Congenital insensitivity to pain (CIP) is an extremely rare inherited disorder characterised by the inability to perceive physical pain from birth, resulting in a number of injuries including self-mutilation, repeated burns and fractures. A number of different genes underlying CIP have been identified over the years and all act principally either to direct development or function of nociceptors. In this dissertation, a number of unrelated families with CIP were recruited and novel missense and splicing mutations in NTRK1, NGF and SCN9A were identified in the cohort. The findings presented in this dissertation demonstrate how mutations in the NTRK1 gene can cause the phenotype of CIP, and increase our knowledge of the functions and the role of key residues of TRKA within the cell. I have verified the importance of NGF in the development of nociceptors and demonstrate the overlapping roles of the precursor proNGF with mature NGF as well as providing insight into the role of proNGF as a neurotrophic molecule, in contrast to the wider consensus. Moreover, I provide further evidence that splicing mutations are also responsible for CIP and highlight that splicing mutations are potentially missed in diagnostic labs. Lastly, I have demonstrated that stem cells can be used to study and generate different types of sensory neurons, indicating a potential use as a suitable platform for investigating monogenic disorders. The identification of novel genes, in addition to the dissection of the residues and pathways of known genes, is essential for the development of new analgesics.

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.

The development, maintenance, and survival of neurons depend on the function of neurotrophins such as nerve growth factor (NGF). One population of neurons that rely heavily on NGF for axonal growth and survival is the postganglionic sympathetic neurons. Trauma or disease resulting in injury to the peripheral nervous system causes an increase in the levels of this neurotrophin. This augmentation promotes the collateral sprouting of postganglionic sympathetic axons into those tissues having elevated levels of NGF. Often, NGF-induced sympathetic sprouting occurs in tissues that are normally innervated by these fibers however, high levels of NGF can also promote sprouting of axons into tissues that are normally devoid of sympathetic fibers, such as the sensory ganglia. When postganglionic sympathetic axons grow into the environment of sensory ganglia, they can converge and wrap around a subset of somata (i.e., cell bodies) belonging to primary sensory neurons. This phenomenon, referred to as sympathosensory plexuses is observed in adult mice and rats following peripheral nerve injury, and is also seen in adult transgenic mice that ectopically over express NGF. The overall aim for this project is to examine the morphological and neurochemical features, as well as the overall consequence of sympathosensory plexuses in nerve-injured adult mice and in adult transgenic mice that over express NGF. We hope that this novel information will add to our understanding of the underlying mechanisms associated with the formation of sympathosensory plexuses that occur following injury. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2013-07-23 18:51:47.902

The development, maintenance, and survival of neurons depend on the function of neurotrophins such as nerve growth factor (NGF). One population of neurons that rely heavily on NGF for axonal growth and survival is the postganglionic sympathetic neurons. Trauma or disease resulting in injury to the peripheral nervous system causes an increase in the levels of this neurotrophin. This augmentation promotes the collateral sprouting of postganglionic sympathetic axons into those tissues having elevated levels of NGF. Often, NGF-induced sympathetic sprouting occurs in tissues that are normally innervated by these fibers however, high levels of NGF can also promote sprouting of axons into tissues that are normally devoid of sympathetic fibers, such as the sensory ganglia. When postganglionic sympathetic axons grow into the environment of sensory ganglia, they can converge and wrap around a subset of somata (i.e., cell bodies) belonging to primary sensory neurons. This phenomenon, referred to as sympathosensory plexuses is observed in adult mice and rats following peripheral nerve injury, and is also seen in adult transgenic mice that ectopically over express NGF. The overall aim for this project is to examine the morphological and neurochemical features, as well as the overall consequence of sympathosensory plexuses in nerve-injured adult mice and in adult transgenic mice that over express NGF. We hope that this novel information will add to our understanding of the underlying mechanisms associated with the formation of sympathosensory plexuses that occur following injury. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2013-07-23 18:51:47.902

