No description available.

No description available.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Although many cellular processes such as cytoskeletal maintenance and synaptic function are disrupted in ALS, the molecular mechanisms by which these defects arise remain poorly understood. TDP-43, an RNA binding protein linked to the majority of ALS cases, is involved in multiple aspects of RNA metabolism. It is hypothesized that TDP-43 may sequester its mRNA targets into cytoplasmic stress granules during disease progression in turn, inhibiting their localization and/or translation. This work uses a Drosophila model of ALS based on TDP-43, to provide evidence for TDP-43’s role in translation regulation of specific mRNA targets. Using a combination of genetic, molecular, and imaging approaches this work has identified TDP-43 induced post-transcriptional alterations in futsch and hsc70-4 mRNAs. First, futsch/MAP1B is a TDP-43 mRNA target altered at the level of mRNA localization and translation. This results in microtubule instability at the NMJ as evidenced by an increased number of satellite boutons and decreased number of Futsch positive loops that are thought to indicate stable synaptic contacts. Furthermore, overexpression of Futsch mitigates defects in microtubule stability and TDP-43 dependent locomotor dysfunction and also increases lifespan. Second, this work shows that synaptic expression of Hsc70-4, a molecular chaperone critical for synaptic vesicle cycling is involved in multiple steps of the synaptic vesicle cycle, is reduced at the NMJ when TDP-43 is overexpressed in motor neurons. Using a combination of electrophysiology and FM1-43 dye uptake assays, this work shows that motor neuron expression of TDP-43 induces defects in synaptic vesicle endocytosis. Third, this work identifies Fragile X Protein (FMRP) as a neuroprotective protein partner of TDP-43. FMRP overexpression remodels RNP granules, extracts TDP-43 from insoluble complexes, and restores the translation of specific TDP-43 targets. Together, these data provides evidence for translation dysregulation underlying microtubule instability and synaptic dysfunction in ALS pathogenesis and identifies restoration of translation via remodeling RNP granules as a neuroprotective strategy to mitigate toxicity.

Axons possess a continuous network of smooth tubular endoplasmic reticulum (ER), extending from the nuclear envelope throughout the neuron to synapses. Mutations affecting proteins with intramembrane hairpin domains that model tubular ER membrane can lead to the axon degenerative disease, hereditary spastic paraplegia (HSP). However, the extent and mechanisms by which HSP proteins contribute to axonal ER organization and dynamics are unclear. To understand these mechanisms, there is a need to visualize axonal ER in wild-type and mutant live axons. I have therefore aimed to develop these tools in Drosophila larvae and adults, and use them to visualize mutant phenotypes. Firstly, I developed a system to visualize fluorescently marked ER in individual axons in adult fly legs, and tested how this can be used to investigate the effects of loss of intramembrane hairpin HSP proteins on ER in adult legs. Secondly, known mutations affecting HSP hairpin proteins reduce the axonal ER network but not severely; I hypothesized that additional HSP ER membrane proteins might contribute to residual tubule formation; these include Arl6IP, also reported to promote ER tubule formation. I generated transgenic flies to overexpress a fluorescently tagged eGFP::Arl6IP1, and found that this fusion protein localizes within axonal ER. To study whether loss of Arl6IP1 function affects axonal ER, I tested the effects of knockdown on this compartment, but found no consistent effects. To achieve stronger loss of function, I also generated a mutant stock that lacked one of the transmembrane domains and showed a slight developmental delay in homozygous Drosophila larvae. Like mutations in a number of other HSP hairpin proteins, this lesion is homozygous viable, and further characterization of its phenotype will help elucidate how Arl6IP1 contributes to modeling the axonal ER network. In conclusion, my work shows the utility of GFP markers of axonal ER, it can facilitate faster screening for other genes that potentially regulate ER structure and for ageing phenotypes that are not apparent in larval stages, and suggests Arl6IP1 as another HSP protein with a role in axonal ER organization.

