Genetic factors influencing the peripheral nervous system in health and disease

Lower motor neurons of the peripheral nervous system are responsible for innervating skeletal muscle and controlling all voluntary movements of the body. Degeneration of motor neurons underlies conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors that influence the form and function of the peripheral nervous system in vivo will be important for our understanding of the neuromuscular system in health and disease. Here, I have studied the effects of three different genes and their respective protein products on the peripheral nervous system: yellow fluorescent protein (YFP), apolipoprotein E (apoE) and Ercc1 (excision repair cross-complementing group 1). YFP has been used as a reporter protein in many fields of research, including as a powerful tool for visualising neurons in mice. It is used under the assumption that it is biologically inert. However, my findings have revealed that YFP expressed in neurons in mice is not inert: it induces a cell stress response at both the RNA and the protein level and alters the time course of dying-back neuropathy. ApoE is a lipid transport protein with three distinct isoforms in humans (apoE2, apoE3 and apoE4), which are known to differentially affect risk and outcome in a number of central nervous system disorders. However, the effects of different apoE isoforms on the peripheral nervous system have yet to be established. I have shown that apoE4 delays peripheral nerve regeneration and subsequent neuromuscular junction reinnervation compared to apoE3, in the absence of any effects on normal form or function, degeneration or developmental plasticity. Ercc1 protein is involved in several DNA repair systems. Ercc1Δ/- mice have reduced levels of functional Ercc1 protein, which leads to a reduced life span and motor abnormalities, potentially due to a build of up DNA damage. Here I have shown that Ercc1Δ/- mice also have increased abnormalities at the neuromuscular junction (an early pathological target in neurodegeneration) with age. These findings contribute significantly to our understanding of the influence of specific genes on the form and function of the peripheral nervous system in health and disease

Investigating the mechanisms underlying cholinergic regulation of dopamine release

Dopamine (DA) is a critical regulator of movement and motivation and disruptions to its signalling underlie various psychomotor disorders, including Parkinson’s disease (PD) and addiction. DA neurons projecting from the midbrain to the striatum form extensive axonal arborisation which are subject to local neuromodulation, gating how action potential firing at the level of DA neuron soma is translated into axonal DA release. Unravelling these mechanisms is therefore fundamental to better understand DA regulation of striatal output in health and disease. Acetylcholine (ACh) is one of the major local modulators of striatal DA release. ACh mainly originates from striatal cholinergic interneurons (ChIs) and dysregulation to striatal cholinergic signalling is also implicated in a range of neuropsychiatric and neurodegenerative disorders. ACh acts at nicotinic ACh receptors (nAChRs) localised on DA axons and modulates DA release in a multiplicity of ways, from directly driving DA release bypassing action potential firing by DA somata, to promoting short-term depression of DA released during burst stimulation. Despite this fundamental role, striatal ACh release dynamics are currently incompletely characterised, mainly due to a lack of tools for direct, real-time measurement of ACh release, only recently filled by the development of genetically encoded fluorescent sensors (GRABACh). In this thesis, I used GRABACh in mouse striatal slices to characterise the major properties of striatal ACh release, a fundamental step to help unravel the mechanisms underlying cholinergic modulation of DA release. I found that ACh in dorsal and ventral striatum is dynamically released according to activity in ChIs and that acetylcholinesterase (AChE) modulates the spatiotemporal dynamics of evoked ACh release and tonic ambient levels of ACh. Furthermore, I show that ACh release is modulated by GABA acting at GABAA and GABAB receptors and inhibited by endogenous DA acting at D2- receptors (D2Rs). I then investigated the complex relationship between changes in ACh release and how these can dynamically gate DA release via nAChRs, combining GRABACh sensors and FCV to measure ACh and DA release in mouse striatal slices. I found that the magnitude of ACh-driven DA release is sensitive to changes in ACh levels but insensitive to activity in ChIs. Furthermore, I found that nAChRs regulate short-term plasticity of DA but not ACh release and are not functionally associated with the calcium binding protein calbindin D-28K in DA axons. Finally, I’ve explored maladaptive alterations in striatal cholinergic signalling in a human a-synuclein overexpressing mouse model of early parkinsonism. In this model, we found lower ACh release and stronger regulation of tonic ACh by AChE. In the early parkinsonian model, we also reported stronger D2R-mediated regulation of ACh release and no changes in tonic GABAergic inhibition. Overall, this thesis aimed at investigating the dynamic interplay between ACh and DA release in the striatum. The data here presented provide a novel characterisation of ACh release dynamics in health and early Parkinson’s disease, together with previously unappreciated aspects of the dynamic gating of DA release by ACh

