Michael L. Garcia

Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice

Dominant mutations in ubiquitously expressed superoxide dismutase (SOD1) cause familial ALS by provoking premature death of adult motor neurons. To test whether mutant damage to cell types beyond motor neurons is required for the onset of motor neuron disease, we generated chimeric mice in which all motor neurons and oligodendrocytes expressed mutant SOD1 at a level sufficient to cause fatal, early-onset motor neuron disease when expressed ubiquitously, but did so in a cellular environment containing variable numbers of non-mutant, non-motor neurons. Despite high-level mutant expression within 100% of motor neurons and oligodendrocytes, in most of these chimeras, the presence of WT non-motor neurons substantially delayed onset of motor neuron degeneration, increasing disease-free life by 50%. Disease onset is therefore non-cell autonomous, and mutant SOD1 damage within cell types other than motor neurons and oligodendrocytes is a central contributor to initiation of motor neuron degeneration.

Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?

Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs). NFs are type IV intermediate filaments (IFs) that can be composed of four subunits, neurofilament heavy (NF-H), neurofilament medium (NF-M), neurofilament light (NF-L), and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP) repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations.

Mechanisms of voltage-gated ion channel regulation: from gene expression to localization

Abstract.The ion channel milieu present in a neuron in large part determines the inherent excitability of a given cell and is responsible for the translation of sensory transduction and synaptic input to axonal output. Intrinsic excitability is a dynamic process subject to multiple levels of regulation from channel gene expression to post-translational modifications that influence channel activity. The goal of this review is to provide an overview of some of the mechanisms by which channels can be modified in order to influence neuronal output. We focus on four levels of regulation: channel gene transcription, alternative splicing of channel transcripts, post-translational modifications that alter channel kinetics (phosphorylation), and subcellular localization and trafficking of channel proteins.

Development and characterization of an SMN2-based intermediate mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is due to the loss of the survival motor neuron gene 1 (SMN1), resulting in motor neuron (MN) degeneration, muscle atrophy and loss of motor function. While SMN2 encodes a protein identical to SMN1, a single nucleotide difference in exon 7 causes most of the SMN2-derived transcripts to be alternatively spliced resulting in a truncated and unstable protein (SMNΔ7). SMA patients retain at least one SMN2 copy, making it an important target for therapeutics. Many of the existing SMA models are very severe, with animals typically living less than 2 weeks. Here, we present a novel intermediate mouse model of SMA based upon the human genomic SMN2 gene. Genetically, this model is similar to the well-characterized SMNΔ7 model; however, we have manipulated the SMNΔ7 transgene to encode a modestly more functional protein referred to as SMN read-through (SMN(RT)). By introducing the SMN(RT) transgene onto the background of a severe mouse model of SMA (SMN2(+/+);Smn(-/-)), disease severity was significantly decreased based upon a battery of phenotypic parameters, including MN pathology and a significant extension in survival. Importantly, there is not a full phenotypic correction, allowing for the examination of a broad range of therapeutics, including SMN2-dependent and SMN-independent pathways. This novel animal model serves as an important biological and therapeutic model for less severe forms of SMA and provides an in vivo validation of the SMN(RT) protein.

Transgenic inactivation of murine myostatin does not decrease the severity of disease in a model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease and is a leading genetic cause of infantile death. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). The presence of a nearly identical copy gene called SMN2 has led to the development of several strategies that are designed to elevate SMN levels, and it is clear that SMN2 is an important modifier gene. However, the possibility exists that SMN-independent strategies to lessen the severity of the SMA phenotype could provide insight into disease development as well as aid in the identification of potential therapeutic targets. Muscle enhancement has been considered an interesting target for a variety of neurodegenerative diseases, including SMA. Previously we have shown in SMA mice that delivery of recombinant follistatin resulted in an extension in survival and a general lessening of disease severity. Follistatin is known to functionally block myostatin (MSTN), a potent inhibitor of muscle development. However, follistatin is a multifaceted protein involved in a variety of cellular pathways. To determine whether MSTN inhibition was the primary pathway associated with the previously reported follistatin results, we generated an animal model of SMA in which Mstn was genetically inactivated. In this report we characterize the novel SMA/Mstn model and demonstrate that Mstn inactivation does not significantly enhance muscle development in neonatal animals, nor does it result in an amelioration of the SMA phenotype.

