The motor neuron disease spinal muscular atrophy (SMA) causes profound muscle weakness that most often leads to early death. At autopsy, SMA is characterized by loss of motor neurons and muscle ...atrophy, but the initial cellular events that precipitate motor unit dysfunction and loss remain poorly characterized. Here, we examined the function and corresponding structure of neuromuscular junction (NMJ) synapses in a mouse model of severe SMA (hSMN2/delta7SMN/mSmn-/-). Surprisingly, most SMA NMJs remained innervated even late in the disease course; however they showed abnormal synaptic transmission. There was a two-fold reduction in the amplitudes of the evoked endplate currents (EPCs), but normal spontaneous miniature EPC (MEPC) amplitudes. These features in combination indicate reduced quantal content. SMA NMJs also demonstrated increased facilitation suggesting a reduced probability of vesicle release. By electron microscopy, we found a decreased density of synaptic vesicles that is likely to contribute to the reduced release probability. In addition to presynaptic defects, there were postsynaptic abnormalities. EPC and MEPC decay time constants were prolonged because of a slowed switch from the fetal acetylcholine receptor (AChR) gamma-subunit to the adult epsilon-subunit. There was also reduced size of AChR clusters and small myofibers, which expressed an immature pattern of myosin heavy chains. Together these results indicate that impaired synaptic vesicle release at NMJs in severe SMA is likely to contribute to failed postnatal maturation of motor units and muscle weakness.
Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose ...at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease.
To determine the molecular basis of multiple respiratory chain complex deficiencies.
We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family.
Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%).
Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.
The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like ...phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition "click" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation.
Although tumor necrosis factor (TNF)-α antagonists are increasingly used in place of nonbiologic comparator medications, their safety profile remains incomplete.
To determine whether initiation of ...TNF-α antagonists compared with nonbiologic comparators is associated with an increased risk of serious infections requiring hospitalization.
Within a US multi-institutional collaboration, we assembled retrospective cohorts (1998-2007) of patients with rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis, psoriatic arthritis, or ankylosing spondylitis (psoriasis and spondyloarthropathies) combining data from Kaiser Permanente Northern California, New Jersey and Pennsylvania Pharmaceutical Assistance programs, Tennessee Medicaid, and national Medicaid/Medicare. TNF-α antagonists and nonbiologic regimens were compared in disease-specific propensity score (PS)-matched cohorts using Cox regression models with nonbiologics as the reference. Baseline glucocorticoid use was evaluated as a separate covariate.
Infections requiring hospitalization (serious infections) during the first 12 months after initiation of TNF-α antagonists or nonbiologic regimens.
Study cohorts included 10,484 RA, 2323 IBD, and 3215 psoriasis and spondyloarthropathies matched pairs using TNF-α antagonists and comparator medications. Overall, we identified 1172 serious infections, most of which (53%) were pneumonia and skin and soft tissue infections. Among patients with RA, serious infection hospitalization rates were 8.16 (TNF-α antagonists) and 7.78 (comparator regimens) per 100 person-years (adjusted hazard ratio aHR, 1.05 95% CI, 0.91-1.21). Among patients with IBD, rates were 10.91 (TNF-α antagonists) and 9.60 (comparator) per 100 person-years (aHR, 1.10 95% CI, 0.83-1.46). Among patients with psoriasis and spondyloarthropathies, rates were 5.41 (TNF-α antagonists) and 5.37 (comparator) per 100 person-years (aHR, 1.05 95% CI, 0.76-1.45). Among patients with RA, infliximab was associated with a significant increase in serious infections compared with etanercept (aHR, 1.26 95% CI, 1.07-1.47) and adalimumab (aHR, 1.23 95% CI, 1.02-1.48). Baseline glucocorticoid use was associated with a dose-dependent increase in infections.
Among patients with autoimmune diseases, compared with treatment with nonbiologic regimens, initiation of TNF-α antagonists was not associated with an increased risk of hospitalizations for serious infections.
NGF controls survival, differentiation, and target innervation of both peptidergic and nonpeptidergic DRG sensory neurons. The common receptor for GDNF family ligands, Ret, is highly expressed in ...nonpeptidergic neurons, but its function during development of these neurons is unclear. Here, we show that expression of Ret and its coreceptors GFRα1 and GFRα2 is dependent on NGF. GFR/Ret signaling, in turn, autoregulates expression of both GFRα1 and GFRα2 and promotes expression of TrpA1, MrgA1, MrgA3, and MrgB4, acquisition of normal neuronal size, axonal innervation of the epidermis, and postnatal extinction of the NGF receptor TrkA. Moreover, NGF controls expression of several other genes characteristic of nonpeptidergic neurons, such as TrpC3, TrpM8, MrgD, and the transcription factor Runx1, via a Ret-independent signaling pathway. These findings support a model in which NGF controls maturation of nonpeptidergic DRG neurons through a combination of GFR/Ret-dependent and -independent signaling pathways.
To understand how the brain processes sensory information to guide behavior, we must know how stimulus representations are transformed throughout the visual cortex. Here we report an open, ...large-scale physiological survey of activity in the awake mouse visual cortex: the Allen Brain Observatory Visual Coding dataset. This publicly available dataset includes the cortical activity of nearly 60,000 neurons from six visual areas, four layers, and 12 transgenic mouse lines in a total of 243 adult mice, in response to a systematic set of visual stimuli. We classify neurons on the basis of joint reliabilities to multiple stimuli and validate this functional classification with models of visual responses. While most classes are characterized by responses to specific subsets of the stimuli, the largest class is not reliably responsive to any of the stimuli and becomes progressively larger in higher visual areas. These classes reveal a functional organization wherein putative dorsal areas show specialization for visual motion signals.
Juvenile amyotrophic lateral sclerosis (ALS4) is a rare autosomal dominant form of juvenile amyotrophic lateral sclerosis (ALS) characterized by distal muscle weakness and atrophy, normal sensation, ...and pyramidal signs. Individuals affected with ALS4 usually have an onset of symptoms at age <25 years, a slow rate of progression, and a normal life span. The ALS4 locus maps to a 1.7-Mb interval on chromosome 9q34 flanked by D9S64 and D9S1198. To identify the molecular basis of ALS4, we tested 19 genes within the ALS4 interval and detected missense mutations (T3I, L389S, and R2136H) in the
Senataxin gene (
SETX). The
SETX gene encodes a novel 302.8-kD protein. Although its function remains unknown, SETX contains a DNA/RNA helicase domain with strong homology to human
RENT1 and
IGHMBP2, two genes encoding proteins known to have roles in RNA processing. These observations of ALS4 suggest that mutations in SETX may cause neuronal degeneration through dysfunction of the helicase activity or other steps in RNA processing.
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