Integrated elemental analysis supports targeting copper perturbations as a therapeutic strategy in multiple sclerosis.

Multiple sclerosis (MS) is a debilitating affliction of the central nervous system (CNS) that involves demyelination of neuronal axons and neurodegeneration resulting in disability that becomes more pronounced in progressive forms of the disease. The involvement of neurodegeneration in MS underscores the need for effective neuroprotective approaches necessitating identification of new therapeutic targets. Herein, we applied an integrated elemental analysis workflow to human MS-affected spinal cord tissue utilising multiple inductively coupled plasma-mass spectrometry methodologies. These analyses revealed shifts in atomic copper as a notable aspect of disease. Complementary gene expression and biochemical analyses demonstrated that changes in copper levels coincided with altered expression of copper handling genes and downstream functionality of cuproenzymes. Copper-related problems observed in the human MS spinal cord were largely reproduced in the experimental autoimmune encephalomyelitis (EAE) mouse model during the acute phase of disease characterised by axonal demyelination, lesion formation, and motor neuron loss. Treatment of EAE mice with the CNS-permeant copper modulating compound CuII(atsm) resulted in recovery of cuproenzyme function, improved myelination and lesion volume, and neuroprotection. These findings support targeting copper perturbations as a therapeutic strategy for MS with CuII(atsm) showing initial promise.

A CCG expansion in ABCD3 causes oculopharyngodistal myopathy in individuals of European ancestry.

Oculopharyngodistal myopathy (OPDM) is an inherited myopathy manifesting with ptosis, dysphagia and distal weakness. Pathologically it is characterised by rimmed vacuoles and intranuclear inclusions on muscle biopsy. In recent years CGG • CCG repeat expansion in four different genes were identified in OPDM individuals in Asian populations. None of these have been found in affected individuals of non-Asian ancestry. In this study we describe the identification of CCG expansions in ABCD3, ranging from 118 to 694 repeats, in 35 affected individuals across eight unrelated OPDM families of European ancestry. ABCD3 transcript appears upregulated in fibroblasts and skeletal muscle from OPDM individuals, suggesting a potential role of over-expression of CCG repeat containing ABCD3 transcript in progressive skeletal muscle degeneration. The study provides further evidence of the role of non-coding repeat expansions in unsolved neuromuscular diseases and strengthens the association between the CGG • CCG repeat motif and a specific pattern of muscle weakness.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Ectopic HCN4 Provides a Target Biomarker for the Genetic Spectrum of mTORopathies.

Background and Objectives: Pathogenic variants in PI3K-AKT-mTOR pathway and GATOR1 complex genes resulting in hyperactivation of mechanistic target of rapamycin (mTOR) complex 1 are a major cause of drug-resistant epilepsy and focal cortical malformations (FCM). Resective neurosurgery is often required to achieve seizure control in patients with mTORopathies due to lack of effectiveness of nonsurgical therapies, including antiseizure medication and mTOR inhibitors. Elevated hyperpolarization-activated cyclic nucleotide-gated potassium channel isoform 4 (HCN4) has been proposed as a key marker in some mTOR-related brain malformations. This study aimed to investigate HCN4 as a biomarker in the brain across the genetic spectrum of mTORopathies in humans. Methods: Our study investigated the relative steady-state levels and cellular localization of HCN4 in resected human brain tissue from 18 individuals with mTORopathies (3 individuals with tuberous sclerosis complex (TSC) due to TSC2 variants, 5 individuals with focal cortical dysplasia type IIA (FCD IIA) due to genetic variants in MTOR, AKT3, and PIK3CA, and 10 individuals with FCD IIB due to variants in TSC1, MTOR, RHEB, DEPDC5, or NPRL3). Results: Elevated HCN4 was observed to be highly restricted to abnormal cell types (dysmorphic neurons and balloon cells) in brain tissue from all mTORopathy tissues (p < 0.0001) compared with those in controls, regardless of genetic cause or variant allele frequency. Elevated HCN4 was not observed in controls or individuals with non-mTOR-related focal epilepsy due to pathogenic variants in ATP1A3, SLC35A2, or FGFR1. Discussion: HCN4 provides a biomarker for the genetic spectrum of mTORopathies and may present a potential therapeutic target for seizure control in mTOR-related epilepsy.

Chronic traumatic encephalopathy neuropathologic change in former Australian rugby players.

