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BackgroundAlzheimer’s disease (AD) is characterized by the accumulation of amyloid-β (Aβ) peptides in intra- and extracellular deposits. How Aβ aggregates perturb the proteome in brains of patients and AD transgenic mouse models, however, remains largely unclear. State-of-the-art mass spectrometry (MS) methods can comprehensively detect proteomic alterations in neurodegenerative disorders, providing relevant insights unobtainable with transcriptomics investigations. Analyses of the relationship between progressive Aβ aggregation and protein abundance changes in brains of 5xFAD transgenic mice have not been reported previously.MethodsWe quantified progressive Aβ aggregation in hippocampus and cortex of 5xFAD mice and controls with immunohistochemistry and biochemical membrane filter assays. Protein changes in different mouse tissues were analysed by MS-based proteomics using label-free quantification (LFQ); resulting MS data were processed using an established pipeline. Results were contrasted with existing proteomic data sets from postmortem AD patient brains. Finally, abundance changes in the candidate marker Arl8b were validated in CSF from AD patients and controls using ELISAs.Results:Experiments revealed a more rapid accumulation of Aβ42 peptides in hippocampus than in cortex of 5xFAD mice, accompanied by many more protein abundance changes in hippocampus than in cortex, indicating that Aβ42 aggregate deposition is associated with brain region-specific proteome perturbations. Generating time-resolved data sets, we defined Aβ aggregate-correlated and anticorrelated proteome changes, a fraction of which was conserved in postmortem AD patient brain tissue, suggesting that proteome changes in 5xFAD mice mimic disease relevant changes in human AD. We detected a positive correlation between Aβ42 aggregate deposition in the hippocampus of 5xFAD mice and the abundance of the lysosome-associated small GTPase Arl8b, which accumulated together with axonal lysosomal membranes in close proximity of extracellular Aβ plaques in 5xFAD brains. Abnormal aggregation of Arl8b was observed in AD brain tissue. Arl8b protein levels were significantly increased in cerebrospinal fluid (CSF) of AD patients, a clinically accessible body fluid.ConclusionsWe report a comprehensive biochemical and proteomic investigation of hippocampal and cortical brain tissue derived from 5xFAD transgenic mice, providing a valuable resource to the neuroscientific community. We identified Arl8b, with significant abundance changes in 5xFAD and AD patient brains. Arl8b might enable the measurement of progressive lysosome accumulation in AD patients and have clinical utility as a candidate biomarker.Data are available via ProteomeXchange with identifier PXD030348.