Neuronal pentraxin with chromo domain (NPCD) belongs to a family of neuronally-expressed pentraxin proteins thought to be involved in synaptic refinement and plasticity. One isoform of Npcd, neuronal pentraxin receptor (NPR), is a type-II transmembrane protein responsible for the clustering of related neuronal pentraxins 1 and 2. However, recently identified cytosolic NPCD isoforms with no known function were discovered through their interaction with the intracellular domain of a receptor protein tyrosine phosphatase PTPRO. PTPRO is a signaling molecule known to be involved in the development of the nervous system. Additionally, upregulated expression of neuronal pentraxins has been implicated in neuronal cytotoxicity and associated with neurodegenerative diseases. Here, we demonstrate that a novel cytosolic NPCD isoform interacts with the BTB-Kelch protein Mayven/KLHL2. This interaction was identified through a yeast two-hybrid screen using the C-terminal pentraxin domain region of NPCD and confirmed through mammalian cell colocalization and co-immunoprecipitation studies. Domain truncation analysis suggests that the kelch domains of Mayven/KLHL2 are responsible for this interaction with NPCD. We also show that Mayven/KLHL2 is capable of interacting with Cullin 3, an integral protein in the Cullin-RING ubiquitin ligase complex. An in-vivo ubiquitylation assay demonstrates that overexpression of Mayven/KLHL2 increases NPCD ubiquitylation, and suggests a novel E3 ubiquitin ligase function of Mayven/KLHL2 with NPCD as its substrate. Furthermore, we observed an increased propensity of overexpressed NPCD to form aggresomes with coexpression of Mayven/KLHL2. As the formation of aggresomes is often associated with protein aggregation and deposition diseases, including a multitude of neurodegenerative diseases, we tested NPCD overexpression and the effects of Mayven/KLHL2 coexpression on neuronal cytotoxicity and apoptosis. Overexpressed NPCD in hippocampal neuron cultures resulted in increased cytotoxicity and apoptosis, further exacerbated by Mayven/KLHL2 coexpression. Our findings report an interaction between NPCD and Mayven/KLHL2, demonstrate a novel role of Mayven/KLHL2 as an E3 ubiquitin ligase, and explore a possible intersection between the ubiquitin-proteasome degradation pathway, neuronal pentraxins, and neurodegenerative disease.

Alcohol abuse is an enormous problem causing death and disability to over 43 million people worldwide each year (WHO). Chronic alcohol consumption also contributes to abnormal brain morphology and significant brain volume loss indicative of neurodegeneration. Until there are effective treatments to alter maladaptive behavioral patterns in alcohol abuse, more research is needed to prevent alcohol-induced toxicity and degeneration. We used C. elegans as a model system to identify genetic modulators of alcohol toxicity and explored whether prolonged alcohol exposure damages the nervous system. In our study, we exposed L4-larval stage worms to varying concentrations of ethanol for three days and found a dose-dependent deficit in crawling. Furthermore, we evaluated degeneration by assessing the health of neurons using fluorescent reporters. Compared to the untreated group, we found that ethanol-exposed worms had a significant neurodegeneration. Previous findings using C. elegans have suggested that the innate immune pathway may protect against neurodegeneration caused by drug toxicity (Schreiber & McIntire, 2012). We find that deletion of either the innate immune gene nsy-1 (orthologous to the mammalian ASK-1 MAPKKK) or pmk-1 (orthologous to the mammalian p38 MAPK) caused hypersensitivity to ethanol toxicity. Conversely, boosting innate immune signaling via gain-of-function mutation in nsy-1 produced resistance to ethanol toxicity and ameliorated ethanol-induced cholinergic degeneration. Our findings indicate that prolonged exposure to ethanol leads to both behavioral impairments and neuronal degeneration in C. elegans and that the ASK1/p38 MAP kinase pathway may play a role in ethanol-induced damage to the nervous system. / text

Nerve growth factor (NGF) represents a new therapeutic strategy for multiple sclerosis (MS) because of its immunomodulatory and neuroprotective activity. To analyze changes in NGF expression experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats. In the dorsal root ganglia (DRG) of animals with EAE, NGF mRNA and protein increased between 18 - 24 days post induction (dpi) during complete neurological recovery. In the spinal cord (SC) of animals with EAE, NGF mRNA and protein expression increased at 15 dpi and 12 dpi, respectively, to reduce EAE-induced disability. We identified the 25 kDa pro-NGF as a biologically active isoform during EAE. EAE SC axons demonstrate a loss or thinning of myelin which correlated with maximal neurological disability. NGF plays a role in minimizing EAE-induced inflammation and myelin damage to promote neurological recovery. NGF may be an “off switch” for a cytokine-neurotrophin signaling triad to govern the extent of myelin damage.

No description available.