AIMS: We applied the 2021 consensus criteria for both chronic traumatic encephalopathy neuropathological change and traumatic encephalopathy syndrome in a small case series of six former elite-level Australian rugby code players. METHODS: Neuropathological assessment of these cases was carried out at the Sydney and Victorian Brain Banks. Clinical data were collected via clinical interviews and health questionnaires completed by the participants and/or their next of kin, and neuropsychological testing was conducted with participants who were capable of completing this testing. RESULTS: All cases exhibited progressive cognitive impairment during life. Chronic traumatic encephalopathy neuropathological change was identified in four out of the six cases. However, coexisting neuropathologies were common, with limbic-predominant age-related TDP-43 encephalopathy and ageing-related tau astrogliopathy seen in all cases, intermediate or high Alzheimer's disease neuropathological change seen in four cases and hippocampal sclerosis seen in two of the six cases. CONCLUSION: The presence of multiple neuropathologies in these cases complicates clinical diagnostic efforts for traumatic encephalopathy syndrome. It will be important for further clinicopathological studies on larger groups to report all neuropathological comorbidities found in cases diagnosed with either chronic traumatic encephalopathy neuropathological change and/or traumatic encephalopathy syndrome.

Evidence for disrupted copper availability in human spinal cord supports CuII(atsm) as a treatment option for sporadic cases of ALS.

The copper compound CuII(atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). CuII(atsm) is neuroprotective in mutant SOD1 mouse models of ALS where its activity is ascribed in part to improving availability of essential copper. However, SOD1 mutations cause only ~ 2% of ALS cases and therapeutic relevance of copper availability in sporadic ALS is unresolved. Herein we assessed spinal cord tissue from human cases of sporadic ALS for copper-related changes. We found that when compared to control cases the natural distribution of spinal cord copper was disrupted in sporadic ALS. A standout feature was decreased copper levels in the ventral grey matter, the primary anatomical site of neuronal loss in ALS. Altered expression of genes involved in copper handling indicated disrupted copper availability, and this was evident in decreased copper-dependent ferroxidase activity despite increased abundance of the ferroxidases ceruloplasmin and hephaestin. Mice expressing mutant SOD1 recapitulate salient features of ALS and the unsatiated requirement for copper in these mice is a biochemical target for CuII(atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for CuII(atsm) involving copper availability may also be pertinent to sporadic cases of ALS.

Microglial ferroptotic stress causes non-cell autonomous neuronal death.

BACKGROUND: Ferroptosis is a form of regulated cell death characterised by lipid peroxidation as the terminal endpoint and a requirement for iron. Although it protects against cancer and infection, ferroptosis is also implicated in causing neuronal death in degenerative diseases of the central nervous system (CNS). The precise role for ferroptosis in causing neuronal death is yet to be fully resolved. METHODS: To elucidate the role of ferroptosis in neuronal death we utilised co-culture and conditioned medium transfer experiments involving microglia, astrocytes and neurones. We ratified clinical significance of our cell culture findings via assessment of human CNS tissue from cases of the fatal, paralysing neurodegenerative condition of amyotrophic lateral sclerosis (ALS). We utilised the SOD1G37R mouse model of ALS and a CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo. RESULTS: We found that sublethal ferroptotic stress selectively affecting microglia triggers an inflammatory cascade that results in non-cell autonomous neuronal death. Central to this cascade is the conversion of astrocytes to a neurotoxic state. We show that spinal cord tissue from human cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Further, we show the molecular correlation between ferroptosis and neurotoxic astrocytes evident in human ALS-affected spinal cord is recapitulated in the SOD1G37R mouse model where treatment with a CNS-permeant ferroptosis inhibitor, CuII(atsm), ameliorated these markers and was neuroprotective. CONCLUSIONS: By showing that microglia responding to sublethal ferroptotic stress culminates in non-cell autonomous neuronal death, our results implicate microglial ferroptotic stress as a rectifiable cause of neuronal death in neurodegenerative disease. As ferroptosis is currently primarily regarded as an intrinsic cell death phenomenon, these results introduce an entirely new pathophysiological role for ferroptosis in disease.

Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Acute leg pain and weakness in pregnancy: A new diagnosis of myotonic dystrophy.