Huntingtin-lowering approaches that target huntingtin expression are a major focus for therapeutic intervention for Huntington’s disease. When the cytosine, adenine and guanine repeat is expanded, the huntingtin pre-mRNA is alternatively processed to generate the full-length huntingtin and HTT1a transcripts. HTT1a encodes the aggregation-prone and highly pathogenic exon 1 huntingtin protein. In evaluating huntingtin-lowering approaches, understanding how the targeting strategy modulates levels of both transcripts and the huntingtin protein isoforms that they encode will be essential. Given the aggregation-propensity of exon 1 huntingtin, the impact of a given strategy on the levels and subcellular location of aggregated huntingtin will need to be determined. We have developed and applied sensitive molecular approaches to monitor the levels of aggregated and soluble huntingtin isoforms in tissue lysates. We have used these, in combination with immunohistochemistry, to map the appearance and accumulation of aggregated huntingtin throughout the CNS of zQ175 mice, a model of Huntington’s disease frequently chosen for preclinical studies. Aggregation analyses were performed on tissues from zQ175 and wild-type mice at monthly intervals from 1 to 6 months of age. We developed three homogeneous time-resolved fluorescence assays to track the accumulation of aggregated huntingtin and showed that two of these were specific for the exon 1 huntingtin protein. Collectively, the homogeneous time-resolved fluorescence assays detected huntingtin aggregation in the 10 zQ175 CNS regions by 1–2 months of age. Immunohistochemistry with the polyclonal S830 anti-huntingtin antibody showed that nuclear huntingtin aggregation, in the form of a diffuse nuclear immunostain, could be visualized in the striatum, hippocampal CA1 region and layer IV of the somatosensory cortex by 2 months. That this diffuse nuclear immunostain represented aggregated huntingtin was confirmed by immunohistochemistry with a polyglutamine-specific antibody, which required formic acid antigen retrieval to expose its epitope. By 6 months of age, nuclear and cytoplasmic inclusions were widely distributed throughout the brain. Homogeneous time-resolved fluorescence analysis showed that the comparative levels of soluble exon 1 huntingtin between CNS regions correlated with those for huntingtin aggregation. We found that soluble exon 1 huntingtin levels decreased over the 6-month period, whilst those of soluble full-length mutant huntingtin remained unchanged, data that were confirmed for the cortex by immunoprecipitation and western blotting. These data support the hypothesis that exon 1 huntingtin initiates the aggregation process in knock-in mouse models and pave the way for a detailed analysis of huntingtin aggregation in response to huntingtin-lowering treatments.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal glutamine (Q) expansion in the huntingtin protein due to elongated CAG repeats in the gene HTT. We used non-integrative episomal plasmids to generate induced pluripotent stem cells (iPSCs) from three individuals affected by HD: CH1 (58Q), and two twin brothers CH3 (44Q) and CH4 (44Q). The iPSC lines exhibited one healthy HTT allele and one with elongated CAG repeats, as confirmed by PCR and sequencing. All iPSC lines expressed pluripotency markers, exhibited a normal karyotype, and generated cells of the three germ layers in vitro.

Huntington’s disease is characterised by CAG expansion in the huntingtin gene above a critical threshold of~35 repeats, resulting in polyglutamine expansion of the huntingtin protein (HTT). The biological role of wildtype HTT and the associated mechanisms of disease pathology caused by expanded HTT remain incompletely understood, in part, due to challenges characterising interactions between HTT and putative binding partners. Here we describe a biochemical toolkit of rationally designed, high-quality recombinant HTT subdomains; one spanning the N-terminal HEAT and bridge domains (NTD) and the second spanning the C-terminal HEAT domain (CTD). Using biophysical methods and cryo-electron microscopy, we show these smaller subdomains are natively folded and can associate to reconstitute a functional full-length HTT structure capable of forming a near native-like complex with 40 kDa HTT-associated protein (HAP40). We report biotin-tagged variants of these subdomains, as well as full-length HTT, that permit immobilisation of each protein for quantitative biophysical assays without impacting protein quality. We demonstrate the CTD alone can form a stable complex when co-expressed with HAP40, which can be structurally resolved. The CTD-HAP40 complex binds the NTD, with a dissociation constant of approximately 10 nM as measured by bio-layer interferometry. We validate the interaction between the CTD and HAP40 using a luciferase two-hybrid assay and use subdomain constructs to demonstrate their respective stabilization of HAP40 in cells. These open-source biochemical tools will enable the wider HD community to study fundamental HTT biology, discover new macromolecular or small-molecule binding partners and map interaction sites across this very large protein.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal length of CAG repeats in the gene HTT, leading to an elongated poly-glutamine (poly-Q) sequence in huntingtin (HTT). We used non-integrative Sendai virus to reprogram fibroblasts from a patient with juvenile onset HD to induced pluripotent stem cells (iPSCs). Reprogrammed iPSCs expressed pluripotency-associated markers, exhibited a normal karyotype, and following directed differentiation generated cell types belonging to the three germ layers. PCR analysis and sequencing confirmed the HD patient-derived iPSC line had one normal HTT allele and one with elongated CAG repeats, equivalent to ≥180Q.

Huntington9s disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene ( HTT ). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J‐protein) that completely suppresses fibrilization of HttExon1Q 48 . The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient‐derived neural cells and on an organismal level in Caenorhabditis elegans . Among the proteins in this chaperone complex, the J‐protein is the concentration‐limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q 97 aggregation and represents a target of future therapeutic avenues for HD.