Identification of Biomolecular Pathways Associated With the Central Nervous System Based Symptoms of Gulf War Illness

The clinical profile of GWI is characterized by the presence the central nervous systems (CNS) symptoms which include memory impairment, anxiety, widespread pain and motor problems. Now, even twenty years later, veterans with GWI continue to suffer from these persistent symptoms. Currently, there is no treatment available for treating GWI, which is largely due to the complexity of the clinical presentation of this illness and the heterogeneity of GW exposures. The main goals of this thesis were to develop and characterize GW agent exposure mouse models that recapitulate the CNS symptoms of GWI and to identify the underlying aberrant biological pathways. Three major objectives were undertaken to accomplish these goals: (1) Two mouse models of GW-agent exposure were developed using combination of the anti-nerve gas treatment pyridostigmine bromide (PB), pesticides (permethrin), an insect repellent (N,N-Diethyl-meta-toluamide) and stress. Neurobehavioral studies show that combined exposure to GW agents produced anxiety and sensorimotor deficits in one mouse model and anxiety and cognitive impairment in the other. Neuropathological studies showed a presence of astrogliosis in both models. (2) Exploratory proteomic studies suggested that lipid-metabolism and immune/inflammation were associated with GW-agent exposure. (3) As lipid dysfunction is upstream of the inflammatory pathways, a lipidomics approach was used to identify the GW-agent exposure dependent lipid profiles. Lipid profiles of mouse models of GW-agent exposure were compared with those of other neurological conditions to identify profiles that were unique to GW-agent exposure. Lipid profiles were interrogated to identify lipid-metabolism pathways that may be amenable to therapeutic targeting. Studies described in this thesis provide novel insight into the pathobiology of GWI and suggest that pathways involved in phosphatidylcholine metabolism might be of therapeutic value in treating the CNS symptoms of GWI

Mechanisms of disease pathogenesis in Spinal Muscular Atrophy

Low levels of survival motor neuron (SMN) protein cause the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA), through mechanisms that are poorly defined. SMN protein is ubiquitously expressed, however the major pathological hallmarks of SMA are focused on the neuromuscular system, including a loss of lower motor neurons in the ventral horn of the spinal cord and atrophy of skeletal muscle. At present there is no cure for SMA. Most research to date has focused on examining how low levels of SMN lead to pathological changes in motor neurons, therefore the contribution of other tissues, for example muscle, remains unclear. In this thesis I have used proteomic techniques to identify intrinsic molecular changes in muscle of SMA mice that contribute to neuromuscular pathology in SMA. I demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomics data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. In addition robust up-regulation of VDAC2 and down-regulation of parvalbumin was confirmed in two mouse models of SMA as well as in patient muscle biopsies. Thus intrinsic pathology of skeletal muscle is an important event in SMA. I then used proteomics to identify individual proteins in skeletal muscle of SMA that report directly on disease status. Two proteins, GRP75 and calreticulin, showed increased expression levels over time in different muscles as well as in skin samples, a more accessible tissue for biopsies in patients. Preliminary results suggest that GRP75 and calreticulin can be detected and measured in SMA patient muscle biopsies. These results show that proteomics provides a powerful platform for biomarker identification in SMA, revealing GRP75 and calreticulin as peripherally accessible potential protein biomarkers capable of reporting on disease progression in muscle as well as in skin samples. Finally I identified a role for ubiquitin-dependent pathways in regulating neuromuscular pathology in SMA. Levels of ubiquitin-like modifier activating enzyme 1 (UBA1) were reduced in spinal cord and skeletal muscle tissue of SMA mice. Dysregulation of UBA1 and subsequently the ubiquitination pathways led to the accumulation of β-catenin. I show here that pharmacological inhibition of β-catenin robustly ameliorates neuromuscular pathology in animal models of SMA. Interestingly, downstream disruption of β-catenin was restricted to the neuromuscular system in SMA mice. Pharmacological inhibition of β-catenin failed to prevent systemic pathology in organs. Thus disruption of ubiquitin homeostasis, with downstream consequences for β-catenin signalling, contributes to the pathogenesis of SMA, thereby highlighting novel therapeutic targets for this disease

