Cryptic inclusions UNCover losses driving neurodegeneration.

Pathology formed by the protein TDP-43 (TAR DNA binding protein 43) is the hallmark of several neurodegenerative diseases. Recent studies by Ma et al. and Brown et al. reveal that loss of TDP-43 function causes inclusion of cryptic exons in specific mRNAs, including the synaptic gene UNC13A, a known genetic risk factor for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These findings suggest new disease mechanisms.

Early activation of cellular stress and death pathways caused by cytoplasmic TDP-43 in the rNLS8 mouse model of ALS and FTD.

TAR DNA binding protein 43 (TDP-43) pathology is a key feature of over 95% of amyotrophic lateral sclerosis (ALS) and nearly half of frontotemporal dementia (FTD) cases. The pathogenic mechanisms of TDP-43 dysfunction are poorly understood, however, activation of cell stress pathways may contribute to pathogenesis. We, therefore, sought to identify which cell stress components are critical for driving disease onset and neurodegeneration in ALS and FTD. We studied the rNLS8 transgenic mouse model, which expresses human TDP-43 with a genetically-ablated nuclear localisation sequence within neurons of the brain and spinal cord resulting in cytoplasmic TDP-43 pathology and progressive motor dysfunction. Amongst numerous cell stress-related biological pathways profiled using qPCR arrays, several critical integrated stress response (ISR) effectors, including CCAAT/enhancer-binding homologous protein (Chop/Ddit3) and activating transcription factor 4 (Atf4), were upregulated in the cortex of rNLS8 mice prior to disease onset. This was accompanied by early up-regulation of anti-apoptotic gene Bcl2 and diverse pro-apoptotic genes including BH3-interacting domain death agonist (Bid). However, pro-apoptotic signalling predominated after onset of motor phenotypes. Notably, pro-apoptotic cleaved caspase-3 protein was elevated in the cortex of rNLS8 mice at later disease stages, suggesting that downstream activation of apoptosis drives neurodegeneration following failure of early protective responses. Unexpectedly, suppression of Chop in the brain and spinal cord using antisense oligonucleotide-mediated silencing had no effect on overall TDP-43 pathology or disease phenotypes in rNLS8 mice. Cytoplasmic TDP-43 accumulation therefore causes very early activation of ISR and both anti- and pro-apoptotic signalling that switches to predominant pro-apoptotic activation later in disease. These findings suggest that precise temporal modulation of cell stress and death pathways may be beneficial to protect against neurodegeneration in ALS and FTD.

Aggregation-prone TDP-43 sequesters and drives pathological transitions of free nuclear TDP-43.

Aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43-related neurodegeneration involves multiple changes to normal physiological TDP-43, which undergoes nuclear depletion, cytoplasmic mislocalisation, post-translational modification, and aberrant liquid-liquid phase separation, preceding inclusion formation. Along with toxic cytoplasmic aggregation, concurrent depletion and dysfunction of normal nuclear TDP-43 in cells with TDP-43 pathology is likely a key potentiator of neurodegeneration, but is not well understood. To define processes driving TDP-43 dysfunction, we used CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of normal nuclear TDP-43 over time in live cells. Oxidative stress stimulated liquid-liquid phase separation of endogenous TDP-43 into droplet-like puncta, or spherical shell-like anisosomes. Further, nuclear RNA-binding-ablated or acetylation-mimicking TDP-43 readily sequestered and depleted free normal nuclear TDP-43 into dynamic anisosomes, in which recruited endogenous TDP-43 proteins remained soluble and highly mobile. Large, phosphorylated inclusions formed by nuclear or cytoplasmic aggregation-prone TDP-43 mutants also caused sequestration, but rendered endogenous TDP-43 immobile and insoluble, indicating pathological transition. These findings suggest that RNA-binding deficiency and post-translational modifications including acetylation exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in neurodegenerative diseases.

Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

Increased prefrontal cortical cells positive for macrophage/microglial marker CD163 along blood vessels characterizes a neuropathology of neuroinflammatory schizophrenia.

Transcript levels of cytokines and SERPINA3 have been used to define a substantial subset (40%) of individuals with schizophrenia with elevated inflammation and worse neuropathology in the dorsolateral prefrontal cortex (DLPFC). In this study, we tested if inflammatory proteins are likewise related to high and low inflammatory states in the human DLFPC in people with schizophrenia and controls. Levels of inflammatory cytokines (IL6, IL1ß, IL18, IL8) and a macrophage marker (CD163 protein) were measured in brains obtained from the National Institute of Mental Health (NIMH) (N = 92). First, we tested for diagnostic differences in protein levels overall, then we determined the percentage of individuals that could be defined as "high" inflammation using protein levels. IL-18 was the only cytokine to show increased expression in schizophrenia compared to controls overall. Interestingly, two-step recursive clustering analysis showed that IL6, IL18, and CD163 protein levels could be used as predictors of "high and low" inflammatory subgroups. By this model, a significantly greater proportion of schizophrenia cases (18/32; 56.25%; SCZ) were identified as belonging to the high inflammatory (HI) subgroup compared to control cases (18/60; 30%; CTRL) [χ2(1) = 6.038, p = 0.014]. When comparing across inflammatory subgroups, IL6, IL1ß, IL18, IL8, and CD163 protein levels were elevated in both SCZ-HI and CTRL-HI compared to both low inflammatory subgroups (all p < 0.05). Surprisingly, TNFα levels were significantly decreased (-32.2%) in schizophrenia compared to controls (p < 0.001), and were most diminished in the SCZ-HI subgroup compared to both CTRL-LI and CTRL-HI subgroups (p < 0.05). Next, we asked if the anatomical distribution and density of CD163+ macrophages differed in those with schizophrenia and high inflammation status. Macrophages were localized to perivascular sites and found surrounding small, medium and large blood vessels in both gray matter and white matter, with macrophage density highest at the pial surface in all schizophrenia cases examined. A higher density of CD163+ macrophages, that were also larger and more darkly stained, was found in the SCZ-HI subgroup (+154% p < 0.05). We also confirmed the rare existence of parenchymal CD163+ macrophages in both high inflammation subgroups (schizophrenia and controls). Brain CD163+ cell density around blood vessels positively correlated with CD163 protein levels. In conclusion, we find a link between elevated interleukin cytokine protein levels, decreased TNFα protein levels, and elevated CD163+ macrophage densities especially along small blood vessels in those with neuroinflammatory schizophrenia.

Changes in cytokine and cytokine receptor levels during postnatal development of the human dorsolateral prefrontal cortex.

In addition to their traditional roles in immune cell communication, cytokines regulate brain development. Cytokines are known to influence neural cell generation, differentiation, maturation, and survival. However, most work on the role of cytokines in brain development investigates rodents or focuses on prenatal events. Here, we investigate how mRNA and protein levels of key cytokines and cytokine receptors change during postnatal development of the human prefrontal cortex. We find that most cytokine transcripts investigated (IL1B, IL18, IL6, TNF, IL13) are lowest at birth and increase between 1.5 and 5 years old. After 5 years old, transcriptional patterns proceeded in one of two directions: decreased expression in teens and young adults (IL1B, p = 0.002; and IL18, p = 0.004) or increased mean expression with maturation, particularly in teenagers (IL6, p = 0.004; TNF, p = 0.002; IL13, p < 0.001). In contrast, cytokine proteins tended to remain elevated after peaking significantly around 3 years of age (IL1B, p = 0.012; IL18, p = 0.026; IL6, p = 0.039; TNF, p < 0.001), with TNF protein being highest in teenagers. An mRNA-only analysis of cytokine receptor transcripts found that early developmental increases in cytokines were paralleled by increases in their ligand-binding receptor subunits, such as IL1R1 (p = 0.033) and IL6R (p < 0.001) transcripts. In contrast, cytokine receptor-associated signaling subunits, IL1RAP and IL6ST, did not change significantly between age groups. Of the two TNF receptors, the 'pro-death' TNFRSF1A and 'pro-survival' TNFRSF1B, only TNFRSF1B was significantly changed (p = 0.028), increasing first in toddlers and again in young adults. Finally, the cytokine inhibitor, IL13, was elevated first in toddlers (p = 0.006) and again in young adults (p = 0.053). While the mean expression of interleukin-1 receptor antagonist (IL1RN) was highest in toddlers, this increase was not statistically significant. The fluctuations in cytokine expression reported here support a role for increases in specific cytokines at two different stages of human cortical development. The first is during the toddler/preschool period (IL1B, IL18, and IL13), and the other occurs at adolescence/young adult maturation (IL6, TNF and IL13).

