor neuron death through pathways similar to those observed in mutant SOD1-ALS. Given the limited neuroprotective treatments currently available that can effectively slow or reverse disease progression, targeting a pathogenic mechanism that features in both familial and sporadic ALS cases represents a promising therapeutic approach for a broader patient population. This review examines the growing body of evidence that supports or challenges the role of misfolded wtSOD1 in the pathophysiology of sporadic ALS and explores the potential implications of this mechanism in disease progression. Understanding how misfolded wtSOD1 contributes to disease pathogenesis provides new opportunities for developing more widely available treatments for this devastating disease.

Cells sense mechanical cues and convert them into biochemical responses to regulate biological processes such as embryonic development, aging, cellular homeostasis, and disease progression. In this study, we introduce a large-scale, systematic approach to identify proteins with mechanosensitive nuclear localization, highlighting their potential roles in mechanotransduction. Among the proteins identified, we focus here on the splicing factor PTBP1. We demonstrate that its nuclear abundance is regulated by mechanical cues such as cell density, size, and extracellular matrix (ECM) stiffness and that PTBP1 medicates the mechanosensitive alternative splicing of the endocytic adapter protein Numb. Furthermore, we show that PTBP1 and Numb alternative splicing is critical for ECM stiffness-induced epithelial cell spreading and proliferation as well as for mesenchymal stem cell differentiation into osteoblasts on a stiff matrix. Our results underscore the emerging role of alternative splicing in mechanotransduction and provide novel mechanistic insights into how matrix stiffness modulates cellular mechanoresponses.

Cells sense and respond to mechanical cues from their environment. Mechanical cues are important for many biological processes, including embryonic development, ageing, cellular homeostasis, and diseases. Cells translate mechanical cues into cellular biochemical signals that govern cellular behaviour, like cell proliferation or migration, via a process called mechanotransduction. However, this process and the proteins involved remain incompletely understood. Here, we present an unbiased and large-scale approach to identify proteins involved in mechanotransduction. The screen revealed that the splicing factor PTBP1 is a novel mechanotransducer. We show that the nuclear localisation of PTBP1 depends on extracellular matrix stiffness, cell density, and the actomyosin-based contractility of the cell. Furthermore, we demonstrate that PTBP1 promotes the mechanosensitive splicing of the adapter protein Numb and that alternative splicing of Numb is crucial for matrix stiffness-induced cell proliferation and mechanomemory. Our results support the idea that changes in alternative splicing are an integral part of mechanotransduction and provide a mechanism by which matrix stiffness regulates cell proliferation and the formation of a mechanomemory in cells. ### Competing Interest Statement The authors have declared no competing interest.

Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid-liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.

Epithelial colonisation is often a critical first step in bacterial pathogenesis, however, different bacterial species utilise several different receptors at the cell membrane to adhere to cells. We have previously demonstrated that interference of the human tetraspanin, CD9, can reduce adherence of multiple species of bacteria to epithelial cells by approximately 50%. However, CD9 does not act as a receptor and is responsible for organising and clustering partner proteins commandeered by bacteria for efficient adherence. CD9 can organise numerous host proteins at the cell membrane but the full interactome has not been delineated. Here, using a novel CD9 proximity-labelling model, we demonstrate a vast and diverse CD9 interactome with 845 significantly enriched proteins associated with CD9 over four hours. These putative proximal proteins were associated with various cellular pathways including cell adhesion, ECM-receptor interactions, endocytosis, SNARE interactions and adherens and tight junctions. Significant and known interactors of CD9 were enriched including β1 integrins and major immunoglobulin superfamily members but also included several known bacterial adherence receptors including CD44, CD46 and CD147. We further demonstrate dynamism of the interactome during infection at three separate time points with two different bacterial species, Neisseria meningitidis and Staphylococcus aureus. During meningococcal infection, 13 unique proximal proteins associated with CD9 were significantly enriched across four hours compared to uninfected cells. However, upon staphylococcal infection far fewer enriched proximal proteins were identified demonstrating that different bacteria require different host factors during CD9-mediated bacterial adherence. Transient knockdown of CD44 and CD147, candidate receptor proteins identified in our screen, significantly reduced staphylococcal and meningococcal adherence respectively. This effect was ablated in the absence of CD9 or if epithelial cells were treated with a CD9-derived peptide demonstrating the association of these proteins during staphylococcal and meningococcal adherence. We demonstrate for the first time the CD9 interactome of epithelial cells and that bacteria hijack these interactions to efficiently adhere to epithelial cells. This process is bacterial species specific, recruiting several different proteins during infection but a host-derived peptide is able to interfere with this process. We have therefore developed a tool that can measure changes within the CD9 interactome after cellular challenge, established a mechanism in which CD9 is used as a universal organiser of bacterial adhesion platforms and demonstrated that this process can be stopped using a CD9-derived peptide. ### Competing Interest Statement The authors have declared no competing interest.