The effects of grape seed procyanidin extract on insulin synthesis and secretion

Las procianidinas son compuestos bioactivos presentes en frutas y vegetales. Aunque se conocen los efectos beneficiosos de estos compuestos en la homeostasis de la glucosa, su acción en la funcionalidad de la célula β no es clara. La presente tesis doctoral se ha centrado en describir los efectos de las procianidinas en la síntesis y secreción de insulina. Nuestros resultados muestran la capacidad de las procianidinas de modificar la funcionalidad de la célula β aumentando la relación insulina plasmática/mRNA, aunque la efectividad del tratamiento depende de la situación fisiológica. En situaciones no patológicas, las procianidinas afectan la insulinemia modificando la síntesis, secreción y/o degradación de la insulina. En situaciones de resistencia a la insulina, el tratamiento crónico con procianidinas disminuye la síntesis y secreción de insulina gracias a su acción limitando el acúmulo de lípidos. En cambio, en un modelo más dañado (obesidad genética), las procianidinas ejercen efectos similares pero no son capaces de mejorar la hipersinulinemia. En conclusión, las procianidinas, en las dosis ensayadas, pueden utilizarse únicamente como compuestos bioactivos limitando la disfuncionalidad de la célula β en sus estados iniciales.Les procianidines són compostos bioactius presents en fruites i vegetals. Tot i que es coneixen els efectes beneficiosos d’aquests compostos en l’homeòstasi de la glucosa, la seva acció en la funcionalitat de la cèl•lulaβ no és clara. La present tesi doctoral s’ha centrat en descriureels efectes de les procianidines en la síntesi i secreció d’insulina. Els nostres resultats mostren la capacitat de les procianidines de modificar la funcionalitat de la cèl•lula β augmentant la relació insulina plasmàtica/mRNA, tot i que l’efectivitat del tractamentdepèn de la situaciófisiològica. En situacions no patològiques, les procianidines afecten la insulinèmia modificant la síntesi, secreciói/o degradació d’insulina. En situacions de resistència a la insulina, el tractamentcrònicamb procianidines disminueix la síntesi i secreció d’insulina gràcies a la seva acció limitant l’acumulació de lípids. En canvi, en un model més danyat (obesitat genètica), les procianidines exerceixen efectes similars però no son capaces de millorar la hiperinsulinèmia. En conclusió, les procianidines, en les dosis assajades, podenutilitzar-seúnicament coma compostos bioactiuslimitant la disfuncionalitat de la cèl•lula β en els seus estats inicials.Procyanidins are bioactive compounds found in fruits and vegetables widely consumed. It has been reported that procyanidins show some beneficial effects on glucose homeostasis, although their effects on β-cell functionality remain unresolved. This doctoral thesis is focus on describing the effects of procyanidins on insulin synthesis and secretion. Our results showed that procyanidins modify β-cell functionality through increasing the plasma insulin/mRNA ratio, although the effectiveness of the treatment depends on the physiological situation. Under non-pathological situation, procyanidins affected insulinaemia by modifying insulin synthesis, secretion and/or degradation activity. Under insulin-resistance situation, chronic procyanidins administration decreased insulin synthesis and secretion, thanks to its lipid-lowering effect. Otherwise in a more damaged model, Zucker fatty rat, procyanidins treatment is not able to reduce insulin plasma levels although they repress insulin expression. In conclusion, procyanidins could be used as bioactive compound to limit β-cell dysfunctions under high-palatable diets, but at the assayed doses, it is not enough to counteract a strong metabolic disruption

Oxidative, inflammatory and vascular factors in Alzheimer's disease

In spite of impressive recent progress, the aetiopathogenesis of Alzheimer’s disease (AD) remains incompletely understood. The distinctive neuropathological features of AD, in particular the plaques and tangles, have been the particular focus of most aetiological theories. It is well accepted that AD is a multifactorial disease, with alterations to a variety of brain structures and cell types, including neurons, glia and the brain vasculature. Studies of risk factors have revealed a diversity of genetic variables that interact with health, diet and lifestyle-related factors in the causation of AD. These factors influence the structure, aggregation and function of a set of proteins that are increasingly the focus of research. The work in this thesis has focused on the pathophysiological aspects of some of these proteins in a number of cellular compartments and brain. Several assays have been established and techniques utilized in the completion of this work, including; differential detergent fractionation of brain tissue, 1D and 2D PAGE, western blotting with chemiluminescence detection, ELISA assays of Abeta 1-40 and 1-42, quantitative ECNI GCMS of o- and m-tyrosine as well as metabolites of the kynurenine pathway, quantitative MALDI-TOF assay of hemorphins and LCMSMS based proteomics, to identify proteins with altered expression levels in AD relative to control brain tissue. A variety of regional differences have been observed in the biochemistry of the AD cortex which are probably the outcome of local response variations to AD pathology. One of the most consistent threads throughout this work has been an apparent resilience of the occipital lobe relative to the other brain regions, as reflected in lower overall levels of oxidative stress and increased levels of proteins associated with metabolic processes, neuronal remodeling and stress reduction