We present a unique case of a 44-year-old woman who presented at 29 weeks' gestation with proximal limb pain and elevated creatine kinase. This occurred in the background of premature cataracts, atrial fibrillation and abnormal liver function. Clinical, pathological and neurodiagnostic findings supported a diagnosis of myotonic dystrophy, confirmed by genetic testing which revealed dystrophia myotonica protein kinase gene expansion. Muscle biopsy found both recent necrotising and chronic myopathic processes. Following delivery, the mother's myalgia resolved and creatine kinase quickly declined. The fetus was diagnosed with congenital myotonic dystrophy. We review the impact of myotonic dystrophy on pregnancy and discuss potential explanations for this patient's clinical course. This case emphasises the importance of considering myotonic dystrophy as a differential diagnosis in the right clinical context and the need for pre-pregnancy assessment and genetic counselling in women with known myotonic dystrophy.

Mitochondria- and NOX4-dependent antioxidant defense mitigates progression to nonalcoholic steatohepatitis in obesity.

Nonalcoholic fatty liver disease (NAFLD) is prevalent in the majority of individuals with obesity, but in a subset of these individuals, it progresses to nonalcoholic steatohepatitis (0NASH) and fibrosis. The mechanisms that prevent NASH and fibrosis in the majority of patients with NAFLD remain unclear. Here, we report that NAD(P)H oxidase 4 (NOX4) and nuclear factor erythroid 2-related factor 2 (NFE2L2) were elevated in hepatocytes early in disease progression to prevent NASH and fibrosis. Mitochondria-derived ROS activated NFE2L2 to induce the expression of NOX4, which in turn generated H2O2 to exacerbate the NFE2L2 antioxidant defense response. The deletion or inhibition of NOX4 in hepatocytes decreased ROS and attenuated antioxidant defense to promote mitochondrial oxidative stress, damage proteins and lipids, diminish insulin signaling, and promote cell death upon oxidant challenge. Hepatocyte NOX4 deletion in high-fat diet-fed obese mice, which otherwise develop steatosis, but not NASH, resulted in hepatic oxidative damage, inflammation, and T cell recruitment to drive NASH and fibrosis, whereas NOX4 overexpression tempered the development of NASH and fibrosis in mice fed a NASH-promoting diet. Thus, mitochondria- and NOX4-derived ROS function in concert to drive a NFE2L2 antioxidant defense response to attenuate oxidative liver damage and progression to NASH and fibrosis in obesity.

Creutzfeldt-Jakob disease surveillance in Australia: update to 31 December 2022.

Creutzfeldt-Jakob disease surveillance in Australia: update to 31 December 2022: Nationwide surveillance of Creutzfeldt-Jakob disease (CJD) and other human prion diseases is performed by the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR). National surveillance encompasses the period since 1 January 1970, with prospective surveillance occurring from 1 October 1993. Over this prospective surveillance period, considerable developments have occurred in pre-mortem diagnostics; in the delineation of new disease subtypes; and in a heightened awareness of prion diseases in healthcare settings. Surveillance practices of the ANCJDR have evolved and adapted accordingly. This report summarises the activities of the ANCJDR during 2022. Since the ANCJDR began offering diagnostic cerebrospinal fluid (CSF) 14-3-3 protein testing in Australia in September 1997, the annual number of referrals has steadily increased. In 2022, a total of 599 domestic CSF specimens were referred for diagnostic testing and 79 persons with suspected human prion disease were formally added to the national register. As of 31 December 2022, just under half of the 79 suspect case notifications (36/79) remain classified as 'incomplete'; 15 cases were classified as 'definite' and 23 as 'probable' prion disease; five cases were excluded through neuropathological examination. For 2022, fifty-five percent of all suspected human-prion-disease-related deaths in Australia underwent neuropathological examination. No cases of variant or iatrogenic CJD were identified. The SARS-CoV-2 pandemic did not affect prion disease surveillance outcomes in Australia during 2022.

A peptide-centric quantitative proteomics dataset for the phenotypic assessment of Alzheimer's disease.