In these experiments, we primarily investigate the role and source of nerve growth factor (NGF) in peripheral tissues undergoing chronic inflammation. It has been previously determined that there is a significant increase in the levels of NGF following prolonged inflammation of the urinary bladder or the colon, and the first of two projects discussed here mimics this increase with transgenic mice which ectopically produce NGF under control of the smooth muscle alpha-actin promoter. It was determined by this increase that a p75-sensitive increase in sympathetic innervation occurs when an abundance of NGF is produced locally in the descending colon. Sensory innervation in the colon was found to come from two unique populations, one of which increased following heightened NGF levels. The urinary bladders of NGF overexpressing mice were determined to have an increase in sensory axonal density. The second project described here features chemically induced colonic inflammation and observes the nervous and growth factor changes as a response. Transgenic reporter mice are used to observe the cellular source of NGF in the descending colon, which unexpectedly was determined to be Dogiel type II (DgII neurons) based on morphological and chemical characteristics. We report the increase in NGF mRNA and protein observed following a brief 5-day colonic inflammation, and note that there is no increase in axonal density observed. The chemical inflammation did, however, induce an increase in axonal varicosities, used as a measure of axon damage. Finally, a heterozygous null-mutation of NGF was made in a line of transgenic mice to observe changes in local sensory neurons, sympathetic innervation or NGF protein production, but no differences between the heterozygous null mutants and age-matched wild-type siblings were observed. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-09-05 12:08:44.326

Ca2+-mediated regulation of gene expression plays an important role in neuronal plasticity. NFAT (Nuclear Factor of Activated T-cells) is a Ca2+/calcineurin (CaN)-dependent transcription factor that has been implicated in a number of neuronal functions including axon outgrowth, presynaptic remodeling and neural survival. NFAT is activated by Ca2+/CaN-dependent dephosphorylation, whereas re-phosphorylation by glycogen synthase kinase-3β (GSK3β) and several other protein kinases deactivates NFAT and triggers its export from the nucleus. In addition to electrically-mediated Ca2+ signals, neurotrophins can potently regulate NFAT function in neurons as well. However the mechanisms of NFAT activation by electrical activity and neurotrophins are not completely understood. In aim 1, I found that electrical stimulation produced a mitochondrial Ca2+ cycling-mediated prolonged [Ca2+]i elevation (plateau), which profoundly affected NFAT activity. The elimination of the [Ca2+]i plateau by blocking mitochondrial Ca2+ uptake or release strongly reduced nuclear import of NFAT. Furthermore, preventing Ca2+ mobilization from mitochondria diminished NFAT-mediated transcription. In aim 2, I found that NGF, a family of neurotrophins, potentiated NFAT-dependent transcription triggered by electrical activity through the TrkA-PI3K-Akt-GSK3β pathway and this effect was mediated primarily by NFATc3. Monitoring NFATc3 movement in DRG neurons in real time showed that NGF slowed the rate of NFATc3 nuclear export, which was mimicked by inhibiting GSK3β, whereas blockade of PI3K prevented this effect. Taken together, I proposed that mitochondrial Ca2+ cycling functions as a novel regulatory mechanism for NFAT activation and NFATc3 serves as an integrator of electrical activity and neurotrophin signaling in the regulation of gene expression in DRG neurons.