Recent structural studies show distinct morphologies for the fibrils of Aβ(1-42) and Aβ(1-40), which are believed not to co-fibrillize. We describe here a novel, structurally-uniform 1 : 1 mixed fibrillar species, which differs from both pure fibrils. It forms preferentially even when Aβ(1-42) : Aβ(1-40) peptides are mixed in a non-stoichiometric ratio.

The deposition of mutant huntingtin (mHTT) protein aggregates in neurons of patients is a pathological hallmark of Huntington’s disease (HD). Previous investigations in cell-free and cell-based disease models showed mHTT exon-1 (mHTTex1) fragments with pathogenic polyglutamine (polyQ) tracts (>40 glutamines) to self-assemble into highly stable, β-sheet-rich protein aggregates with a fibrillar morphology. HD knock-in mouse models have not been extensively studied with regard to mHTT aggregation. They endogenously produce full-length mHTT with a pathogenic polyQ tract as well as mHTTex1 fragments. Here, we demonstrate that seeding-competent, fibrillar mHTT aggregates can be readily detected in brains of zQ175 knock-in HD mice. To do this, we applied a highly sensitive FRET-based protein amplification assay that is capable of detecting seeding-competent mHTT aggregate species down to the femtomolar range. Furthermore, we show that fibrillar structures with an average length of ∼200 nm can be enriched with aggregate-specific mouse and human antibodies from zQ175 mouse brain extracts through immunoprecipitations, confirming that such structures are formed in vivo. Together these studies indicate that small, fibrillar, seeding-competent mHTT structures are prominent aggregate species in brains of zQ175 mice.

Previously evidence was presented that the single-nucleotide polymorphism rs1344706 located in an intronic region of the ZNF804A gene is associated with reduced transcript levels in fetal brains. This genetic variation in the gene encoding the zinc-finger protein ZNF804A is associated with schizophrenia (SZ) and bipolar disorder. Currently, the molecular and cellular function of ZNF804A is unclear. Here, we generated a high-confidence protein-protein interaction (PPI) network for ZNF804A using a combination of yeast two-hybrid and bioluminescence-based PPI detection assays, directly linking 12 proteins to the disease-associated target protein. Among the top hits was the signal transducer and activator of transcription 2 (STAT2), an interferon-regulated transcription factor. Detailed mechanistic studies revealed that STAT2 binds to the unstructured N-terminus of ZNF804A. This interaction is mediated by multiple short amino acid motifs in ZNF804A but not by the conserved C2H2 zinc-finger domain, which is also located at the N-terminus. Interestingly, investigations in HEK293 cells demonstrated that ZNF804A and STAT2 both co-translocate from the cytoplasm into the nucleus upon interferon (IFN) treatment. Furthermore, a concentration-dependent effect of ZNF804A overproduction on STAT2-mediated gene expression was observed using a luciferase reporter, which is under the control of an IFN-stimulated response element (ISRE). Together these results indicate the formation of ZNF804A:STAT2 protein complex and its translocation from the cytoplasm into the nucleus upon IFN stimulation, suggesting that it may function as a signal transducer that activates IFN-mediated gene expression programs.

Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease., Leigh syndrome (LS) is a severe neurometabolic disorder which lacks effective models. Here, the authors developed human neuronal models of LS carrying mutations in SURF1 which show impaired neuronal morphogenesis due to metabolic deficiencies.

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the Huntingtin gene. As disease-modifying therapies for HD are being developed, peripheral blood cells may be used to indicate disease progression and to monitor treatment response. In order to investigate whether gene expression changes can be found in the blood of individuals with HD that distinguish them from healthy controls, we performed transcriptome analysis by next-generation sequencing (RNA-seq). We detected a gene expression signature consistent with dysregulation of immune-related functions and inflammatory response in peripheral blood from HD cases vs. controls, including induction of the interferon response genes, IFITM3, IFI6 and IRF7. Our results suggest that it is possible to detect gene expression changes in blood samples from individuals with HD, which may reflect the immune pathology associated with the disease.

