Mapping the degradation pathway of a disease-linked aspartoacylase variant

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.

Feline Spongy Encephalopathy With a Mutation in the ASPA Gene

Canavan disease is an autosomal recessive leukodystrophy caused by mutations in the gene encoding aspartoacylase (ASPA), which hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. A similar feline neurodegenerative disease associated with a mutation in the ASPA gene is reported herein. Comprehensive clinical, genetic, and pathological analyses were performed on 4 affected cats. Gait disturbance and head tremors initially appeared at 1 to 19 months of age. These cats eventually exhibited dysstasia and seizures and died at 7 to 53 months of age. Magnetic resonance imaging of the brain revealed diffuse symmetrical intensity change of the cerebral cortex, brainstem, and cerebellum. Gas chromatography–mass spectrometry analysis of urine showed significant excretion of NAA. Genetic analysis of the 4 affected cats identified a missense mutation (c.859G>C) in exon 6 of the ASPA gene, which was not detected in 4 neurologically intact cats examined as controls. Postmortem analysis revealed vacuolar changes predominantly distributed in the gray matter of the cerebrum and brain stem as well as in the cerebellar Purkinje cell layer. Immunohistochemically, these vacuoles were surrounded by neurofilaments and sometimes contained MBP- and Olig2-positive cells. Ultrastructurally, a large number of intracytoplasmic vacuoles containing mitochondria and electron-dense granules were detected in the cerebral cortex. All 4 cats were diagnosed as spongy encephalopathy with a mutation in the ASPA gene, a syndrome analogous to human Canavan disease. The histopathological findings suggest that feline ASPA deficiency induces intracytoplasmic edema in neurons and oligodendrocytes, resulting in spongy degeneration of the central nervous system.

Cobalt deficiency in cattle and its impact on production

ABSTRACT: Two outbreaks of cobalt deficiency in beef cattle were diagnosed in Midwestern Brazil. We discuss the clinical, epidemiological, pathological features, therapeutic measures, and impact aspects of the production system associated with these outbreaks occurring outbreaks in two farms of extensive cattle raising-system in the state of Mato Grosso do Sul. Seven affected cattle were euthanized and necropsied. Tissues for histopathology and microelements dosage were secured. At Farm A, 3100 cattle of all ages got sick, and 396 died; at Farm B, 148 were affected, and 110 died. In both farms, cattle were fed the same mineral supplement. The main clinical signs were weight loss and weakness, even though a good supply of forage was available in the paddocks. Many cattle stop grazing and chew at tree barks, wood chips from fence posts, and bones. In addition to the deaths, there was a compromised growth, and the reproductive rates fell sharply. The necropsied cattle were thin, with rough hair coat and pale mucous membranes. The liver was diffusely orange and showed a lobular pattern. The bone marrow was gelatinous and diffusely yellow. Histological changes included hemosiderosis in the liver and spleen, hepatocellular vacuolar degeneration, and myeloid and erythroid hypoplasia of the bone marrow. In the white matter of four cattle’s brains, the myelin sheath was markedly distended (spongy degeneration). Proliferative parasitic abomasitis was observed in three cattle. The presumptive diagnosis was based on the association of the clinical picture, the necropsy findings, and the ruling out of other possible causes. The diagnosis was confirmed by the favorable response to treatment with cobalt and vitamin B12 orally and by mineral supplementation.

Evolutionarily conserved chaperone-mediated proteasomal degradation of a disease-linked aspartoacylase variant

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we examine the stability and degradation pathway of ASPA C152W. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution prevents ASPA from reaching its stable native conformation, presumably by impacting on de novo folding. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. We propose that Hsp110 is a potential therapeutic target for misfolding ASPA variants that trigger Canavan disease due to excessive degradation.

A Missense Variant in SCN8A in Alpine Dachsbracke Dogs Affected by Spinocerebellar Ataxia

Spinocerebellar ataxias is an umbrella term for clinically- and neuropathologically-heterogeneous early-onset hereditary neurodegenerative diseases affecting several dog breeds. The purpose of this study is to identify the causative genetic variant associated with ataxia, tremor, and loss of balance in Alpine Dachsbracke dogs. We investigated two related litters in which four cases were reported. Neuropathology of two dogs revealed spongy degeneration associated with axonal degeneration. Combined genetic linkage and autozygosity analyses in four cases and eight related controls showed one critical disease-associated interval on chromosomes 27. Private whole-genome sequence variants of one ataxia case against 600 unrelated controls revealed one protein-changing variant within the critical interval in the SCN8A gene (c.4898G>T; p.Gly1633Val). Perfect segregation with the phenotype was confirmed by genotyping >200 Alpine Dachsbracke dogs. SCN8A encodes a voltage-gated sodium channel and the missense variant was predicted deleterious by three different in silico prediction tools. Pathogenic variants in SCN8A were previously reported in humans with ataxia, pancerebellar atrophy, and cognitive disability. Furthermore, cerebellar ataxia syndrome in the ‘jolting’ mutant mice is caused by a missense variant in Scn8a. Therefore, we considered the SCN8A:c.4898G>T variant to be the most likely cause for recessively inherited spinocerebellar ataxia in Alpine Dachsbracke dogs.

Correlation of MRI with the Neuropathologic Changes in Two Cats with Bromethalin Intoxication

ABSTRACT Two cats were presented with multifocal neurological signs. One cat’s signs progressed over 2 wk; the other cat progressed over 5 days. Examinations were consistent with a process involving the prosencephalon, vestibular system, and general proprioceptive/upper motor neuron systems. MRI of the brain and cervical spinal cord reveal widespread T2 hyperintensity of the white matter. Affected areas included the cerebrum, cerebral peduncles, corticospinal tracts of the pons and medulla, and the cerebellum. T2 hyperintensity was present in all funiculi of the spinal cord. Diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) maps were consistent with cytotoxic or intramyelinic edema. Differential diagnosis included toxic or metabolic/degenerative leukoencephalopathies. Necropsies revealed widespread spongy degeneration of the central nervous system white matter. Toxicologic assays of liver specimens revealed desmethylbromethalin, a metabolite of bromethalin. Bromethalin is a rodenticide that causes uncoupling of oxidative phosphorylation. Antemortem diagnosis is challenging. DWI and ADC maps were instrumental in narrowing the differential diagnosis and raised the index of suspicion for bromethalin. Bromethalin intoxication should be considered in all animals with a progressive course of multifocal neurologic deficits. MRI, specifically, DWI and ADC maps, may serve as a biomarker of cytotoxic or intramyelinic edema associated with spongiform leukoencephalomyelopathy.