Muscle pathology without severe nerve pathology in a new mouse model of Charcot–Marie–Tooth disease type 2E

Mutations in neurofilament light (NF-L) have been linked to Charcot-Marie-Tooth disease type 2E (CMT2E) in humans. To provide insight into disease pathogenesis, we developed a novel line of CMT2E mice that constitutively express human NF-L (hNF-L) with a glutamic acid to lysine mutation at position 397 (hNF-L(E397K)). This new line of mice developed signs consistent with CMT2E patients. Disease signs were first observed at 4 months in hNF-L(E397K) mice, and consisted of aberrant hind limb posture, digit deformities, reduced voluntary locomotor activity, reduced motor nerve conduction velocities (MNCVs) and muscle atrophy. Reduced voluntary locomotor activity and muscle pathology occurred without significant denervation, and hNF-L(E397K) mice showed relatively mild signs of nerve pathology. Nerve pathology in hNF-L(E397K) mice was characterized by ectopic accumulations of phosphorylated NFs in motor neuron cell bodies as early as 1 month. Moreover, NF organization was altered in motor and sensory roots, with small motor axons being most affected. Peak axonal diameter was reduced for small motor axons prior to and after the onset of overt phenotypes, whereas large motor axons were affected only after onset, which correlated with reduced MNCVs. Additionally, there was a small reduction in the number of sensory axons in symptomatic hNF-L(E397K) mice. hNF-L(E397K) mice are a novel line of CMT2E mice that recapitulate many of the overt phenotypes observed in CMT2E patients and hNF-L(P22S) mice. The cellular pathology observed in hNF-L(E397K) mice differed from that recently reported in hNF-L(P22S) mice, suggesting that overt CMT2E phenotypes may arise through different cellular mechanisms.

Expansion of neurofilament medium C terminus increases axonal diameter independent of increases in conduction velocity or myelin thickness.

Maturation of the peripheral nervous system requires specification of axonal diameter, which, in turn, has a significant influence on nerve conduction velocity. Radial axonal growth initiates with myelination, and is dependent upon the C terminus of neurofilament medium (NF-M). Molecular phylogenetic analysis in mammals suggested that expanded NF-M C termini correlated with larger-diameter axons. We used gene targeting and computational modeling to test this new hypothesis. Increasing the length of NF-M C terminus in mice increased diameter of motor axons without altering neurofilament subunit stoichiometry. Computational modeling predicted that an expanded NF-M C terminus extended farther from the neurofilament core independent of lysine-serine-proline (KSP) phosphorylation. However, expansion of NF-M C terminus did not affect the distance between adjacent neurofilaments. Increased axonal diameter did not increase conduction velocity, possibly due to a failure to increase myelin thickness by the same proportion. Failure of myelin to compensate for larger axonal diameters suggested a lack of plasticity during the processes of myelination and radial axonal growth.

X-linked spinal muscular atrophy in mice caused by autonomous loss of ATP7A in the motor neuron

ATP7A is a copper‐transporting P‐type ATPase that is essential for cellular copper homeostasis. Loss‐of‐function mutations in the ATP7A gene result in Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia and failure to thrive, due to systemic copper deficiency. Most recently, rare missense mutations in ATP7A that do not impact systemic copper homeostasis have been shown to cause X‐linked spinal muscular atrophy type 3 (SMAX3), a distal hereditary motor neuropathy. An understanding of the mechanistic and pathophysiological basis of SMAX3 is currently lacking, in part because the disease‐causing mutations have been shown to confer both loss‐ and gain‐of‐function properties to ATP7A, and because there is currently no animal model of the disease. In this study, the Atp7a gene was specifically deleted in the motor neurons of mice, resulting in a degenerative phenotype consistent with the clinical features in affected patients with SMAX3, including the progressive deterioration of gait, age‐dependent muscle atrophy, denervation of neuromuscular junctions and a loss of motor neuron cell bodies. Taken together, these data reveal autonomous requirements for ATP7A that reveal essential roles for copper in the maintenance and function of the motor neuron, and suggest that SMAX3 is caused by a loss of ATP7A function that specifically impacts the spinal motor neuron. Copyright

Distal to proximal development of peripheral nerves requires the expression of neurofilament heavy

At the initiation of radial growth, neurofilaments are likely to consist primarily of neurofilament light and medium as neurofilament heavy expression is developmentally delayed. To better understand the role of neurofilament heavy in structuring axons, axonal diameter and neurofilament organization were measured in proximal and distal segments of the sciatic nerve and along the entire length of the phrenic nerve. Deletion of neurofilament heavy reduced axonal diameters and neurofilament number in proximal nerve segments. However, neurofilament spacing was greater in proximal versus distal phrenic nerve segments. Taken together, these results suggest that loss of neurofilament heavy reduces radial growth in proximal axonal segments by reducing the accumulation of neurofilaments. As neurofilament heavy expression is developmentally delayed, these results suggest that without neurofilament heavy, the neurofilament network is established in a distal to proximal gradient perhaps to allow distal axonal segments to develop prior to proximal segments.

The Wallerian degeneration slow (Wlds) gene does not attenuate disease in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal alpha-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld(s)) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld(s) on the phenotype of a mouse model of SMA. We found that Wld(s) does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.