SMN Depleted Mice Offer a Robust and Rapid Onset Model of Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn2B/- mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn2B/- mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn2B/- mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). METHODS: Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. RESULTS: The Smn2B/- mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced ß-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn2B/- mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. CONCLUSIONS: The Smn2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.

UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy.

Deafferentation of motor neurons as a result of defective sensory-motor connectivity is a critical early event in the pathogenesis of spinal muscular atrophy, but the underlying molecular pathways remain unknown. We show that restoration of ubiquitin-like modifier-activating enzyme 1 (UBA1) was sufficient to correct sensory-motor connectivity in the spinal cord of mice with spinal muscular atrophy. Aminoacyl-tRNA synthetases, including GARS, were identified as downstream targets of UBA1. Regulation of GARS by UBA1 occurred via a non-canonical pathway independent of ubiquitylation. Dysregulation of UBA1/GARS pathways in spinal muscular atrophy mice disrupted sensory neuron fate, phenocopying GARS-dependent defects associated with Charcot-Marie-Tooth disease. Sensory neuron fate was corrected following restoration of UBA1 expression and UBA1/GARS pathways in spinal muscular atrophy mice. We conclude that defective sensory motor connectivity in spinal muscular atrophy results from perturbations in a UBA1/GARS pathway that modulates sensory neuron fate, thereby highlighting significant molecular and phenotypic overlap between spinal muscular atrophy and Charcot-Marie-Tooth disease.

Cellular and Molecular Anatomy of the Human Neuromuscular Junction.

The neuromuscular junction (NMJ) plays a fundamental role in transferring information from lower motor neuron to skeletal muscle to generate movement. It is also an experimentally accessible model synapse routinely studied in animal models to explore fundamental aspects of synaptic form and function. Here, we combined morphological techniques, super-resolution imaging, and proteomic profiling to reveal the detailed cellular and molecular architecture of the human NMJ. Human NMJs were significantly smaller, less complex, and more fragmented than mouse NMJs. In contrast to mice, human NMJs were also remarkably stable across the entire adult lifespan, showing no signs of age-related degeneration or remodeling. Super-resolution imaging and proteomic profiling revealed distinctive distribution of active zone proteins and differential expression of core synaptic proteins and molecular pathways at the human NMJ. Taken together, these findings reveal human-specific cellular and molecular features of the NMJ that distinguish them from comparable synapses in other mammalian species.

Molecular analysis of axonal-intrinsic and glial-associated co-regulation of axon degeneration.

Wallerian degeneration is an active program tightly associated with axonal degeneration, required for axonal regeneration and functional recovery after nerve damage. Here we provide a functional molecular foundation for our undertstanding of the complex non-cell autonomous role of glial cells in the regulation of axonal degeneration. To shed light on the complexity of the molecular machinery governing axonal degeneration we employ a multi-model, unbiased, in vivo approach combining morphological assesment and quantitative proteomics with in silico-based higher order functional clustering to genetically uncouple the intrinsic and extrinsic processes governing Wallerian degeneration. Highlighting a pivotal role for glial cells in the early stages fragmenting the axon by a cytokinesis-like process and a cell autonomous stage of axonal disintegration associated to mitochondrial dysfunction.

Proteomic mapping of differentially vulnerable pre-synaptic populations identifies regulators of neuronal stability in vivo.

Synapses are an early pathological target in many neurodegenerative diseases ranging from well-known adult onset conditions such as Alzheimer and Parkinson disease to neurodegenerative conditions of childhood such as spinal muscular atrophy (SMA) and neuronal ceroid lipofuscinosis (NCLs). However, the reasons why synapses are particularly vulnerable to such a broad range of neurodegeneration inducing stimuli remains unknown. To identify molecular modulators of synaptic stability and degeneration, we have used the Cln3 -/- mouse model of a juvenile form of NCL. We profiled and compared the molecular composition of anatomically-distinct, differentially-affected pre-synaptic populations from the Cln3 -/- mouse brain using proteomics followed by bioinformatic analyses. Identified protein candidates were then tested using a Drosophila CLN3 model to study their ability to modify the CLN3-neurodegenerative phenotype in vivo. We identified differential perturbations in a range of molecular cascades correlating with synaptic vulnerability, including valine catabolism and rho signalling pathways. Genetic and pharmacological targeting of key 'hub' proteins in such pathways was sufficient to modulate phenotypic presentation in a Drosophila CLN3 model. We propose that such a workflow provides a target rich method for the identification of novel disease regulators which could be applicable to the study of other conditions where appropriate models exist.

Understanding the molecular consequences of inherited muscular dystrophies: advancements through proteomic experimentation.

INTRODUCTION: Proteomic techniques offer insights into the molecular perturbations occurring in muscular-dystrophies (MD). Revisiting published datasets can highlight conserved downstream molecular alterations, which may be worth re-assessing to determine whether their experimental manipulation is capable of modulating disease severity. AREAS COVERED: Here, we review the MD literature, highlighting conserved molecular insights warranting mechanistic investigation for therapeutic potential. We also describe a workflow currently proving effective for efficient identification of biomarkers & therapeutic targets in other neurodegenerative conditions, upon which future MD proteomic investigations could be modelled. Expert commentary: Studying disease models can be useful for identifying biomarkers and model specific degenerative cascades, but rarely offer translatable mechanistic insights into disease pathology. Conversely, direct analysis of human samples undergoing degeneration presents challenges derived from complex chronic degenerative molecular processes. This requires a carefully planed & reproducible experimental paradigm accounting for patient selection through to grouping by disease severity and ending with proteomic data filtering and processing.

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.

Label-free quantitative proteomic profiling identifies disruption of ubiquitin homeostasis as a key driver of Schwann cell defects in spinal muscular atrophy.

Low levels of survival of motor neuron (SMN) protein cause the neuromuscular disease spinal muscular atrophy (SMA), characterized by degeneration of lower motor neurons and atrophy of skeletal muscle. Recent work demonstrated that low levels of SMN also trigger pathological changes in Schwann cells, leading to abnormal axon myelination and disrupted deposition of extracellular matrix proteins in peripheral nerve. However, the molecular pathways linking SMN depletion to intrinsic defects in Schwann cells remained unclear. Label-free proteomics analysis of Schwann cells isolated from SMA mouse peripheral nerve revealed widespread changes to the Schwann cell proteome, including disruption to growth/proliferation, cell death/survival, and molecular transport pathways. Functional clustering analyses revealed significant disruption to a number of proteins contributing to ubiquitination pathways, including reduced levels of ubiquitin-like modifier activating enzyme 1 (Uba1). Pharmacological suppression of Uba1 in Schwann cells was sufficient to reproduce the defective myelination phenotype seen in SMA. These findings demonstrate an important role for SMN protein and ubiquitin-dependent pathways in maintaining Schwann cell homeostasis and provide significant additional experimental evidence supporting a key role for ubiquitin pathways and, Uba1 in particular, in driving SMA pathogenesis across a broad range of cells and tissues.

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to ß-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.