Elucidating regulators and biomarkers of synaptic stability during neurodegeneration

Synapses are an early pathological target in a wide range of neurodegenerative conditions including adult-onset Alzheimer’s and Parkinson’s, and diseases of childhood such as spinal muscular atrophy and neuronal ceroid lipofuscinoses (NCLs). However, our understanding of the mechanisms regulating the stability of synapses and their exceptional vulnerability to neurodegenerative stimuli remains in its infancy. To address this, we have used the NCLs to model the molecular alterations underpinning synaptic vulnerability. Our primary objective is to identify novel regulators of synaptic stability as well as highlight novel therapeutic targets which may prove effective across multiple neurodegenerative conditions where synapses are an early pathological target. The NCLs, are the most frequent autosomal-recessive disease of childhood. There are currently 14 individual genes whose mutations result in similar phenotypes including blindness, cognitive/motor deficits, seizures and premature death. This suggests that despite the difference in the initiating mutation and the degenerative processes across this collective group are likely to impact on overlapping pathways. Focusing on two murine models of NCL; one with an infantile onset - CLN1 disease (Ppt1-/-) and one with a juvenile onset - CLN3 disease (Cln3-/-) we made use of the temporo-spatial synaptic vulnerability pattern in these mice to plan proteomic and in silico analyses. This pipeline was utilised to identify perturbed protein candidates and pathways correlating with differential regional synaptic vulnerability. This ultimately allowed the generation of a list of candidate proteins, some of which were relevant to human NCL as they were altered in post mortem brain samples. Interestingly, many of the correlative candidates also appear to show conserved alterations in both NCL forms examined and other neurodegenerative diseases. Next, candidates were genetically and/or pharmacologically targeted to study their modulatory effects on neuronal stability in vivo. This was done using CLN3 Drosophila as a rapid screening assay and led to the successful characterisation of a subset of candidates as either enhancers or suppressors of the CLN3-induced phenotype in vivo. As well as identifying regulators of neuronal stability, following a similar pipeline, we identified a set of putative biomarkers of disease progression in muscle and blood in the Ppt1- /- mice, a subset of which appeared conserved in Cln3-/- mice. One of these conserved candidates presented the same directionality of change in human post mortem brain samples, indicating its relevance to the human NCL. Following this workflow from spatio-temporal profiling of murine synaptic populations, to in silico analyses and in vivo phenotypic assessment, we demonstrate that we can identify multiple protein candidates capable of modulating neuronal stability in vivo and identified putative biomarkers that tracked disease progression

The rat striatum responds to nigro-striatal degeneration via the increased expression of proteins associated with growth and regeneration of neuronal circuitry

BACKGROUND: Idiopathic Parkinson's disease is marked by degeneration of dopamine neurons projecting from the substantia nigra to the striatum. Although proteins expressed by the target striatum can positively affect the viability and growth of dopaminergic neurons, very little is known about the molecular response of the striatum as nigro-striatal denervation progresses. Here, iTRAQ labelling and MALDI TOF/TOF mass spectrometry have been used to quantitatively compare the striatal proteome of rats before, during, and after 6-OHDA induced dopamine denervation. RESULTS: iTRAQ analysis revealed the differential expression of 50 proteins at 3 days, 26 proteins at 7 days, and 34 proteins at 14 days post-lesioning, compared to the unlesioned striatum. While the denervated striatum showed a reduced expression of proteins associated with the loss of dopaminergic input (e.g., TH and DARPP-32), there was an increased expression of proteins associated with regeneration and growth of neurites (e.g., GFAP). In particular, the expression of guanine deaminase (GDA, cypin) - a protein known to be involved in dendritic branching - was significantly increased in the striatum at 3, 7 and 14 days post-lesioning (a finding verified by immunohistochemistry). CONCLUSIONS: Together, these findings provide evidence to suggest that the response of the normal mammalian striatum to nigro-striatal denervation includes the increased expression of proteins that may have the capacity to facilitate repair and growth of neuronal circuitry

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

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 β-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

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term “western blot” suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many “optimized” western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies

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.</p