Alzheimer's disease (AD) is a looming public health disaster with limited interventions. Alzheimer's is a complex disease that can present with or without causative mutations and can be accompanied by a range of age-related comorbidities. This diverse presentation makes it difficult to study molecular changes specific to AD. To better understand the molecular signatures of disease we constructed a unique human brain sample cohort inclusive of autosomal dominant AD dementia (ADD), sporadic ADD, and those without dementia but with high AD histopathologic burden, and cognitively normal individuals with no/minimal AD histopathologic burden. All samples are clinically well characterized, and brain tissue was preserved postmortem by rapid autopsy. Samples from four brain regions were processed and analyzed by data-independent acquisition LC-MS/MS. Here we present a high-quality quantitative dataset at the peptide and protein level for each brain region. Multiple internal and external control strategies were included in this experiment to ensure data quality. All data are deposited in the ProteomeXchange repositories and available from each step of our processing.

Pre-targeting amyloid-ß with antibodies for potential molecular imaging of Alzheimer's disease.

With the aim of developing the concept of pretargeted click chemistry for the diagnosis of Alzheimer's disease two antibodies specific for amyloid-ß were modified to incorporate trans-cyclooctene functional groups. Two bis(thiosemicarbazone) compounds with pendant 1,2,4,5-tetrazine functional groups were prepared and radiolabelled with positron emitting copper-64. The new copper-64 complexes rapidly react with the trans-cyclooctene functionalized antibodies in a bioorthogonal click reaction and cross the blood-brain barrier in mice.

Severe NAD(P)HX Dehydratase (NAXD) Neurometabolic Syndrome May Present in Adulthood after Mild Head Trauma.

We have previously reported that pathogenic variants in a key metabolite repair enzyme NAXD cause a lethal neurodegenerative condition triggered by episodes of fever in young children. However, the clinical and genetic spectrum of NAXD deficiency is broadening as our understanding of the disease expands and as more cases are identified. Here, we report the oldest known individual succumbing to NAXD-related neurometabolic crisis, at 32 years of age. The clinical deterioration and demise of this individual were likely triggered by mild head trauma. This patient had a novel homozygous NAXD variant [NM_001242882.1:c.441+3A>G:p.?] that induces the mis-splicing of the majority of NAXD transcripts, leaving only trace levels of canonically spliced NAXD mRNA, and protein levels below the detection threshold by proteomic analysis. Accumulation of damaged NADH, the substrate of NAXD, could be detected in the fibroblasts of the patient. In agreement with prior anecdotal reports in paediatric patients, niacin-based treatment also partly alleviated some clinical symptoms in this adult patient. The present study extends our understanding of NAXD deficiency by uncovering shared mitochondrial proteomic signatures between the adult and our previously reported paediatric NAXD cases, with reduced levels of respiratory complexes I and IV as well as the mitoribosome, and the upregulation of mitochondrial apoptotic pathways. Importantly, we highlight that head trauma in adults, in addition to paediatric fever or illness, may precipitate neurometabolic crises associated with pathogenic NAXD variants.

GM-CSF antibodies in artificial stone associated silicoproteinosis: A case report and literature review.

Pulmonary alveolar proteinosis (PAP) is a rare lung disease where there is accumulation of surfactant in the alveoli. It can be classified based on the underlying aetiology into three categories: primary, secondary and congenital. Autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF-Ab) are a key diagnostic feature of autoimmune PAP. High intensity occupational exposure and inhalation of toxic particles such as silica can cause a form of secondary PAP called acute silicoproteinosis. We describe a 26-year-old stone benchtop fabricator with silicoproteinosis following daily exposure to high levels of silica who had elevated serum GM-CSF-Ab. We discuss the role of GM-CSF-Ab in cases of PAP with occupational inhalational exposure and the challenges in its interpretation.

Cardiovascular Magnetic Resonance for Rejection Surveillance After Cardiac Transplantation.