Leucine rich repeat kinase 2 (LRRK2) is a 286 kDa protein expressed in a variety of tissues and cell types, including neuronal tissue and innate immune cells. Mutations in LRRK2 have been linked to inflammatory diseases, most notably Crohn’s disease and Parkinson’s disease. Further to this, LRRK2 expression is induced by innate immune stimuli, and can be phosphorylated by Myd88 directed TLR signalling. Functional experiments were performed using macrophages from WT and LRRK2 knockout mice. Many phenotypes and interactions have been described for LRRK2 in a neuronal or in vitro context; therefore experiments in macrophages were specifically designed to investigate these phenotypes and interactions in an innate immune context. LRRK2 interacts with a range of small GTPase proteins called Rabs, which coordinate and carry out vesicular trafficking, including that of innate immune receptors. Further interactions have been shown with clathrin-mediated endocytic machinery and phagocytic machinery; including cytoskeletal components actin and tubulin. Accordingly, the role of LRRK2 in the expression, membrane localisation, and ligand-induced endocytosis of the innate immune receptors such as TLR4 were assayed. TLR4 plays an important role in immune responses to alpha-synuclein, an immunogenic protein aggregate that accumulates as part of Parkinson’s disease pathology, making it a particularly interesting target for this assay. No effect was shown for LRRK2 on TLR4 expression or receptor mediated endocytosis, so attention was focused upon LRRK2 cytoskeletal interactions. An unclear role of LRRK2 has been described in phagocytosis. Application of LRRK2 KO macrophages in a series of systematic phagocytosis assays was used to demonstrate and clarify that there is no role of LRRK2 in the phagocytosis of simple beads, opsonised material, or complex bacterial targets expressing a range of immunogenic molecules such as LPS. A genome wide approach was applied to further investigate the role of LRRK2 in TLR4 mediated signalling, as well as NOD2 mediated signalling. Comparison of LPS responses between WT and LRRK2 KO genotype macrophages identified a role of LRRK2 in modulating transcription of a range of chemokines and chemokine receptors. This indicates a specific role of LRRK2 in regulating chemotaxis in LPS stimulated cells. Knockout of LRRK2 resulted in a complete reversal of the regulation of the expression of EPAC1, a cAMP inducible protein working in parallel with a previously described LRRK2 interacting protein PKA. EPAC1 acts, at least in part, via Ca2+ signalling. Modulation of signalling through pathways such as Ca2+, Wnt and cAMP appear as a theme in results described in this transcriptomic experiment. A parallel metabolomic approach allowed analysis of ceramide levels in resting and innate immune stimulated macrophages. Ceramides are lipid molecules able to activate the NLRP3 inflammasome, as well as modulate alpha-synuclein pathology via ceramide metabolomic products. In contrast to results described in neuronal tissue, LRRK2 has no effect on ceramide levels in resting macrophages, however stimulation of NOD2 via MDP resulted in a dramatic LRRK2 specific increase in ceramide levels. Together, these results indicate a role of LRRK2 in activated innate immune cells.

Alzheimer’s disease (AD) is neuropathologically characterized by excessive beta -amyloid (Abeta)plaques and neurofibrillary tangles composed of hyperphosphorylated tau in the brain. Although the etiology of genetic cases of AD has been attributed to mutations in presenilin and amyloid precursor protein (APP) genes, in most sporadic cases of AD, the etiology is still unknown and various predisposing factors could contribute to the pathology of AD. Predominant among these possible predisposing factors that have been implicated in AD are age, hypertension, traumatic brain injury, diabetes, chronic neuroinflammation, alteration in calcium levels and oxidative stress. Since both inflammation and altered calcium levels are implicated in the pathogenesis of AD, we wanted to study the effect of altered levels of calcium on inflammation and the subsequent effect of selective calcium channel blockers on the production of pro-inflammatory cytokines and chemokines. Our hypothesis is that Abeta depending on it conformation, may contribute to altered levels of intracellular calcium in neurons and glial cells. We wanted to determine which conformation of Abeta was most pathogenic in terms of increasing inflammation and calcium influx and further elucidate the possibility of a link between altered calcium levels and inflammation. In addition, we wanted to test whether calcium channel blockers could inhibit the inflammation mediated by the most pathogenic form of Abeta by antagonizing the calcium influx triggered by Abeta. Our results in human glial and neuronal cells demonstrate that the high molecular weight oligomers are the most potent at stimulating the release of pro-inflammatory cytokines IL-6 and IL-8 as well as increasing intracellular levels of calcium compared to other conformations of Abeta. Further, L-type calcium channel blockers and calmodulin kinase inhibitors are able to significantly reduce the levels of IL-6 and IL-8. These results suggest that Abeta induced alteration of intracellular calcium levels contributes to its pro-inflammatory effect.