A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity.

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.

Optical control of muscle function by transplantation of stem cell-derived motor neurons in mice.

Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell-derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.

The legs at odd angles (Loa) mutation in cytoplasmic dynein ameliorates mitochondrial function in SOD1G93A mouse model for motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute approximately 10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15-20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1(G93A). In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1(G93A) motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1(G93A) motor neurons.

Modification of superoxide dismutase 1 (SOD1) properties by a GFP tag--implications for research into amyotrophic lateral sclerosis (ALS).

BACKGROUND: Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts. METHODOLOGY/PRINCIPAL FINDINGS: Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.

Mutant glycyl-tRNA synthetase (Gars) ameliorates SOD1(G93A) motor neuron degeneration phenotype but has little affect on Loa dynein heavy chain mutant mice.

BACKGROUND: In humans, mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system, and clinical phenotypes ranging from Charcot-Marie-Tooth neuropathy to a severe infantile form of spinal muscular atrophy. GARS is ubiquitously expressed and may have functions in addition to its canonical role in protein synthesis through catalyzing the addition of glycine to cognate tRNAs. METHODOLOGY/PRINCIPAL FINDINGS: We have recently described a new mouse model with a point mutation in the Gars gene resulting in a cysteine to arginine change at residue 201. Heterozygous Gars(C201R/+) mice have locomotor and sensory deficits. In an investigation of genetic mutations that lead to death of motor and sensory neurons, we have crossed the Gars(C201R/+) mice to two other mutants: the TgSOD1(G93A) model of human amyotrophic lateral sclerosis and the Legs at odd angles mouse (Dync1h1(Loa)) which has a defect in the heavy chain of the dynein complex. We found the Dync1h1(Loa/+);Gars(C201R/+) double heterozygous mice are more impaired than either parent, and this is may be an additive effect of both mutations. Surprisingly, the Gars(C201R) mutation significantly delayed disease onset in the SOD1(G93A);Gars(C201R/+) double heterozygous mutant mice and increased lifespan by 29% on the genetic background investigated. CONCLUSIONS/SIGNIFICANCE: These findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains.

Focal distortion of the nuclear envelope by huntingtin aggregates revealed by lamin immunostaining.

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine repeat tract in the huntingtin protein. Polyglutamine-expanded huntingtin forms intranuclear as well as perinuclear inclusion bodies. Perinuclear aggregates formed by polyglutamine-expanded proteins are associated with a characteristic indentation of the nuclear envelope. We examined the nuclear envelope in cells containing huntingtin aggregates using immunostaining for lamin B1, a major component of the nuclear lamina. Laser confocal microscopy analysis revealed that huntingtin aggregates in a juxtanuclear position were associated with a clear focal distortion in the nuclear envelope in cells transfected with polyglutamine-expanded huntingtin. Lamin B1 distribution was not altered by aggregates of polyglutamine-expanded ataxin-1, that are exclusively intranuclear. Thus lamin immunocytochemistry demonstrates clearly the depression of the nuclear envelope resulting from the formation of perinuclear aggregates by polyglutamine-expanded huntingtin. Lamin immunocytochemistry would be of value to monitor the state of the nuclear envelope in experimental paradigms aimed at establishing the significance of perinuclear aggregates of pathogenic proteins.