NRF2 (NFE2L2) is a cytoprotective transcription factor associated with >60 human diseases, adverse drug reactions and therapeutic resistance. To provide insight into the complex regulation of NRF2 responses, 1962 predicted NRF2-partner interactions were systematically tested to generate an experimentally defined high-density human NRF2 interactome. Verification and conditional stratification of 46 new NRF2 partners was achieved by co-immunoprecipitation and the novel integration of quantitative data from dual luminescence-based co-immunoprecipitation (DULIP) assays and live-cell fluorescence cross-correlation spectroscopy (FCCS). The functional impact of new partners was then assessed in genetically edited loss-of-function (NRF2−/−) and disease-related gain-of-function (NRF2T80K and KEAP1−/−) cell-lines. Of the new partners investigated >77% (17/22) modified NRF2 responses, including partners that only exhibited effects under disease-related conditions. This experimentally defined binary NRF2 interactome provides a new vision of the complex molecular networks that govern the modulation and consequence of NRF2 activity in health and disease., Graphical abstract Image 1, Highlights • 1,962 predicted NRF2 partners were tested and 46 new binary partners were identified. • 77% of tested new NRF2 partners modulate ARE-mediated gene expression. • Some novel partners only exhibit functional effects under disease-like conditions. • Quantitative evidence that KEAP1 can modulate ARE-transactivation independently of NRF2. • New NRF2 binding partners reveal 130 novel disease gene associations.

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human dopaminergic (DA) neurons, which are crucial for the development and maintenance of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human DA neurons from induced pluripotent stem cells (iPSCs). The assay, dubbed mitochondrial neuronal health (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DA neurons decreases MMP and branching complexity in a dose-dependent manner. The toxic effect of ethanol on DA neurons was already detectable after 1 hour of exposure, and occurred similarly in DA neurons derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate drugs modulating acute ethanol toxicity in human DA neurons. Among these drugs, flavoxate and disulfiram influenced mitochondrial neuronal health independently from ethanol, leading to amelioration and worsening, respectively. Altogether, we developed an HT assay to probe human mitochondrial neuronal health and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of mitochondrial neuronal health and toxicity in live human neurons.

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson’s disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-β amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c’s activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits.

Huntington’s disease (HD) is a monogenic neurodegenerative disorder with one causative gene, huntingtin (HTT). Yet, HD pathobiology is multifactorial, suggesting that cellular factors influence disease progression. Here, we define HTT protein-protein interactions (PPIs) perturbed by the mutant protein with expanded polyglutamine in the mouse striatum, a brain region with selective HD vulnerability. Using metabolically labeled tissues and immunoaffinity purification-mass spectrometry, we establish that polyglutamine-dependent modulation of HTT PPI abundances and relative stability starts at an early stage of pathogenesis in a Q140 HD mouse model. We identify direct and indirect PPIs that are also genetic disease modifiers using in-cell two-hybrid and behavioral assays in HD human cell and Drosophila models, respectively. Validated, disease-relevant mHTT-dependent interactions encompass mediators of synaptic neurotransmission (SNAREs and glutamate receptors) and lysosomal acidification (V-ATPase). Our study provides a resource for understanding mHTT-dependent dysfunction in cortico-striatal cellular networks partly through impaired synaptic communication and endosomal-lysosomal system. A record of this paper’s Transparent Peer Review process is included in the Supplemental Information.

Numerous genetic methods facilitate the detection of binary protein-protein interactions (PPIs) by exogenous overexpression, which can lead to false results. Here, we describe CellFIE, a CRISPR- and cell fusion-based PPI detection method, which enables the mapping of interactions between endogenously tagged two-hybrid proteins. We demonstrate the specificity and reproducibility of CellFIE in a matrix mapping approach, validating the interactions of VCP with ASPL and UBXD1, and the self-interaction of TDP-43 under endogenous conditions. Furthermore, we show that CellFIE can be used to quantify changes of endogenous PPIs upon stress induction or drug treatment. For the first time, CellFIE facilitates systematic mapping of interactions between endogenously tagged proteins and represents a novel tool to characterize PPIs in live cells under dynamic conditions.

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here, we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.

Summary Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer’s and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases., Graphical Abstract, Highlights • Inactivation of MOAG-2/LIR-3 reduces polyglutamine aggregation • MOAG-2/LIR-3 regulates Pol III-mediated transcription of small non-coding RNAs • Polyglutamine mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol • Polyglutamine converts MOAG-2/LIR-3 into an aggregation-promoting factor, The cellular mechanisms that drive polyglutamine aggregation are poorly understood. Sin et al. show that polyglutamine relocates MOAG-2/LIR-3 from the nucleus to the cytosol, thereby converting this protein into an aggregation-promoting factor to drive protein aggregation and toxicity.

Objective Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic β-cells. Methods A β-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation. Results The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co-localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from β-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from β-cell-specific Arfrp1 knockout mice. Conclusion Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting., Highlights • β-cell specific deletion of the trans-Golgi residing small GTPase ARFRP1 leads to elevated blood glucose levels in mice. • GOPC is a newly identified ARFRP1 dependent scaffolding protein. • ARFRP1 and GOPC are required for glucose-stimulated insulin secretion from pancreatic β-cells.

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the Huntingtin gene. As disease-modifying therapies for HD are being developed, peripheral blood cells may be used to indicate disease progression and to monitor treatment response. In order to investigate whether gene expression changes can be found in the blood of individuals with HD that distinguish them from healthy controls, we performed transcriptome analysis by next-generation sequencing (RNA-seq). We detected a gene expression signature consistent with dysregulation of immune-related functions and inflammatory response in peripheral blood from HD cases vs. controls, including induction of the interferon response genes, IFITM3, IFI6 and IRF7. Our results suggest that it is possible to detect gene expression changes in blood samples from individuals with HD, which may reflect the immune pathology associated with the disease.

Pathological aggregation of amyloid‐β (Aβ) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender‐specific pathologies in the APP23 AD‐like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender‐specific effect of lack of IL12p40, the shared subunit of interleukin (IL)‐12 and IL‐23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aβ plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aβ1–40 without changes in Aβ plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL‐12/IL‐23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD., Abrogating IL‐12/IL‐23 signaling ameliorates amyloidogenesis gender‐specifically in APP23 mice, indicating therapeutic potential of targeting IL‐12/IL‐23 in Alzheimer's disease and highlighting the importance of gender considerations in AD.

Huntington’s disease is caused by the expansion of a CAG repeat within exon 1 of the HTT gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington’s disease, that were known to abrogate somatic instability in Huntington’s disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the HTT gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 HTT with 90 CAGs, a mutation that causes juvenile Huntington’s disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington’s disease patient’s blood, but which has been detected in post-mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG)90 mice and that this correlated with the onset of transcriptional dysregulation. Whereas in R6/2(CAG)200 mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG)90 brains have different properties to those in the R6/2(CAG)200 mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilized by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG)200 brains polymerize into larger structures that appear as inclusion bodies. These data emphasize that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes., Huntingtin aggregates in the brains of Huntington’s disease transgenic mice with 90 or 200 CAGs have different subcellular localization, appearance and molecular properties. In these mice, aggregated huntingtin precedes transcriptional dysregulation in neuronal nuclei and the rate of disease progression correlated with the accumulation of aggregated huntingtin in the cytoplasm., Graphical Abstract Graphical Abstract

The self-assembly of the 42-residue amyloid-β peptide, Aβ42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aβ42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aβ42 oligomers. These structures exhibit low β-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, β-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aβ42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aβ42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aβ42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, β-sheet-rich Aβ42 fibrils in AD, while structures with low β-sheet content may be less relevant.