miR-23a suppression accelerates functional decline in the rNLS8 mouse model of TDP-43 proteinopathy.

Skeletal muscle dysfunction may contribute to the progression and severity of amyotrophic lateral sclerosis (ALS). In the present study, we characterized the skeletal muscle pathophysiology in an inducible transgenic mouse model (rNLS8) that develops a TAR-DNA binding protein (TDP-43) proteinopathy and ALS-like neuropathology and disease progression; representative of >90% of all familial and sporadic ALS cases. As we previously observed elevated levels of miR-23a in skeletal muscle of patients with familial and sporadic ALS, we also investigated the effect of miR-23a suppression on skeletal muscle pathophysiology and disease severity in rNLS8 mice. Five weeks after disease onset TDP-43 protein accumulation was observed in tibialis anterior (TA), quadriceps (QUAD) and diaphragm muscle lysates and associated with skeletal muscle atrophy. In the TA muscle TDP-43 was detected in muscle fibres that appeared atrophied and angular in appearance and that also contained ß-amyloid aggregates. These fibres were also positive for neural cell adhesion molecule (NCAM), but not embryonic myosin heavy chain (eMHC), indicating TDP-43/ ß-amyloid localization in denervated muscle fibres. There was an upregulation of genes associated with myogenesis and NMJ degeneration and a decrease in the MURF1 atrophy-related protein in skeletal muscle. Suppression of miR-23a impaired rotarod performance and grip strength and accelerated body weight loss during early stages of disease progression. This was associated with increased AchRα mRNA expression and decreased protein levels of PGC-1α. The TDP-43 proteinopathy-induced impairment of whole body and skeletal muscle functional performance is associated with muscle wasting and elevated myogenic and NMJ stress markers. Suppressing miR-23a in the rNLS8 mouse model of ALS contributes to an early acceleration of disease progression as measured by decline in motor function.

Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal cells.

Protein aggregates that result in inclusion formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1 (HSF1)-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or a pathogenic form of polyglutamine-expanded huntingtin (Htt72Q). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR might contribute to the development of inclusion pathology in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.

NSAID use and unnatural deaths after cancer diagnosis: a nationwide cohort study in Sweden.

