Amyotrophic Lateral Sclerosis Is Associated with Hypolipidemia at the Presymptomatic Stage in Mice

OBJECTIVE: To demonstrate that hypolipidemia is a typical feature of the mouse model of amyotrophic lateral sclerosis (ALS) and to assess the association between hypolipidemia and disease stage, dietary intake, and sex. METHODS: We compared daily dietary intake, body weight, and serumlipid and glucose levels in ALS mice and wild-type controls at different stages of the disease. FINDINGS: Total cholesterol low-density lipoprotein (LDL) and LDL/high-density lipoprotein (HDL) ratio were significantly lower in ALS mice compared with controls. Subgroup analysis revealed that the incidence of hypolipidemia was significantly greater in male, but not female, ALS mice compared with control mice and that hypolipidemia was present at the presymptomatic stage of the disease. This hypolipidemia can be found without a decrease in the serum levels of other energy sources, such as glucose, in the presymptomatic stage. CONCLUSIONS: Hypolipidemia is present at the presymptomatic stage of the ALS mouse model in the absence of malnutrition, significant neuromuscular degeneration or regeneration, and respiratory difficulty. Our findings suggest that hypolipidemia might be associated with the pathomechanism of ALS and/or lipid-specific metabolism rather than simply an epiphenomenon of neuromuscular degeneration or energy imbalance

The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis

International audienceMutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS

Musculotopic organization of the motor neurons supplying the mouse hindlimb muscles: a quantitative study using Fluoro-Gold retrograde tracing

We have mapped the motor neurons (MNs) supplying the major hindlimb muscles of transgenic (C57/BL6J-ChAT-EGFP) and wild-type (C57/BL6J) mice. The fluorescent retrograde tracer Fluoro-Gold was injected into 19 hindlimb muscles. Consecutive transverse spinal cord sections were harvested, the MNs counted, and the MN columns reconstructed in 3D. Three longitudinal MN columns were identified. The dorsolateral column extends from L4 to L6 and consists of MNs innervating the crural muscles and the foot. The ventrolateral column extends from L1 to L6 and accommodates MNs supplying the iliopsoas, gluteal, and quadriceps femoris muscles. The middle part of the ventral horn hosts the central MN column, which extends between L2–L6 and consists of MNs for the thigh adductor, hamstring, and quadratus femoris muscles. Within these longitudinal columns, the arrangement of the different MN groups reflects their somatotopic organization. MNs innervating muscles developing from the dorsal (e.g., quadriceps) and ventral muscle mass (e.g., hamstring) are situated in the lateral and medial part of the ventral gray, respectively.MN pools belonging to proximal muscles (e.g., quadratus femoris and iliopsoas) are situatedventral to those supplying more distal ones (e.g., plantar muscles). Finally, MNs innervatingflexors (e.g., posterior crural muscles) are more medial than those belonging to extensors ofthe same joint (e.g., anterior crural muscles). These data extend and modify the MN maps in the recently published atlas of the mouse spinal cord and may help when assessing neuronal loss associated with MN diseases

A comparison of in vitro properties of resting SOD1 transgenic microglia reveals evidence of reduced neuroprotective function

<p>Abstract</p> <p>Background</p> <p>Overexpression of mutant copper/zinc superoxide dismutase (<it>SOD1</it>) in rodents has provided useful models for studying the pathogenesis of amyotrophic lateral sclerosis (ALS). Microglia have been shown to contribute to ALS disease progression in these models, although the mechanism of this contribution remains to be elucidated. Here, we present the first evidence of the effects of overexpression of mutant (TG G93A) and wild type (TG WT) human <it>SOD1 </it>transgenes on a set of functional properties of microglia relevant to ALS progression, including expression of integrin β-1, spreading and migration, phagocytosis of apoptotic neuronal cell debris, and intracellular calcium changes in response to an inflammatory stimulus.</p> <p>Results</p> <p>TG SOD1 G93A but not TG SOD1 WT microglia had lower expression levels of the cell adhesion molecule subunit integrin β-1 than their NTG control cells [NTG (G93A) and NTG (WT), respectively, 92.8 ± 2.8% on TG G93A, 92.0 ± 6.6% on TG WT, 100.0 ± 1.6% on NTG (G93A), and 100.0 ± 2.7% on NTG (WT) cells], resulting in decreased spreading ability, with no effect on ability to migrate. Both TG G93A and TG WT microglia had reduced capacity to phagocytose apoptotic neuronal cell debris (13.0 ± 1.3% for TG G93A, 16.5 ± 1.9% for TG WT, 28.6 ± 1.8% for NTG (G93A), and 26.9 ± 2.8% for NTG (WT) cells). Extracellular stimulation of microglia with ATP resulted in smaller increase in intracellular free calcium in TG G93A and TG WT microglia relative to NTG controls (0.28 ± 0.02 μM for TG G93A, 0.24 ± 0.03 μM for TG WT, 0.39 ± 0.03 μM for NTG (G93A), and 0.37 ± 0.05 μM for NTG (WT) microglia).</p> <p>Conclusions</p> <p>These findings indicate that, under resting conditions, microglia from mutant <it>SOD1 </it>transgenic mice have a reduced capacity to elicit physiological responses following tissue disturbances and that higher levels of stimulatory signals, and/or prolonged stimulation may be necessary to initiate these responses. Overall, resting mutant <it>SOD1</it>-overexpressing microglia may have reduced capacity to function as sensors of disturbed tissue/cellular homeostasis in the CNS and thus have reduced neuroprotective function.</p

An ALS-Linked Mutant SOD1 Produces a Locomotor Defect Associated with Aggregation and Synaptic Dysfunction When Expressed in Neurons of Caenorhabditis elegans

The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking

Exendin-4 Ameliorates Motor Neuron Degeneration in Cellular and Animal Models of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS

Knockdown of the Drosophila Fused in Sarcoma (FUS) Homologue Causes Deficient Locomotive Behavior and Shortening of Motoneuron Terminal Branches

Mutations in the fused in sarcoma/translated in liposarcoma gene (FUS/TLS, FUS) have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). FUS is an RNA-binding protein that is normally localized in the nucleus, but is mislocalized to the cytoplasm in ALS, and comprises cytoplasmic inclusions in ALS-affected areas. However, it is still unknown whether the neurodegeneration that occurs in ALS is caused by the loss of FUS nuclear function, or by the gain of toxic function due to cytoplasmic FUS aggregation. Cabeza (Caz) is a Drosophila orthologue of human FUS. Here, we generated Drosophila models with Caz knockdown, and investigated their phenotypes. In wild-type Drosophila, Caz was strongly expressed in the central nervous system of larvae and adults. Caz did not colocalize with a presynaptic marker, suggesting that Caz physiologically functions in neuronal cell bodies and/or their axons. Fly models with neuron-specific Caz knockdown exhibited reduced climbing ability in adulthood and anatomical defects in presynaptic terminals of motoneurons in third instar larvae. Our results demonstrated that decreased expression of Drosophila Caz is sufficient to cause degeneration of motoneurons and locomotive disability in the absence of abnormal cytoplasmic Caz aggregates, suggesting that the pathogenic mechanism underlying FUS-related ALS should be ascribed more to the loss of physiological FUS functions in the nucleus than to the toxicity of cytoplasmic FUS aggregates. Since the Caz-knockdown Drosophila model we presented recapitulates key features of human ALS, it would be a suitable animal model for the screening of genes and chemicals that might modify the pathogenic processes that lead to the degeneration of motoneurons in ALS