© The Author(s) 2019., Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes., This work was supported by a Claudia Adams Barr Award to S.G.C., a Fonds de la Recherche Scientifique (F.R.S.- FNRS)-Télévie Grant and a Wallonia-Brussels International (WBI)-World Excellence Fellowship to J.O., NHGRI grants U41HG001715 awarded to M.V., D.E.H. and M.A.C, and P50HG004233 awarded to M.V. We acknowledge the support of Labex IBEID (10- LABX-0062). M.V. is a Chercheur Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

The hexameric ring structure of the type II AAA+ ATPases is considered as stable and permanent. Recently, the UBX domain-containing cofactors Arabidopsis thaliana PUX1 and human alveolar soft part sarcoma locus (ASPL) were reported to bind and disassemble the cognate AAA+ ATPases AtCDC48 and human p97. Here, we present two crystal structures related to these complexes: a truncated AtCDC48 (AtCDC48-ND1) and a hybrid complex containing human p97-ND1 and the UBX domain of plant PUX1 (p97-ND1:PUX1-UBX). These structures reveal close similarity between the human and plant AAA+ ATPases, but also highlight differences between disassembling and non-disassembling AAA+ ATPase cofactors. Based on an AtCDC48 disassembly assay with PUX1 and known crystal structures of the p97-bound human cofactor ASPL, we propose a general ATPase disassembly model. Thus, our structural and biophysical investigations provide detailed insight into the mechanism of AAA+ ATPase disassembly by UBX domain cofactors and suggest a general mode of regulating the cellular activity of these molecular machines.

Mutations are at the root of many human diseases. Still, we largely do not exactly understand how they trigger pathogenesis. One, more recent, hypothesis has been that they comprehensively perturb protein-protein interaction (PPI) networks and significantly alter key biological processes. Under this premise, many rare genetic disorders with Mendelian inheritance, like Huntington's disease and several spinocerebellar ataxias, are likely to be caused by complex genotype-phenotype relationships involving abnormal PPIs. These altered PPI networks and their effects on cellular pathways are poorly understood at the molecular level. In this review, we focus on PPIs that are perturbed by the expanded pathogenic polyglutamine tract in huntingtin (HTT), the protein which, in its mutated form, leads to the autosomal dominant, neurodegenerative Huntington's disease. One aspect of perturbed mutant HTT interactions is the formation of abnormal protein species such as fibrils or large neuronal inclusions as a result of homotypic and heterotypic aberrant molecular interactions. This review focuses on abnormal PPIs that are associated with the assembly of mutant HTT aggregates in cells and their potential relevance in disease. Furthermore, the mechanisms and pathobiological processes that may contribute to phenotype development, neuronal dysfunction and toxicity in Huntington's disease brains are also discussed. This article is part of the Special Issue "Proteomics".

Elaboration of neuronal processes is an early step in neuronal development. Guidance cues must work closely with intracellular trafficking pathways to direct expanding axons and dendrites to their target neurons during the formation of neuronal networks. However, how such coordination is achieved remains incompletely understood. Here, we characterize an interaction between fasciculation and elongation protein zeta 1 (FEZ1), an adapter involved in synaptic protein transport, and collapsin response mediator protein (CRMP)1, a protein that functions in growth cone guidance, at neuronal growth cones. We show that similar to CRMP1 loss-of-function mutants, FEZ1 deficiency in rat hippocampal neurons causes growth cone collapse and impairs axonal development. Strikingly, FEZ1-deficient neurons also exhibited a reduction in dendritic complexity stronger than that observed in CRMP1-deficient neurons, suggesting that the former could partake in additional developmental signaling pathways. Supporting this, FEZ1 colocalizes with VAMP2 in developing hippocampal neurons and forms a separate complex with deleted in colorectal cancer (DCC) and Syntaxin-1 (Stx1), components of the Netrin-1 signaling pathway that are also involved in regulating axon and dendrite development. Significantly, developing axons and dendrites of FEZ1-deficient neurons fail to respond to Netrin-1 or Netrin-1 and Sema3A treatment, respectively. Taken together, these findings highlight the importance of FEZ1 as a common effector to integrate guidance signaling pathways with intracellular trafficking to mediate axo-dendrite development during neuronal network formation.

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Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.