Diversas líneas de evidencia asignan al factor de crecimiento neuronal (NGF), expresado en el espacio endoneural por células de Schwann activadas tras una lesión nerviosa, un papel en el mecanismo del dolor neuropático periférico. Se ha sugerido, pues, que el NGF podría promover sprouting en la zona de la lesión y generar cambios plásticos en la primera neurona sensorial y sus proyecciones centrales, colaborando de este modo al inicio del dolor neuropático. El objetivo general de esta investigación fue evaluar estos posibles mecanismos. Para realizar el estudio se utilizó un abordaje metodológico directo: la administración endoneural de NGF o vehículo. Se utilizaron ratas Sprague-Dawley macho de 250 a 300 g, cuyos nervios ciáticos izquierdos fueron inyectados con 10 µl de solución. Se realizaron pruebas conductuales de dolor neuropático estímulo-dependiente (hiperalgesia térmica y alodinia mecánica), de dolor inflamatorio (prueba de la formalina), se realizó un estudio histológico del nervio ciático inyectado y un estudio inmunohistoquímico para los péptidos sensoriales SP y péptido relacionado al gen de la calcitonina (calcitonin gene-related peptide, CGRP) en el ganglio de la raíz dorsal (dorsal root ganglion, DRG) y el asta posterior de la médula espinal. La administración endoneural de NGF (1 a 30 ng) produjo hiperalgesia térmica, pero no alodinia mecánica. Además, el NGF produjo desmielinización, una modesta degeneración de fibras e indujo la formación de brotes axonales. Estos efectos conductuales e histológicos del NGF fueron comparables y vinculables en cuanto a su relación dosis-efecto como en cuanto a su curso temporal, lo que sugiere que ambos efectos pueden estar relacionados. La administración endoneural de 30 ng de NGF produjo un aumento significativo tanto del porcentaje de células medianas a grandes que contienen CGRP en el DRG, como del área ocupada por fibras que contienen CGRP en las láminas III y IV del asta medular posterior, que corresponden a neuronas sensoriales medianas a grandes. Ambos efectos fueron limitados en el tiempo, prolongándose por 5 días. No se observaron cambios en el contenido de SP ni en el DRG ni en la médula espinal. Finalmente, la administración de 30 ng de NGF produjo un aumento en la fase 2 de la prueba de la formalina el día 3 posinyección de NGF, pero no los días 5 ni 10. En conjunto, estos resultados otorgan un importante apoyo a las hipótesis que asignan un papel al NGF endoneural en el inicio del dolor neuropático. Tanto la hiperalgesia como los efectos histológicos del ciático inyectado y los cambios en la expresión de CGRP han sido descritos en modelos animales de dolor neuropático. Estos resultados además ilustran acerca de los efectos del NGF sobre axones no lesionados, lo que debe ser tomado en cuenta antes de pensar en un eventual uso terapéutico del NGF, que ha sido propuesto por otros autores. Finalmente, los resultados de la prueba de la formalina colaboran a la comprensión de la escasamente conocida relación entre el dolor neuropático y el dolor inflamatorio. / Neuroimmune interactions and neuropathic pain: role of nerve growth factorSeveral lines of evidence suggest that nerve growth factor (NGF), expressed in the endoneurial milieu by activated Schwann cells, has an important role on peripheral neuropathic pain mechanism. Specifically, it has been hypothesized that NGF acts on neuropathic pain by inducing axonal sprouting at the injury site and by generating plastic changes at primary sensory neurons and their central processes. The main objective of this investigation was to evaluate these possible mechanisms. For this study a direct methodological approach was used: the endoneurial administration of NGF or vehicle. Male Sprague-Dawley rats of 250 to 300 g were injected on their left sciatic nerves with 10 µl of solution. Behavioral tests were conducted to evaluate thermal hyperalgesia and mechanical allodynia as neuroapthic pain behaviors, and also to evaluate inflammatory pain (formalin test). Additionally, a histological study of the injected sciatic nerve and an immunohistochemical study for sensory neuropeptides SP and calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG) and the dorsal horn were also carried out. Endoneurial administration of NGF (1 to 30 ng) produced thermal hyperalgesia, but not mechanical allodynia. Moreover, NGF produced demielinization, sporadic fiber degeneration, and induced axonal sprouting. The behavioural and histological effects of NGF were both dose-dependent, and the timecourses of both effects were similar. NGF administration increased the percentage of medium- to large- sized CGRP-positive neuron profiles, and increased CGRP content in laminae III and IV of the dorsal horn, the area occupied by medium to large neuron processes. Both effects were time-limited, lasting for 5 days. NGF did not induce changes on SP content nor in the DRG neither in the dorsal horn. Finally, 30 ng of NGF produced an increase in the phase 2 of formalin test on day 3, but not on subsequent days. Taken together, these results give an important support to the hypotheses that propose that endoneurial NGF has an important role in the onset of neuropathic pain. Thermal hyperalgesia, histological effects on the sciatic nerve and CGRP-expression changes have been also described using animal models of neuropathic pain. Otherwise, these results illustrate about the effects of NGF on uninjured axons. This has to be considered before applying neurotrophic factors therapeutically, as it has been suggested by other authors. Finally, the formalin test results collaborate to the understanding of the largely unknown relation between neuropathic pain and inflammatory pain.

No description available.

The activity of the growth cone is necessary for neuron axon elongation during neuron development and regeneration. This is a highly dynamic process of cytoskeletal recorganization that requires a significant amount of energy provided by the mitochondria. The localization of a sufficient number of mitochondria at the growth cone is essential to support its activity during neuron development and regeneration. Both promotion and inhibition of the motility of growth cones can be induced by intrinsic factors of neuron itself such as cytoskeleton dynamics and motor protein activity, as well as extracellular molecules in the vicinity of the neuron such as nerve growth factor (NGF) and components of the extracellular matrix. The proposed hypothesis is that some of these factors have an impact on the localization and morphology of mitochondria. My work in this project is aimed to determine which of these factors have the greatest impact on mitochondria in neurons. Using sensory neurons isolate / Biomedical Sciences