Elevated PGC-1α Activity Sustains Mitochondrial Biogenesis and Muscle Function without Extending Survival in a Mouse Model of Inherited ALS

SummaryThe transcriptional coactivator PGC-1α induces multiple effects on muscle, including increased mitochondrial mass and activity. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, adult-onset neurodegenerative disorder characterized by selective loss of motor neurons and skeletal muscle degeneration. An early event is thought to be denervation-induced muscle atrophy accompanied by alterations in mitochondrial activity and morphology within muscle. We now report that elevation of PGC-1α levels in muscles of mice that develop fatal paralysis from an ALS-causing SOD1 mutant elevates PGC-1α-dependent pathways throughout disease course. Mitochondrial biogenesis and activity are maintained through end-stage disease, accompanied by retention of muscle function, delayed muscle atrophy, and significantly improved muscle endurance even at late disease stages. However, survival was not extended. Therefore, muscle is not a primary target of mutant SOD1-mediated toxicity, but drugs increasing PGC-1α activity in muscle represent an attractive therapy for maintaining muscle function during progression of ALS

Mitochondrial Isolation and Purification from Mouse Spinal Cord.

Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in the study of mitochondria following the discoveries that mitochondria are central to the process of programmed cell death and that mitochondrial dysfunctions are implicated in numerous diseases including a wide range of neurological disorders such as Parkinson's disease, Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis. In order to identify and study changes in mitochondrial function related to specific neurological conditions the mitochondria are often isolated from the compartment of the central nervous system most affected during disease. Here, we describe a protocol for the isolation of mitochondria from mouse spinal cord, a compartment of the central nervous system that is significantly affected in neuromuscular diseases such as amyotrophic lateral sclerosis. This method relies on differential centrifugation to separate the mitochondria from the other subcellular compartments

Mitochondrial Isolation and Purification from Mouse Spinal Cord

Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in the study of mitochondria following the discoveries that mitochondria are central to the process of programmed cell death and that mitochondrial dysfunctions are implicated in numerous diseases including a wide range of neurological disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis. In order to identify and study changes in mitochondrial function related to specific neurological conditions the mitochondria are often isolated from the compartment of the central nervous system most affected during disease. Here, we describe a protocol for the isolation of mitochondria from mouse spinal cord, a compartment of the central nervous system that is significantly affected in neuromuscular diseases such as amyotrophic lateral sclerosis. This method relies on differential centrifugation to separate the mitochondria from the other subcellular compartments

Misfolded SOD1 is not a primary component of sporadic ALS

A common feature of inherited and sporadic ALS is accumulation of abnormal proteinaceous inclusions in motor neurons and glia. SOD1 is the major protein component accumulating in patients with SOD1 mutations, as well as in mutant SOD1 mouse models. ALS-linked mutations of SOD1 have been shown to increase its propensity to misfold and/or aggregate. Antibodies specific for monomeric or misfolded SOD1 have detected misfolded SOD1 accumulating predominantly in spinal cord motor neurons of ALS patients with SOD1 mutations. We now use seven different conformationally sensitive antibodies to misfolded human SOD1 (including novel high affinity antibodies currently in pre-clinical development) coupled with immunohistochemistry, immunofluorescence and immunoprecipitation to test for the presence of misfolded SOD1 in high quality human autopsy samples. Whereas misfolded SOD1 is readily detectable in samples from patients with SOD1 mutations, it is below detection limits for all of our measures in spinal cord and cortex tissues from patients with sporadic or non-SOD1 inherited ALS. The absence of evidence for accumulated misfolded SOD1 supports a conclusion that SOD1 misfolding is not a primary component of sporadic ALS

Fused in sarcoma (FUS) protein lacking nuclear localization signal (NLS) and major RNA binding motifs triggers proteinopathy and severe motor phenotype in transgenic mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43(Q331K) and TDP-43(M337V)), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43(Q331K), but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43(Q331K) enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage