hESC- and hiPSC-derived Schwann cells are molecularly comparable and functionally equivalent.

Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial, we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally, both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent.

Inborn errors of amino acid metabolism - from underlying pathophysiology to therapeutic advances.

Amino acids are organic molecules that serve as basic substrates for protein synthesis and have additional key roles in a diverse array of cellular functions, including cell signaling, gene expression, energy production and molecular biosynthesis. Genetic defects in the synthesis, catabolism or transport of amino acids underlie a diverse class of diseases known as inborn errors of amino acid metabolism. Individually, these disorders are rare, but collectively, they represent an important group of potentially treatable disorders. In this Clinical Puzzle, we discuss the pathophysiology, clinical features and management of three disorders that showcase the diverse clinical presentations of disorders of amino acid metabolism: phenylketonuria, lysinuric protein intolerance and homocystinuria due to cystathionine ß-synthase (CBS) deficiency. Understanding the biochemical perturbations caused by defects in amino acid metabolism will contribute to ongoing development of diagnostic and management strategies aimed at improving the morbidity and mortality associated with this diverse group of disorders.

GADD45A is a protective modifier of neurogenic skeletal muscle atrophy.

Neurogenic muscle atrophy is the loss of skeletal muscle mass and function that occurs with nerve injury and in denervating diseases, such as amyotrophic lateral sclerosis. Aside from prompt restoration of innervation and exercise where feasible, there are currently no effective strategies for maintaining skeletal muscle mass in the setting of denervation. We conducted a longitudinal analysis of gene expression changes occurring in atrophying skeletal muscle and identified growth arrest and DNA damage-inducible A (Gadd45a) as a gene that shows one of the earliest and most sustained increases in expression in skeletal muscle after denervation. We evaluated the role of this induction using genetic mouse models and found that mice lacking GADD45A showed accelerated and exacerbated neurogenic muscle atrophy, as well as loss of fiber type identity. Our genetic analyses demonstrate that, rather than directly contributing to muscle atrophy as proposed in earlier studies, GADD45A induction likely represents a protective negative feedback response to denervation. Establishing the downstream effectors that mediate this protective effect and the pathways they participate in may yield new opportunities to modify the course of muscle atrophy.

Cellular and molecular features of neurogenic skeletal muscle atrophy.

Neurogenic atrophy refers to the loss of muscle mass and function that results directly from injury or disease of the peripheral nervous system. Individuals with neurogenic atrophy may experience reduced functional status and quality of life and, in some circumstances, reduced survival. Distinct pathological findings on muscle histology can aid in diagnosis of a neurogenic cause for muscle dysfunction, and provide indicators for the chronicity of denervation. Denervation induces pleiotypic responses in skeletal muscle, and the molecular mechanisms underlying neurogenic muscle atrophy appear to share common features with other causes of muscle atrophy, including activation of FOXO transcription factors and corresponding induction of ubiquitin-proteasomal and lysosomal degradation. In this review, we provide an overview of histologic features of neurogenic atrophy and a summary of current understanding of underlying mechanisms.

Longitudinal RNA-Seq analysis of acute and chronic neurogenic skeletal muscle atrophy.

Skeletal muscle is a highly adaptable tissue capable of changes in size, contractility, and metabolism according to functional demands. Atrophy is a decline in mass and strength caused by pathologic loss of myofibrillar proteins, and can result from disuse, aging, or denervation caused by injury or peripheral nerve disorders. We provide a high-quality longitudinal RNA-Seq dataset of skeletal muscle from a cohort of adult C57BL/6J male mice subjected to tibial nerve denervation for 0 (baseline), 1, 3, 7, 14, 30, or 90 days. Using an unbiased genomics approach to identify gene expression changes across the entire longitudinal course of muscle atrophy affords the opportunity to (1) establish acute responses to denervation, (2) detect pathways that mediate rapid loss of muscle mass within the first week after denervation, and (3) capture the molecular phenotype of chronically atrophied muscle at a stage when it is largely resistant to recovery.

The astrocytic transporter SLC7A10 (Asc-1) mediates glycinergic inhibition of spinal cord motor neurons.

SLC7A10 (Asc-1) is a sodium-independent amino acid transporter known to facilitate transport of a number of amino acids including glycine, L-serine, L-alanine, and L-cysteine, as well as their D-enantiomers. It has been described as a neuronal transporter with a primary role related to modulation of excitatory glutamatergic neurotransmission. We find that SLC7A10 is substantially enriched in a subset of astrocytes of the caudal brain and spinal cord in a distribution corresponding with high densities of glycinergic inhibitory synapses. Accordingly, we find that spinal cord glycine levels are significantly reduced in Slc7a10-null mice and spontaneous glycinergic postsynaptic currents in motor neurons show substantially diminished amplitudes, demonstrating an essential role for SLC7A10 in glycinergic inhibitory function in the central nervous system. These observations establish the etiology of sustained myoclonus (sudden involuntary muscle movements) and early postnatal lethality characteristic of Slc7a10-null mice, and implicate SLC7A10 as a candidate gene and auto-antibody target in human hyperekplexia and stiff person syndrome, respectively.

D-serine in glia and neurons derives from 3-phosphoglycerate dehydrogenase.

d-Serine is an endogenous ligand for NMDARs generated from l-serine by the enzyme serine racemase (Srr). Both neuronal and glial localizations have been reported for d-serine and Srr. 3-Phosphoglycerate dehydrogenase is an exclusively astrocytic enzyme that catalyzes the first committed step of l-serine biosynthesis. Using transgenic mice expressing enhanced green fluorescent protein under the Srr promoter and mice with targeted deletion of Srr or 3-Phosphoglycerate dehydrogenase, we demonstrate predominantly neuronal sources of d-serine dependent on astrocytic supply of l-serine. These findings clarify the cellular basis for the regulation of NMDAR neurotransmission by d-serine.

Serine racemase deletion protects against cerebral ischemia and excitotoxicity.

D-Serine, formed from L-serine by serine racemase (SR), is a physiologic coagonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role for D-serine in NMDA receptor-mediated neurotoxicity and stroke. Brain cultures of SR-deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knock-out mice, NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity.

GOSPEL: a neuroprotective protein that binds to GAPDH upon S-nitrosylation.

We recently reported a cell death cascade whereby cellular stressors activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The nuclear GAPDH/Siah complex augments p300/CBP-associated acetylation of nuclear proteins, including p53, which mediate cell death. We report a 52 kDa cytosolic protein, GOSPEL, which physiologically binds GAPDH, in competition with Siah, retaining GAPDH in the cytosol and preventing its nuclear translocation. GOSPEL is neuroprotective, as its overexpression prevents NMDA-glutamate excitotoxicity while its depletion enhances death in primary neuron cultures. S-nitrosylation of GOSPEL at cysteine 47 enhances GAPDH-GOSPEL binding and the neuroprotective actions of GOSPEL. In intact mice, virally delivered GOSPEL selectively diminishes NMDA neurotoxicity. Thus, GOSPEL may physiologically regulate the viability of neurons and other cells.

Glutamatergic regulation of serine racemase via reversal of PIP2 inhibition.

D-serine is a physiologic coagonist with glutamate at NMDA-subtype glutamate receptors. As D-serine is localized in glia, synaptically released glutamate presumably stimulates the glia to form and release D-serine, enabling glutamate/D-serine cotransmission. We show that serine racemase (SR), which generates D-serine from L-serine, is physiologically inhibited by phosphatidylinositol (4,5)-bisphosphate (PIP2) presence in membranes where SR is localized. Activation of metabotropic glutamate receptors (mGluR5) on glia leads to phospholipase C-mediated degradation of PIP2, relieving SR inhibition. Thus mutants of SR that cannot bind PIP2 lose their membrane localizations and display a 4-fold enhancement of catalytic activity. Moreover, mGluR5 activation of SR activity is abolished by inhibiting phospholipase C.

Nitric oxide S-nitrosylates serine racemase, mediating feedback inhibition of D-serine formation.

Serine racemase (SR) generates D-serine, a coagonist with glutamate at NMDA receptors. We show that SR is physiologically S-nitrosylated leading to marked inhibition of enzyme activity. Inhibition involves interactions with the cofactor ATP reflecting juxtaposition of the ATP-binding site and cysteine-113 (C113), the site for physiological S-nitrosylation. NMDA receptor physiologically enhances SR S-nitrosylation by activating neuronal nitric-oxide synthase (nNOS). These findings support a model whereby postsynaptic stimulation of nitric-oxide (NO) formation feeds back to presynaptic cells to S-nitrosylate SR and decrease D-serine availability to postsynaptic NMDA receptors.

Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation.

The neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) results from integral deletions of the subtelomeric repeat D4Z4 on chromosome 4q. A disruption of chromatin structure affecting gene expression is thought to underlie the pathophysiology. The global gene expression profiling of mature muscle tissue presented here provides the first insight into an FSHD-specific defect in myogenic differentiation. FSHD expression profiles generated by oligonucleotide microarrays were compared with those from normal muscle as well as other types of muscular dystrophies (DMD, aSGD) in order to determine FSHD-specific changes. In addition, matched biopsies (affected and unaffected muscle) from individuals with FSHD served to monitor expression changes during the progression of the disease as well as to diminish non-specific changes resulting from individual variability. Among genes altered in an FSHD-specific and highly significant manner, many are involved in myogenic differentiation and suggest a partial block in the normal differentiation program. Indeed, many of the transcripts affected in FSHD represent direct targets of the transcription factor MyoD. Additional mis-expressed genes confirm a diminished capacity to buffer oxidative stress, as demonstrated in FSHD myoblasts. This enhanced vulnerability of proliferative stage myoblasts to reactive oxygen species is also disease-specific, further implicating a defect in FSHD muscle satellite cells. Importantly, none of the genes localizing to the FSHD region at 4q35 were found to exhibit a significantly altered pattern of expression in FSHD muscle. This finding was corroborated by expression analysis of FSHD muscle using a custom cDNA microarray containing 51 genes and ESTs from the 4q35 region. Disruptions in FSHD myogenesis and oxidative capacity may therefore not arise from a position effect mechanism as has been previously suggested, but rather from a global effect on gene regulation. Improper nuclear localization of 4qter is discussed as an alternative model for FSHD gene regulation and pathogenesis.