Histone Deacetylase 1 (HDAC1) removes acetyl groups from lysine residues on core histones, a critical step in regulating chromatin accessibility. Despite histone deacetylation being an apparently repressive activity, suppression of HDACs causes both up- and downregulation of gene expression. Here we exploited the degradation tag (dTAG) system to rapidly degrade HDAC1 in mouse embryonic stem cells (ESCs) lacking its paralog, HDAC2. The dTAG system allowed specific degradation and removal of HDAC1 in <1 h (100x faster than genetic knockouts). This rapid degradation caused increased histone acetylation in as little as 2 h, with H2BK5 and H2BK11 being the most sensitive. The majority of differentially expressed genes following 2 h of HDAC1 degradation were upregulated (275 genes up versus 15 down) with increased proportions of downregulated genes observed at 6 h (1153 up versus 443 down) and 24 h (1146 up versus 967 down), respectively. Upregulated genes showed increased H2BK5ac and H3K27ac around their transcriptional start site (TSS). In contrast, decreased acetylation and chromatin accessibility of super-enhancers was linked to the most strongly downregulated genes. These findings suggest a paradoxical role for HDAC1 in the maintenance of histone acetylation levels at critical enhancer regions required for the pluripotency-associated gene network.

Breast tumours are embedded in a collagen I-rich extracellular matrix (ECM) network, where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required for cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required macropinocytosis-dependent ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the up-regulation of the intracellular levels of several amino acids, most notably tyrosine and phenylalanine. This resulted in elevated tyrosine catabolism on ECM under starvation, leading to increased fumarate levels, potentially feeding into the tricarboxylic acid (TCA) cycle. Interestingly, this pathway was required for ECM-dependent cell growth and invasive cell migration under amino acid starvation, as the knockdown of p-hydroxyphenylpyruvate hydroxylase-like protein (HPDL), the third enzyme of the pathway, opposed cell growth and motility on ECM in both 2D and 3D systems, without affecting cell proliferation on plastic. Finally, high HPDL expression correlated with poor prognosis in breast cancer patients. Collectively, our results highlight that the ECM in the tumour microenvironment (TME) represents an alternative source of nutrients to support cancer cell growth by regulating phenylalanine and tyrosine metabolism.

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a senescence-associated secretory phenotype (SASP) that propagates the stress response to bystander cells. Thus, acute senescence is a powerful tumor suppressor. Salmonella enterica hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (TxSCs) and proteomics of their secretome identify the factors as Wnt5a, INHBA, and GDF15. Wnt5a establishes a positive feedback loop, driving INHBA and GDF15 expression. In fibroblasts, Wnt5a and INHBA mediate autocrine senescence in TxSCs and paracrine senescence in naive cells. Wnt5a synergizes with GDF15 to increase Salmonella invasion. Intestinal TxSCs undergo apoptosis without Wnt5a, which is required for establishing intestinal TxSCs. The study reveals how an innate defense against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections.

Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here, we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and could potentially deposit histone octamer onto nucleosome-depleted regions of DNA.