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.

Evidence for decreased copper associated with demyelination in the corpus callosum of cuprizone-treated mice.

Demyelination within the central nervous system (CNS) is a significant feature of debilitating neurological diseases such as multiple sclerosis and administering the copper-selective chelatorcuprizone to mice is widely used to model demyelination in vivo. Conspicuous demyelination within the corpus callosum is generally attributed to cuprizone's ability to restrict copper availability in this vulnerable brain region. However, the small number of studies that have assessed copper in brain tissue from cuprizone-treated mice have produced seemingly conflicting outcomes, leaving the role of CNS copper availability in demyelination unresolved. Herein we describe our assessment of copper concentrations in brain samples from mice treated with cuprizone for 40 d. Importantly, we applied an inductively coupled plasma mass spectrometry methodology that enabled assessment of copper partitioned into soluble and insoluble fractions within distinct brain regions, including the corpus callosum. Our results show that cuprizone-induced demyelination in the corpus callosum was associated with decreased soluble copper in this brain region. Insoluble copper in the corpus callosum was unaffected, as were pools of soluble and insoluble copper in other brain regions. Treatment with the blood-brain barrier permeant copper compound CuII(atsm) increased brain copper levels and this was most pronounced in the soluble fraction of the corpus callosum. This effect was associated with significant mitigation of cuprizone-induced demyelination. These results provide support for the involvement of decreased CNS copper availability in demyelination in the cuprizone model. Relevance to human demyelinating disease is discussed.

Fundamental Neurochemistry Review: Copper availability as a potential therapeutic target in progressive supranuclear palsy: Insight from other neurodegenerative diseases.

Since the first description of Parkinson's disease (PD) over two centuries ago, the recognition of rare types of atypical parkinsonism has introduced a spectrum of related PD-like diseases. Among these is progressive supranuclear palsy (PSP), a neurodegenerative condition that clinically differentiates through the presence of additional symptoms uncommon in PD. As with PD, the initial symptoms of PSP generally present in the sixth decade of life when the underpinning neurodegeneration is already significantly advanced. The causal trigger of neuronal cell loss in PSP is unknown and treatment options are consequently limited. However, converging lines of evidence from the distinct neurodegenerative conditions of PD and amyotrophic lateral sclerosis (ALS) are beginning to provide insights into potential commonalities in PSP pathology and opportunity for novel therapeutic intervention. These include accumulation of the high abundance cuproenzyme superoxide dismutase 1 (SOD1) in an aberrant copper-deficient state, associated evidence for altered availability of the essential micronutrient copper, and evidence for neuroprotection using compounds that can deliver available copper to the central nervous system. Herein, we discuss the existing evidence for SOD1 pathology and copper imbalance in PSP and speculate that treatments able to provide neuroprotection through manipulation of copper availability could be applicable to the treatment of PSP.

Transdermal Application of Soluble CuII(atsm) Increases Brain and Spinal Cord Uptake Compared to Gavage with an Insoluble Suspension.

CuII(atsm) is a blood-brain barrier permeant copper(II) compound that is under investigation in human clinical trials for the treatment of neurodegenerative diseases of the central nervous system (CNS). Imaging in humans by positron emission tomography shows the compound accumulates in affected regions of the CNS in patients. Most therapeutic studies to date have utilised oral administration of CuII(atsm) in an insoluble form, as either solid tablets or a liquid suspension. However, two pre-clinical studies have demonstrated disease-modifying outcomes following transdermal application of soluble CuII(atsm) prepared in dimethyl sulphoxide. Whether differences in the method of administration lead to different degrees of tissue accumulation of the compound has never been examined. Here, we compare the two methods of administration in wild-type mice by assessing changes in tissue concentrations of copper. Both administration methods resulted in elevated copper concentrations in numerous tissues, with the largest increases evident in the liver, brain and spinal cord. In all instances where treatment with CuII(atsm) resulted in elevated tissue copper, transdermal application of soluble CuII(atsm) led to higher concentrations of copper. In contrast to CuII(atsm), an equivalent dose of copper(II) chloride resulted in minimal changes to tissue copper concentrations, regardless of the administration method. Data presented herein provide quantitative insight to transdermal application of soluble CuII(atsm) as a potential alternative to oral administration of the compound in an insoluble formulation.

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 previously acquired 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.

Sex-dependent effects of amyloid precursor-like protein 2 in the SOD1-G37R transgenic mouse model of MND.

Motor neurone disease (MND) is a neurodegenerative disorder characterised by progressive destruction of motor neurons, muscle paralysis and death. The amyloid precursor protein (APP) is highly expressed in the central nervous system and has been shown to modulate disease outcomes in MND. APP is part of a gene family that includes the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) genes. In the present study, we investigated the role of APLP2 in MND through the examination of human spinal cord tissue and by crossing APLP2 knockout mice with the superoxide dismutase 1 (SOD1-G37R) transgenic mouse model of MND. We found the expression of APLP2 is elevated in the spinal cord from human cases of MND and that this feature of the human disease is reproduced in SOD1-G37R mice at the End-stage of their MND-like phenotype progression. APLP2 deletion in SOD1-G37R mice significantly delayed disease progression and increased the survival of female SOD1-G37R mice. Molecular and biochemical analysis showed female SOD1-G37R:APLP2-/- mice displayed improved innervation of the neuromuscular junction, ameliorated atrophy of muscle fibres with increased APP protein expression levels in the gastrocnemius muscle. These results indicate a sex-dependent role for APLP2 in mutant SOD1-mediated MND and further support the APP family as a potential target for further investigation into the cause and regulation of MND.

Copper-ATSM as a Treatment for ALS: Support from Mutant SOD1 Models and Beyond.

The blood-brain barrier permeant, copper-containing compound, CuII(atsm), has successfully progressed from fundamental research outcomes in the laboratory through to phase 2/3 clinical assessment in patients with the highly aggressive and fatal neurodegenerative condition of amyotrophic lateral sclerosis (ALS). The most compelling outcomes to date to indicate potential for disease-modification have come from pre-clinical studies utilising mouse models that involve transgenic expression of mutated superoxide dismutase 1 (SOD1). Mutant SOD1 mice provide a very robust mammalian model of ALS with high validity, but mutations in SOD1 account for only a small percentage of ALS cases in the clinic, with the preponderant amount of cases being sporadic and of unknown aetiology. As per other putative drugs for ALS developed and tested primarily in mutant SOD1 mice, this raises important questions about the pertinence of CuII(atsm) to broader clinical translation. This review highlights some of the challenges associated with the clinical translation of new treatment options for ALS. It then provides a brief account of pre-clinical outcomes for CuII(atsm) in SOD1 mouse models of ALS, followed by an outline of additional studies which report positive outcomes for CuII(atsm) when assessed in cell and mouse models of neurodegeneration which do not involve mutant SOD1. Clinical evidence for CuII(atsm) selectively targeting affected regions of the CNS in patients is also presented. Overall, this review summarises the existing evidence which indicates why clinical relevance of CuII(atsm) likely extends beyond the context of cases of ALS caused by mutant SOD1.