BACKGROUND: Endomyocardial biopsy (EMB) is the gold standard method for surveillance of acute cardiac allograft rejection (ACAR) despite its invasive nature. Cardiovascular magnetic resonance (CMR)-based myocardial tissue characterization allows detection of myocarditis. The feasibility of CMR-based surveillance for ACAR-induced myocarditis in the first year after heart transplantation is currently undescribed. METHODS: CMR-based multiparametric mapping was initially assessed in a prospective cross-sectional fashion to establish agreement between CMR- and EMB-based ACAR and to determine CMR cutoff values between rejection grades. A prospective randomized noninferiority pilot study was then undertaken in adult orthotopic heart transplant recipients who were randomized at 4 weeks after orthotopic heart transplantation to either CMR- or EMB-based rejection surveillance. Clinical end points were assessed at 52 weeks. RESULTS: Four hundred one CMR studies and 354 EMB procedures were performed in 106 participants. Forty heart transplant recipients were randomized. CMR-based multiparametric assessment was highly reproducible and reliable at detecting ACAR (area under the curve, 0.92; sensitivity, 93%; specificity, 92%; negative predictive value, 99%) with greater specificity and negative predictive value than either T1 or T2 parametric CMR mapping alone. High-grade rejection occurred in similar numbers of patients in each randomized group (CMR, n=7; EMB, n=8; P=0.74). Despite similarities in immunosuppression requirements, kidney function, and mortality between groups, the rates of hospitalization (9 of 20 [45%] versus 18 of 20 [90%]; odds ratio, 0.091; P=0.006) and infection (7 of 20 [35%] versus 14 of 20 [70%]; odds ratio, 0.192; P=0,019) were lower in the CMR group. On 15 occasions (6%), patients who were randomized to the CMR arm underwent EMB for clarification or logistic reasons, representing a 94% reduction in the requirement for EMB-based surveillance. CONCLUSIONS: A noninvasive CMR-based surveillance strategy for ACAR in the first year after orthotopic heart transplantation is feasible compared with EMB-based surveillance. REGISTRATION: HREC/13/SVH/66 and HREC/17/SVH/80. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12618000672257.

Type 2 immune polarization is associated with cardiopulmonary disease in preterm infants.

Postnatal maturation of the immune system is poorly understood, as is its impact on illnesses afflicting term or preterm infants, such as bronchopulmonary dysplasia (BPD) and BPD-associated pulmonary hypertension. These are both cardiopulmonary inflammatory diseases that cause substantial mortality and morbidity with high treatment costs. Here, we characterized blood samples collected from 51 preterm infants longitudinally at five time points, 20 healthy term infants at birth and age 3 to 16 weeks, and 5 healthy adults. We observed strong associations between type 2 immune polarization in circulating CD3+CD4+ T cells and cardiopulmonary illness, with odds ratios up to 24. Maternal magnesium sulfate therapy, delayed hepatitis B vaccination, and increasing fetal, but not maternal, chorioamnionitis severity were associated with attenuated type 2 polarization. Blocking type 2 mediators such as interleukin-4 (IL-4), IL-5, IL-13, or signal transducer and activator of transcription 6 (STAT6) in murine neonatal cardiopulmonary disease in vivo prevented changes in cell type composition, increases in IL-1ß and IL-13, and losses of pulmonary capillaries, but not gains in larger vessels. Thereby, type 2 blockade ameliorated lung inflammation, protected alveolar and vascular integrity, and confirmed the pathological impact of type 2 cytokines and STAT6. In-depth flow cytometry and single-cell transcriptomics of mouse lungs further revealed complex associations between immune polarization and cardiopulmonary disease. Thus, this work advances knowledge on developmental immunology and its impact on early life disease and identifies multiple therapeutic approaches that may relieve inflammation-driven suffering in the youngest patients.

Does Data-Independent Acquisition Data Contain Hidden Gems? A Case Study Related to Alzheimer's Disease.

One of the potential benefits of using data-independent acquisition (DIA) proteomics protocols is that information not originally targeted by the study may be present and discovered by subsequent analysis. Herein, we reanalyzed DIA data originally recorded for global proteomic analysis to look for isomerized peptides, which occur as a result of spontaneous chemical modifications to long-lived proteins. Examination of a large set of human brain samples revealed a striking relationship between Alzheimer's disease (AD) status and isomerization of aspartic acid in a peptide from tau. Relative to controls, a surprising increase in isomer abundance was found in both autosomal dominant and sporadic AD samples. To explore potential mechanisms that might account for these observations, quantitative analysis of proteins related to isomerization repair and autophagy was performed. Differences consistent with reduced autophagic flux in AD-related samples relative to controls were found for numerous proteins, including most notably p62, a recognized indicator of autophagic inhibition. These results suggest, but do not conclusively demonstrate, that lower autophagic flux may be strongly associated with loss of function in AD brains. This study illustrates that DIA data may contain unforeseen results of interest and may be particularly useful for pilot studies investigating new research directions. In this case, a promising target for future investigations into the therapy and prevention of AD has been identified.