Biology subjects

Crouch, P. J.

Publications and source records attributed to Crouch, P. J..

5 recordsLinked to original sources

bioRxiv2025

The structure, redox chemistry and motor neuron toxicity of heterodimeric zinc-deficient SOD1-Implications for the toxic gain of function observed in ALS.

A subset of familial cases of amyotrophic lateral sclerosis (fALS) are caused by mutations to copper, zinc superoxide dismutase (Cu, Zn SOD1). There are over 200 mutations to SOD1 that have been associated with fALS and the majority of these mutations are dominantly inherited. Thus, individuals are heterozygous and express both wild-type SOD1 and the mutant form of the protein. Paradoxically, when rodent models are produced that mimic the co-expression of wild-type SOD1 with mutant fALS SOD1 the motor neuron disease accelerates. Previously, we have shown that the loss of zinc from an SOD1 kills cultured motor neurons due to a gained, redox activity catalyzed by the active-site copper. Furthermore, motor neuron toxicity of zinc-deficient SOD1 is enhanced by wild-type Cu, Zn SOD1. Because SOD1 exists as a non-covalent dimer, the enhanced toxicity might result from stabilization of the heterodimeric interface between zinc-deficient SOD1 and Cu, Zn-SOD1. However, experimentation with the heterodimer is difficult because SOD1 subunits exchange in minutes. To better characterize the role of dimer stabilization on the enhanced toxicity of fALS mutant SOD1 by wild type SOD1, we genetically tethered a zinc-deficient SOD1 subunit with a Cu, Zn SOD1 subunit with a 16-residue linker. The x-ray structure of the tethered heterodimer shows that zinc-deficient subunit adopts a wild-type-like conformation and is not misfolded. The heterodimer intermediate also produced peroxynitrite from nitric oxide, and the tethered SOD1 was strikingly toxic to primary cultures of motor neurons. This work supports the concept that zinc-deficient SOD1 is a likely toxic intermediate in ALS. Furthermore, the wild-type allele in human familial-SOD1 ALS patients may physically contribute to the dominant inheritance of SOD1 mutations through heterodimer formation.

biochemistry↗

bioRxiv2024

Impaired maturation of wild-type superoxide dismutase 1 associated with neurodegeneration in Parkinson disease brain and a novel murine model

Atypical wild-type superoxide dismutase 1 (SOD1) protein misfolding and deposition occurs specifically within the degenerating substantia nigra pars compacta (SNc) in Parkinson disease. Mechanisms driving the formation of this pathology and relationship with SNc dopamine neuron health, are yet to be fully understood. We applied proteomic mass spectrometry and synchrotron-based biometal quantification to post-mortem brain tissues from the SNc of Parkinson disease patients and age-matched controls to uncover key factors underlying the formation of wild-type SOD1 pathology in this disorder. We then engineered brain copper deficiency and upregulated SOD1 protein levels in a novel mouse strain, termed the SOCK mouse, to verify their involvement in the development of Parkinson-like wild-type SOD1 pathology and their impact on dopamine neuron health. Soluble SOD1 protein in the degenerating Parkinson disease SNc exhibited altered post-translational modifications, which may underlie changes to the enzymatic activity and aggregation of the protein in this region. These include decreased copper binding, dysregulation of physiological glycosylation, and atypical oxidation and glycation of key SOD1 amino acid residues. We demonstrated that the biochemical profile introduced in SOCK mice promotes the same post-translational modifications and the development of Parkinson-like wild-type SOD1 pathology in the midbrain and cortex. This pathology accumulates progressively with age and is accompanied by nigrostriatal degeneration and dysfunction, which occur in the absence of -synuclein deposition. These mice do not exhibit weight loss nor spinal cord motor neuron degeneration, distinguishing them from transgenic mutant SOD1 mouse models. This study provides the first in vivo evidence that mismetallation and altered post-translational modifications precipitates wild-type SOD1 misfolding, dysfunction and deposition in the Parkinson disease brain, which may contribute to SNc dopamine neuron degeneration. Our data position this pathology as a novel drug target for this disorder, with a particular focus on therapies capable of correcting alterations to SOD1 post-translational modifications.

neuroscience↗

bioRxiv2024

Characterization of the skeletal muscle arginine methylome in health and disease reveals remodeling in Amyotrophic Lateral Sclerosis

Arginine methylation is a protein post-translational modification important for the development of skeletal muscle mass and function. Despite this, our understanding of the regulation of arginine methylation under settings of health and disease remains largely undefined. Here, we investigated the regulation of arginine methylation in skeletal muscles in response to exercise and hypertrophic growth, and in diseases involving metabolic dysfunction and atrophy. We report a limited regulation of arginine methylation under physiological settings that promote muscle health, such as during growth and acute exercise, nor in disease models of insulin resistance. In contrast, we saw a significant remodeling of asymmetric dimethylation in models of atrophy characterized by the loss of innervation, including in muscle biopsies from patients with amyotrophic lateral sclerosis (ALS). Mass spectrometry-based quantification of the proteome and asymmetric arginine dimethylome of skeletal muscle from individuals with ALS revealed the largest compendium of protein changes with the identification of 793 regulated proteins, and novel site-specific changes in asymmetric dimethyl arginine (aDMA) of key sarcomeric and cytoskeletal proteins. Finally, we show that in vivo overexpression of PRMT1 and aDMA resulted in increased fatigue resistance and functional recovery in mice. Our study provides evidence for asymmetric dimethylation as a regulator of muscle pathophysiology and presents a valuable proteomics resource and rationale for numerous methylated and non-methylated proteins, including PRMT1, to be pursued for therapeutic development in ALS.

physiology↗

bioRxiv2022

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

systems biology↗

bioRxiv2022

Microglial ferroptotic stress causes non-cell autonomous neuronal death

BackgroundFerroptosis 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. MethodsTo 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). Finally, we utilised the SOD1G37R mouse model of ALS and a novel CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo. ResultsWe 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 cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Moreover, a molecular correlation between ferroptosis and neurotoxic astrocytes evident in ALS-affected spinal cord is recapitulated in the SOD1G37R mouse model where treatment with the novel, CNS-permeant ferroptosis inhibitor, CuII(atsm), ameliorated these markers and was neuroprotective. ConclusionsBy 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.

neuroscience↗