BACKGROUND: Cancer patients experience increased risk of death from accident and suicide. Cognitive impairment induced by cancer-related inflammation and stress-related psychiatric symptoms may be underlying mechanisms. We therefore studied the association between use of nonsteroidal anti-inflammatory drugs (NSAIDs) and risk of these outcomes. METHODS: Following a cohort of 388,443 cancer patients diagnosed between October 2005 and December 2014 in Sweden, we ascertained dispense of aspirin or non-aspirin NSAIDs from 3 months before cancer diagnosis onward and defined the on-medication period as from date of drug dispense until the prescribed dosage was consumed. Follow-up time outside medicated periods and time from unexposed patients were defined as off-medication periods. We used Cox models to estimate hazard ratios (HRs) of death due to suicide or accident, by comparing the on-medication periods with off-medication periods. RESULTS: In total, 29.7% of the cancer patients had low-dose aspirin dispensed and 29.1% had non-aspirin NSAIDs dispensed. Patients with aspirin use were more likely to be male than patients without aspirin use. Compared with off-medication periods, there was a 22% lower risk of accidental death (N = 651; HR 0.78, 95% confidence interval [CI]: 0.70 to 0.87) during on-medication periods with aspirin. The use of aspirin was not associated with risk of suicide (N = 59; HR 0.96, 95% CI: 0.66 to 1.39). No association was noted between use of non-aspirin NSAIDs and the risk of suicide (N = 13; HR 0.95, 95% CI: 0.42 to 2.18) or accidental death (N = 59; HR 0.92, 95% CI: 0.68 to 1.26). CONCLUSIONS: Intake of low-dose aspirin after cancer diagnosis was associated with a lower risk of unnatural deaths among cancer patients.

Cancer-related cognitive impairment in patients with newly diagnosed aggressive lymphoma undergoing standard chemotherapy: a longitudinal feasibility study.

PURPOSE: Cancer-related cognitive impairment (CRCI) is a recognised adverse consequence of cancer and its treatment. This study assessed the feasibility of collecting longitudinal data on cognition in patients with newly diagnosed, aggressive lymphoma undergoing standard therapy with curative intent via self-report, neuropsychological assessment, peripheral markers of inflammation, and neuroimaging. An exploration and description of patterns of cancer-related cognitive impairment over the course of treatment and recovery was also undertaken and will be reported separately. METHODS: Eligible participants completed repeated measures of cognition including self-report and neuropsychological assessment, and correlates of cognition including blood cell-based inflammatory markers, and neuroimaging at three pre-specified timepoints, time 1 (T1) - pre-treatment (treatment naïve), time 2 (T2) - mid-treatment, and time 3 (T3) - 6 to 8 weeks post-completion of treatment. RESULTS: 30/33 eligible patients (91%, 95% CI: 76%, 97%) were recruited over 10 months. The recruitment rate was 3 patients/month (95% CI: 2.0, 4.3 patients/month). Reasons for declining included feeling overwhelmed and rapid treatment commencement. Mean age was 57 years (SD = 17 years) and 16/30 (53%) were male. Most patients (20/30, 67%) had diffuse large B cell lymphoma or Hodgkin lymphoma (4/30, 13%). The neuroimaging sub-study was optional, 11/30 participants (37%) were eligible to take part, and all agreed. The remaining 19 participants were ineligible as their diagnostic PET/CT scan was completed prior. Retention and compliance with all assessments were 89 to 100% at all timepoints. Only one participant was withdrawn due to disease progression. CONCLUSIONS: Findings from this study including excellent recruitment, retention, and compliance rates demonstrate it is feasible to longitudinally assess cognition in people with newly diagnosed aggressive lymphoma during their initial treatment and recovery to inform the development of future research to improve patient experiences and cognitive outcomes. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12619001649101.

Trajectory of change in brain complement factors from neonatal to young adult humans.

Immune system components also regulate synapse formation and refinement in neurodevelopment. The complement pathway, associated with cell lysis and phagocytosis, is implicated in synaptic elimination. Aberrant adolescent synaptic pruning may underpin schizophrenia onset; thus, changes in cortical complement activity during human development are of major interest. Complement is genetically linked to schizophrenia via increased C4 copy number variants, but the developmental trajectory of complement expression in the human brain is undetermined. As complement increases during periods of active synaptic engulfment in rodents, we hypothesized that complement expression would increase during postnatal development in humans, particularly during adolescence. Using human postmortem prefrontal cortex, we observed that complement activator (C1QB and C3) transcripts peaked in early neurodevelopment, and were highest in toddlers, declining in teenagers (all ANCOVAs between F = 2.41 -3.325, p = .01-0.05). We found that C4 protein was higher at 1-5 years (H = 16.378, p = .012), whereas C3 protein levels were unchanged with age. The microglial complement receptor subunit CD11b increased in mRNA early in life and peaked in the toddler brain (ANCOVA: pH, F = 4.186, p = .003). Complement inhibitors (CD46 and CD55) increased at school age, but failed to decrease like complement activators (both ANCOVAs, F > 4.4, p < .01). These data suggest the activation of complement in the human prefrontal cortex occurs between 1 and 5 years. We did not find evidence of induction of complement factors during adolescence and instead found increased or sustained levels of complement inhibitor mRNA at maturation. Dysregulation of these typical patterns of complement may predispose the brain to neurodevelopmental disorders such as autism or schizophrenia.

