Early signs of motoneuron vulnerability in a disease model system: Characterization of transverse slice cultures of spinal cord isolated from embryonic ALS mice.
ABSTRACT Mutations in the SOD1 gene are associated with familial amyotrophic lateral sclerosis. The mechanisms by which these mutations lead to cell loss within the spinal cord ventral horns are unknown. In the present report we used the G93A transgenic mouse model of amyotrophic lateral sclerosis to develop and characterize an in vitro tool for the investigation of subtle alterations of spinal tissue prior to frank neuronal degeneration. To this aim, we developed organotypic slice cultures from wild type and G93A embryonic spinal cords. We combined immunocytochemistry and electron microscopy techniques to compare wild type and G93A spinal cord tissues after 14 days of growth under standard in vitro conditions. By SMI32 and choline acetyl transferase immunostaining, the distribution and morphology of motoneurons were compared in the two culture groups. Wild type and mutant cultures displayed no differences in the analyzed parameters as well as in the number of motoneurons. Similar results were observed when glial fibrillary acidic protein and myelin basic protein-positive cells were examined. Cell types within the G93A slice underwent maturation and slices could be maintained in culture for at least 3 weeks when prepared from embryos. Electron microscopy investigation confirmed the absence of early signs of mitochondria vacuolization or protein aggregate formation in G93A ventral horns. However, a significantly different ratio between inhibitory and excitatory synapses was present in G93A cultures, when compared with wild type ones, suggesting the expression of subtle synaptic dysfunction in G93A cultured tissue. When compared with controls, G93A motoneurons exhibited increased vulnerability to AMPA glutamate receptor-mediated excitotoxic stress prior to clear disease appearance. This in vitro disease model may thus represent a valuable tool to test early mechanisms contributing to motoneuron degeneration and potential therapeutic molecular interventions.
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ABSTRACT: Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin upregulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious and disease conditions remains unknown, but may be related to differential expression of spliced isoforms. To better understand peripherin function, we performed a yeast two-hybrid screen on a mouse brain cDNA library using an assembly-incompetent peripherin isoform, Per-61, as bait. We identified new peripherin interactors with roles in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding and mitochondrial metabolism. We focused on the interaction of Per-61 and the constitutive isoform, Per-58, with SNAP25 interacting protein 30 (SIP30), a neuronal protein involved in SNAP receptor-dependent exocytosis. We found that peripherin and SIP30 interacted through coiled-coil domains and co-localized in cytoplasmic aggregates in SW-13vim(-) cells. Interestingly, Per-61 and Per-58 differentially altered the subcellular distribution of SIP30 and SNAP25 in primary motor neurons. Our findings suggest a novel role of peripherin in vesicle trafficking.This article is protected by copyright. All rights reserved.Journal of Neurochemistry 08/2014; · 4.24 Impact Factor
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ABSTRACT: Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast-iPS-motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.Cell Death & Disease 02/2014; 5:e1096. · 5.18 Impact Factor
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ABSTRACT: The increasing number of mouse models of human degenerative and injury related diseases that affect motor behavior raises the importance of in vivo methodologies allowing measurement of physiological and behavioral changes over an extended period of time in individual animals. A method that provides long-term measurements of muscle denervation and its behavioral consequences in individual mice for several months is presented in this article. The method is applied to mSod1G93A mice, which model human amyotrophic lateral sclerosis (ALS). The denervation process of gastrocnemius and soleus muscles in mSod1G93A mice is demonstrated for up to three months. The data suggest that as muscle denervation progresses, massive behavioral compensation occurs within the spinal cord that allows animals to walk almost normally until late ages. Only around the age of 84 days, is the first sign of abnormal movement during walking behavior detected as an abnormal TA activity profile that is manifested in subtle but abnormal swing movement during walking. Additionally, this method can be used with other mouse models of human diseases, such as spinal cord injury, intracerebral hemorrhage, Parkinson's diseases, and spinal muscular atrophy.Journal of Neurophysiology 10/2013; · 3.04 Impact Factor