Circadian clock gene aryl hydrocarbon receptor nuclear translocator-like polymorphisms are associated with seasonal affective disorder: An Indian family study.

BACKGROUND AND AIM: Polymorphisms in aryl hydrocarbon receptor nuclear translocator-like (ARNTL) gene, the key component of circadian clock manifests circadian rhythm abnormalities. As seasonal affective disorder (SAD) is associated with disrupted circadian rhythms, the main objective of this study was to screen an Indian family with SAD for ARNTL gene polymorphisms. MATERIALS AND METHODS: In this study, 30 members of close-knit family with SAD, 30 age- and sex-matched controls of the same caste with no prior history of psychiatric illness and 30 age- and sex-matched controls belonging to 17 different castes with no prior history of psychiatric illness were genotyped for five different single nucleotide polymorphisms (SNPs) in ARNTL gene by TaqMan allele-specific genotyping assay. STATISTICAL ANALYSIS: Statistical significance was assessed by more powerful quasi-likelihood score test-XM. RESULTS: Most of the family members carried the risk alleles and we observed a highly significant SNP rs2279287 (A/G) in ARNTL gene with an allelic frequency of 0.75. CONCLUSIONS: Polymorphisms in ARNTL gene disrupt circadian rhythms causing SAD and genetic predisposition becomes more deleterious in the presence of adverse environment.

Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology.

Cu, Zn superoxide dismutase (SOD1) has been detected within spinal cord mitochondria of mutant SOD1 transgenic mice, a model of familial ALS. The copper chaperone for SOD1 (CCS) provides SOD1 with copper, facilitates the conversion of immature apo-SOD1 to a mature holoform, and influences in yeast the cytosolic/mitochondrial partitioning of SOD1. To determine how CCS affects G93A-SOD1-induced disease, we generated transgenic mice overexpressing CCS and crossed them to G93A-SOD1 or wild-type SOD1 transgenic mice. Both CCS transgenic mice and CCS/wild-type-SOD1 dual transgenic mice are neurologically normal. In contrast, CCS/G93A-SOD1 dual transgenic mice develop accelerated neurological deficits, with a mean survival of 36 days, compared with 242 days for G93A-SOD1 mice. Immuno-EM and subcellular fractionation studies on the spinal cord show that G93A-SOD1 is enriched within mitochondria in the presence of CCS overexpression. Our results indicate that CCS overexpression in G93A-SOD1 mice produces severe mitochondrial pathology and accelerates disease course.

Novel mutations that enhance or repress the aggregation potential of SOD1.

Mutations in SOD1 cause FALS by a gain of function likely related to protein misfolding and aggregation. SOD1 mutations encompass virtually every domain of the molecule, making it difficult to identify motifs important in SOD1 aggregation. Zinc binding to SOD1 is important for structural integrity, and is hypothesized to play a role in mutant SOD1 aggregation. To address this question, we mutated the unique zinc binding sites of SOD1 and examined whether these changes would influence SOD1 aggregation. We generated single and multiple mutations in SOD1 zinc binding residues (H71, H80 and D83) either alone or in combination with an aggregate forming mutation (A4V) known to cause disease. These SOD1 mutants were assayed for their ability to form aggregates. Using an in vitro cellular SOD1 aggregation assay, we show that combining A4V with mutations in non-zinc binding domains (G37R or G85R) increases SOD1 aggregation potential. Mutations at two zinc binding residues (H71G and D83G) also increase SOD1 aggregation potential. However, an H80G mutation at the third zinc binding residue decreases SOD1 aggregation potential even in the context of other aggregate forming SOD1 mutations. These results demonstrate that various mutations have different effects on SOD1 aggregation potential and that the H80G mutation appears to uniquely act as a dominant inhibitor of SOD1 aggregation.

Aggregate formation in the spinal cord of mutant SOD1 transgenic mice is reversible and mediated by proteasomes.

Cu,Zn superoxide dismutase (SOD1) mutations cause one form of familial amyotrophic lateral sclerosis by a toxic gain of function that may be related to abnormal protein folding and aggregate formation. However, the processing pathways involved in SOD1 aggregate generation within spinal cord remain unclear. We have now developed an experimental system for studying SOD1 aggregate formation and clearance in intact spinal cord tissue. Here we demonstrate that the formation of SOD1-positive aggregates in G93A SOD1 transgenic mouse spinal cord tissue involves proteasome-mediated proteolysis. Organotypic spinal cord slices from 9-day-old transgenic mice expressing G93A SOD1 develop SOD1 aggregates with proteasome inhibition. In contrast, SOD1 aggregates do not form in spinal cord slices from wild type mice or transgenic mice overexpressing wild type SOD1 following proteasome inhibition. Furthermore, SOD1 aggregate formation within G93A SOD1 spinal cord is both sensitive to small changes in overall proteasome activity and reversible with the restoration of proteasome function. Our results also establish that adult mouse spinal cord exhibits a relative deficiency in proteasome activity compared with non-CNS tissue that may help explain the propensity of spinal cord to form SOD1-positive aggregates.

Aggregate formation in Cu,Zn superoxide dismutase-related proteins.

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.

Disease progression in a transgenic model of familial amyotrophic lateral sclerosis is dependent on both neuronal and non-neuronal zinc binding proteins.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause one form of familial amyotrophic lateral sclerosis, a progressive disorder of motor neurons leading to weakness and death of affected individuals. Experiments using both transgenic mice expressing mutant SOD1 and SOD1 knock-out mice have demonstrated that disease is caused by a toxic gain of function and not by a loss of normal SOD1 activity. Precise mechanisms underlying mutant SOD1 toxicity are unclear but may involve abnormal interactions between zinc and SOD1. The metallothioneins (MTs) represent a family of zinc binding proteins that can function as zinc chaperones for apo-SOD1 in vitro. We hypothesized that manipulation of metallothioneins in vivo might alter the disease phenotype of transgenic mice expressing G93A SOD1 and therefore crossed this line with MT-I and MT-II or MT-III knock-out mice. G93A SOD1 mice deficient of either MT-I and MT-II or MT-III exhibited significant reductions in survival compared with G93A SOD1 mice. In addition, motor dysfunction was markedly accelerated in G93A SOD1 mice deficient in metallothioneins with regard to onset (MT-I and MT-II) or progression (MT-III). These results indicate that the disease course in G93A SOD1 mice is dependent on levels of metallothionein expression. Because MT-I and MT-II are expressed in glia whereas MT-III is found in neurons, these results also indicate that primary changes within non-neuronal cells can affect mutant SOD1-induced disease and do so in ways distinct from primary neuronal changes.