Mislocalisation of TDP-43 to the cytoplasm causes cortical hyperexcitability and reduced excitatory neurotransmission in the motor cortex.

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease pathologically characterised by mislocalisation of the RNA-binding protein TAR-DNA-binding protein 43 (TDP-43) from the nucleus to the cytoplasm. Changes to neuronal excitability and synapse dysfunction in the motor cortex are early pathological changes occurring in people with ALS and mouse models of disease. To investigate the effect of mislocalised TDP-43 on the function of motor cortex neurons we utilised mouse models that express either human wild-type (TDP-43WT ) or nuclear localisation sequence-deficient TDP-43 (TDP-43ΔNLS ) on an inducible promoter that enriches expression to forebrain neurons. Pathophysiology was investigated through immunohistochemistry and whole-cell patch-clamp electrophysiology. Thirty days expression of TDP-43ΔNLS in adult mice did not cause any changes in the number of CTIP2-positive neurons in the motor cortex. However, at this time-point, the expression of TDP-43ΔNLS drives intrinsic hyperexcitability in layer V excitatory neurons of the motor cortex. This hyperexcitability occurs concomitantly with a decrease in excitatory synaptic input to these cells and fluctuations in both directions of ionotropic glutamate receptors. This pathophysiology is not present with TDP-43WT expression, demonstrating that the localisation of TDP-43 to the cytoplasm is crucial for the altered excitability phenotype. This study has important implications for the mechanisms of toxicity of one of the most notorious proteins linked to ALS, TDP-43. We provide the first evidence that TDP-43 mislocalisation causes aberrant synaptic function and a hyperexcitability phenotype in the motor cortex, linking some of the earliest dysfunctions to arise in people with ALS to mislocalisation of TDP-43.

Riluzole does not ameliorate disease caused by cytoplasmic TDP-43 in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease commonly treated with riluzole, a small molecule that may act via modulation of glutamatergic neurotransmission. However, riluzole only modestly extends lifespan for people living with ALS, and its precise mechanisms of action remain unclear. Most ALS cases are characterised by accumulation of cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43), and understanding the effects of riluzole in models that closely recapitulate TDP-43 pathology may provide insights for development of improved therapeutics. We therefore investigated the effects of riluzole in female transgenic mice that inducibly express nuclear localisation sequence (NLS)-deficient human TDP-43 in neurons (NEFH-tTA/tetO-hTDP-43ΔNLS, 'rNLS8', mice). Riluzole treatment from the first day of hTDP-43ΔNLS expression did not alter disease onset, weight loss or performance on multiple motor behavioural tasks. Riluzole treatment also did not alter TDP-43 protein levels, solubility or phosphorylation. Although we identified a significant decrease in GluA2 and GluA3 proteins in the cortex of rNLS8 mice, riluzole did not ameliorate this disease-associated molecular phenotype. Likewise, riluzole did not alter the disease-associated atrophy of hindlimb muscle in rNLS8 mice. Finally, riluzole treatment beginning after disease onset in rNLS8 mice similarly had no effect on progression of late-stage disease or animal survival. Together, we demonstrate specific glutamatergic receptor alterations and muscle fibre-type changes reminiscent of ALS in female rNLS8 mice, but riluzole had no effect on these or any other disease phenotypes. Future targeting of pathways related to accumulation of TDP-43 pathology may be needed to develop better treatments for ALS.