Copine III interacts with ERBB2 and promotes tumor cell migration

Breast cancer is the most prevalent form of cancer in females: one of nine women develops breast cancer during her lifetime and it is predicted that one in 27 women will die as a result of this disease. Moreover, it is anticipated that with almost 30 % of females affected, breast cancer will be the most frequently diagnosed cancer in 2009 (www.cancer.org). Given these facts, much time and resources have been provided to research in the breast cancer area. The ErbB2 receptor tyrosine kinase is one of the most-studied oncogenes in breast cancer as amplification and overexpression of the ERBB2 gene is known to occur in up to 25 % of all affected patients and is correlated with a highly aggressive disease and poor patient prognosis. Our study focused on signaling molecules interacting with the C-terminal regulatory region of the ErbB2 receptor. We used T47D breast cancer cells metabolically labeled with SILAC to identify binding partners of the pTyr1248 site of ErbB2. Using a peptide affinity pull-down approach followed by quantitative mass spectrometry, we identified Copine III as a novel interaction partner of ErbB2-pTyr1248. Copine III belongs to a family of Ca2+-dependent phospholipid binding proteins that is conserved from plants to humans. All copines carry two C2 domains followed by an A domain, similar to the von Willebrand A domain of integrins, in their C-terminus. Although Copine III is ubiquitously expressed, to date it has not been assigned a function downstream of ErbB2. In this study we first analyzed the biochemical properties of Copine III and its interaction with ErbB2. We show that Copine III is a cytoplasmic protein that localizes to the nucleus and the plasma membrane in a Ca2+-dependent manner and upon stimulation of the cells with the ErbB ligand heregulin (HRG). We used FRET acceptor photobleaching to show that Copine III and ErbB2 not only co-localize in HRG-stimulated breast cancer cells, but also interact at the plasma membrane. This co-localization is blocked when the cells are treated with the ErbB2 inhibitor AEE788, implying that Copine III only interacts with phosphorylated active ErbB2. The second goal of my studies was to place Copine III within a signaling pathway downstream of ErbB2. For this, we again used SILAC together with quantitative mass spectrometry and identified the scaffolding protein RACK1 as a binding partner of Copine III. We were able to show that Copine III, RACK1 and the adaptor molecule Shc form a complex with ErbB2 in HRG-stimulated cells. RACK1 has been implicated in focal-adhesion mediated cell migration and here we demonstrate that Copine III localizes to focal adhesions and is required for ErbB2-dependent cell migration. Moreover, knock-down of Copine III affects Src kinase activity and the subsequent phosphorylation of focal adhesion kinase, resulting in the observed defects in cellular migration. Thus Copine III is an important effector molecule in ErbB2-mediated cell migration. Finally, we analyzed Copine III expression in the broader context of cancer, looking at carcinomas of the breast, prostate and ovary. In a set of 49 breast cancer tumor samples, 10 of the 11 cases with ERBB2 amplification display elevated levels of Copine III. This connected well with the protein expression levels of Copine III in a panel of breast cancer cell lines that also correlated with ErbB2 amplification. In published ovarian and prostate transcriptome studies, Copine III mRNA levels are upregulated in cancer as compared to normal tissue. Based on these findings, we performed immunohistochemistry (IHC) stainings of Copine III on breast, prostate and ovarian tissue microarrays. While some Copine III staining was evident in normal breast, normal prostate and ovarian tissues have very low levels of Copine-III. Strikingly, tumors of all three types showed higher Copine III levels. To summarize, we present Copine III here for the first time as an interaction partner of the ErbB2 receptor. Copine III interacts with ErbB2 in a Ca2+- and HRG-dependent manner and is required for tumor cell migration. Furthermore, Copine III levels were found to be upregulated in tissue microarrays of breast, ovarian and prostate tumor tissue as compared to normal tissue. Together, these findings imply a biological function for Copine III in cancer progression and suggest that further studies into the functions of Copine III are merited