Workflow for Rapidly Extracting Biological Insights from Complex, Multicondition Proteomics Experiments with WGCNA and PloGO2.

We describe a useful workflow for characterizing proteomics experiments incorporating many conditions and abundance data using the popular weighted gene correlation network analysis (WGCNA) approach and functional annotation with the PloGO2 R package, the latter of which we have extended and made available to Bioconductor. The approach can use quantitative data from labeled or label-free experiments and was developed to handle multiple files stemming from data partition or multiple pairwise comparisons. The WGCNA approach can similarly produce a potentially large number of clusters of interest, which can also be functionally characterized using PloGO2. Enrichment analysis will identify clusters or subsets of proteins of interest, and the WGCNA network topology scores will produce a ranking of proteins within these clusters or subsets. This can naturally lead to prioritized proteins to be considered for further analysis or as candidates of interest for validation in the context of complex experiments. We demonstrate the use of the package on two published data sets using two different biological systems (plant and human plasma) and proteomics platforms (sequential window acquisition of all theoretical fragment-ion spectra (SWATH) and tandem mass tag (TMT)): an analysis of the effect of drought on rice over time generated using TMT and a pediatric plasma sample data set generated using SWATH. In both, the automated workflow recapitulates key insights or observations of the published papers and provides additional suggestions for further investigation. These findings indicate that the data set analysis using WGCNA combined with the updated PloGO2 package is a powerful method to gain biological insights from complex multifaceted proteomics experiments.

ERp57 is protective against mutant SOD1-induced cellular pathology in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder and mutations in superoxide dismutase 1 (SOD1) account for 20% of familial ALS cases. The aetiology of ALS remains unclear, but protein misfolding, endoplasmic reticulum (ER) stress and neuronal apoptosis are implicated. We previously established that protein disulphide isomerase (PDIA1) is protective against ER stress and apoptosis in neuronal cells expressing mutant SOD1, and recently mutations in PDIA1 and related PDI family member endoplasmic reticulum protein 57 (ERp57/PDIA3), were associated with ALS. Here, we examined whether ERp57 is also protective against mutant SOD1 or whether distinct specificity exists amongst individual PDI family members. Neuronal cells co-expressing SOD1 and ERp57 were examined for inclusion formation, ER stress, ubiquitin proteasome system (UPS) dysfunction and apoptosis. Over-expression of ERp57 inhibited inclusion formation, ER stress, UPS dysfunction and apoptosis, whereas silencing of ERp57 expression enhanced mutant SOD1 inclusion formation, ER stress and toxicity, indicating a protective role for ERp57 against SOD1 misfolding. ERp57 also inhibited the formation of mutant SOD1 inclusions and apoptosis in primary cortical neurons, thus confirming results obtained from cell lines. ERp57 partially co-localized with TAR DNA-binding protein-43 (TDP-43)-positive inclusions in spinal cords from sporadic ALS patients, thus linking ERp57 to protein misfolding in human sporadic disease. Our results therefore imply that ERp57 has a protective role against pathological events induced by mutant SOD1 and they link ERp57 to the misfolding of TDP-43. This study therefore has implications for the design of novel therapeutics based on the activities of the PDI family of proteins.

Aspirin and other non-steroidal anti-inflammatory drugs and depression, anxiety, and stress-related disorders following a cancer diagnosis: a nationwide register-based cohort study.

BACKGROUND: Cancer patients have a highly increased risk of psychiatric disorders following diagnosis, compared with cancer-free individuals. Inflammation is involved in the development of both cancer and psychiatric disorders. The role of non-steroidal anti-inflammatory drugs (NSAIDs) in the subsequent risk of psychiatric disorders after cancer diagnosis is however unknown. METHODS: We performed a cohort study of all patients diagnosed with a first primary malignancy between July 2006 and December 2013 in Sweden. Cox proportional hazards models were used to assess the association of NSAID use during the year before cancer diagnosis with the risk of depression, anxiety, and stress-related disorders during the first year after cancer diagnosis. RESULTS: Among 316,904 patients identified, 5613 patients received a diagnosis of depression, anxiety, or stress-related disorders during the year after cancer diagnosis. Compared with no use of NSAIDs, the use of aspirin alone was associated with a lower rate of depression, anxiety, and stress-related disorders (hazard ratio [HR], 0.88; 95% confidence interval [CI], 0.81 to 0.97), whereas the use of non-aspirin NSAIDs alone was associated with a higher rate (HR, 1.24; 95% CI, 1.15 to 1.32), after adjustment for sociodemographic factors, comorbidity, indications for NSAID use, and cancer characteristics. The association of aspirin with reduced rate of depression, anxiety, and stress-related disorders was strongest for current use (HR, 0.84; 95% CI, 0.75 to 0.93), low-dose use (HR, 0.88; 95% CI, 0.80 to 0.98), long-term use (HR, 0.84; 95% CI, 0.76 to 0.94), and among patients with cardiovascular disease (HR, 0.81; 95% CI, 0.68 to 0.95) or breast cancer (HR, 0.74; 95% CI, 0.56 to 0.98). CONCLUSION: Pre-diagnostic use of aspirin was associated with a decreased risk of depression, anxiety, and stress-related disorders during the first year following cancer diagnosis.

Nuclear factor kappa B activation appears weaker in schizophrenia patients with high brain cytokines than in non-schizophrenic controls with high brain cytokines.

BACKGROUND: High inflammation status despite an absence of known infection characterizes a subpopulation of people with schizophrenia who suffer from more severe cognitive deficits, less cortical grey matter, and worse neuropathology. Transcripts encoding factors upstream of nuclear factor kappa B (NF-κB), a major transcriptional activator for the synthesis of pro-inflammatory cytokines, are increased in the frontal cortex in schizophrenia compared to controls. However, the extent to which these changes are disease-specific, restricted to those with schizophrenia and high-neuroinflammatory status, or caused by loss of a key NF-κB inhibitor (HIVEP2) found in schizophrenia brain, has not been tested. METHODS: Post-mortem prefrontal cortex samples were assessed in 141 human brains (69 controls and 72 schizophrenia) and 13 brains of wild-type mice and mice lacking HIVEP2 (6 wild-type, 7 knockout mice). Gene expression of pro-inflammatory cytokines and acute phase protein SERPINA3 was used to categorize high and low neuroinflammation biotype groups in human samples via cluster analysis. Expression of 18 canonical and non-canonical NF-κB pathway genes was assessed by qPCR in human and mouse tissue. RESULTS: In humans, we found non-canonical upstream activators of NF-κB were generally elevated in individuals with neuroinflammation regardless of diagnosis, supporting NF-κB activation in both controls and people with schizophrenia when cytokine mRNAs are high. However, high neuroinflammation schizophrenia patients had weaker (or absent) transcriptional increases of several canonical upstream activators of NF-κB as compared to the high neuroinflammation controls. HIVEP2 mRNA reduction was specific to patients with schizophrenia who also had high neuroinflammatory status, and we also found decreases in NF-κB transcripts typically induced by activated microglia in mice lacking HIVEP2. CONCLUSIONS: Collectively, our results show that high cortical expression of pro-inflammatory cytokines and low cortical expression of HIVEP2 in a subset of people with schizophrenia is associated with a relatively weak NF-κB transcriptional signature compared to non-schizophrenic controls with high cytokine expression. We speculate that this comparatively milder NF-κB induction may reflect schizophrenia-specific suppression possibly related to HIVEP2 deficiency in the cortex.

Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis and frontotemporal dementia and transgenic TDP-43 mice.

OBJECTIVE: Since the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia. METHODS: Whole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology. RESULTS: No causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease. CONCLUSIONS: Genetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.

Regional, cellular and species difference of two key neuroinflammatory genes implicated in schizophrenia.

The transcription factor nuclear factor kappa B (NF-κB) regulates the expression of many inflammatory genes that are overexpressed in a subset of people with schizophrenia. Transcriptional reduction in one NF-κB inhibitor, Human Immunodeficiency Virus Enhancer Binding Protein 2 (HIVEP2), is found in the brain of patients, aligning with evidence of NF-κB over-activity. Cellular co-expression of HIVEP2 and cytokine transcripts is a prerequisite for a direct effect of HIVEP2 on pro-inflammatory transcription, and we do not know if changes in HIVEP2 and markers of neuroinflammation are occurring in the same brain cell type. We performed in situ hybridisation on postmortem dorsolateral prefrontal cortex tissue to map and compare the expression of HIVEP2 and Serpin Family A Member 3 (SERPINA3), one of the most consistently increased inflammatory genes in schizophrenia, between schizophrenia patients and controls. We find that HIVEP2 expression is neuronal and is decreased in almost all grey matter cortical layers in schizophrenia patients with neuroinflammation, and that SERPINA3 is increased in the dorsolateral prefrontal cortex grey matter and white matter in the same group of patients. We are the first to map the upregulation of SERPINA3 to astrocytes and to some neurons, and find evidence to suggest that blood vessel-associated astrocytes are the main cellular source of SERPINA3 in the schizophrenia cortex. We show that a lack of HIVEP2 in mice does not cause astrocytic upregulation of Serpina3n but does induce its transcription in neurons. We speculate that HIVEP2 downregulation is not a direct cause of astrocytic pro-inflammatory cytokine synthesis in schizophrenia but may contribute to neuronally-mediated neuroinflammation.

Leucine competes with kynurenine for blood-to-brain transport and prevents lipopolysaccharide-induced depression-like behavior in mice.

Inflammation activates indoleamine 2,3-dioxygenase (IDO) which metabolizes tryptophan into kynurenine. Circulating kynurenine is transported into the brain by the large amino transporter LAT1 at the level of the blood-brain barrier. We hypothesized that administration of leucine that has a high affinity for LAT1 should prevent the entry of kynurenine into the brain and attenuate the formation of neurotoxic kynurenine metabolites. To test whether leucine could prevent inflammation-induced depression-like behavior, mice were treated with lipopolysaccharide (LPS, 0.83 mg/kg IP) or saline and treated with L-leucine (50 mg/kg, IP) or vehicle administered before and 6 h after LPS. Depression-like behavior was measured by increased duration of immobility in the forced swim test and decreased sucrose preference. Leucine decreased brain kynurenine levels, blocked LPS-induced depression-like behavior and had antidepressant-like effects in control mice. Leucine had no effect of its own on sickness behavior and neuroinflammation. To confirm that leucine acts by interfering with the transport of kynurenine into the brain, mice were injected with L-leucine (300 mg/kg, IP) immediately before kynurenine (33 mg/kg IP) and brain kynurenine and depression-like behavior were measured 3 h later. Leucine did prevent the entry of exogenous kynurenine into the brain and abrogated depression-like behavior measured by increased duration of immobility in the forced swim test. Additional experiments using an in vitro model of the blood-brain barrier confirmed that kynurenine competes with leucine at the level of the amino acid transporter LAT1 for brain uptake. These experiments also revealed that efflux was the dominant direction of kynurenine transport and was largely independent of LAT1 and leucine, which explains why leucine could block brain uptake of kynurenine without affecting brain clearance. These findings demonstrate that leucine has antidepressant properties vis-à-vis inflammation-induced depression and one mechanism for this is by blocking the ability of kynurenine to enter the brain.