A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of MSc (Med) Johannesburg, 2011 / Peripheral sensory neuropathy is a common neurological complication of antiretroviral therapy, typically occurring within 6-months of starting Highly Active Antiretroviral Therapy (HAART) which includes stavudine. Therefore, the primary aim of the study was to determine the 6-month incidence of ATN in patients free of neuropathy and beginning stavudine-based HAART for the first time. Also, we examined whether initiating stavudine-based HAART altered the symptoms of patients who had a pre-existing, virus-mediated distal symmetrical polyneuropathy (HIV-DSP). Seventy-five HIV-positive patients were screened for neuropathy, at the Chris-Hani Baragwanath Hospital, using the AIDS Clinical Trials Group neuropathy screening tool. The bilateral presence of atleast one sign (decreased vibration sense in the great toe or absent ankle reflex) and one symptom (pain, paraesthesia or numbness) in the feet was indicative of neuropathy. On recruitment, 52 patients presented without neuropathy and 13 patients presented with HIV-DSP. After 3- months of follow-up (n=46), 23% (10/46) of patients had developed peripheral neuropathy, and by 6-months (n=44), 41% (18/44) of patients had developed neuropathy. Greater disease severity was the only risk factor significantly associated with the development of neuropathy. Eleven (61%) of the 18 patients that developed neuropathy, developed painful symptomatic neuropathy, and only 6 (55%) of these patients were receiving treatment for symptom relief. In patients with HIV-DSP, numbness was the most common symptom reported at baseline and was the only symptom to reduce in frequency across the 6-months. In conclusion, we found that the development of neuropathy is common in the first 6-months of patients initiating stavudine-based HAART.

No description available.

R-loop is a specific nucleic acid structure, and that forms during RNA transcription. It comprises an RNA:DNA hybrid and displaced ssDNA. R-loops are prevalent and dynamic in the mammalian genome occupying up to 5%. R-loops are known to act as modulators of genome dynamics. They regulate a variety of gene transcription mechanisms and influence genomic stability. Dysregulation of R-loop is linked to a variety of human diseases. For this reason, protein factors involved in DNA and RNA metabolism are known to mediate R-loop resolution. Dysfunction of these factors resulted in aberrant R-loop accumulation, often resulting in transcription disruption and genomic instability as reported in tumorigenesis and a number of genetic disorders, including trinucleotide repeat-associated diseases and neurological disorders. Here I describe the roles of Senataxin (SETX) and Replication protein A (RPA) complex in nucleolar R-loop resolution. I demonstrate their function in nucleolar homeostasis and in particular in RNA polymerase I mediated rRNA transcription and the maintenance of nucleolar structure.This dissertation is composed of three sessions. Section 1: I review nucleolar transcription that generate a complex network of RNAs, and their contribution to nucleolar R-loops formation, with multiple roles in maintaining nucleolar homeostasis. Section 2: I describe novel roles of Replication Protein A in nucleolar homeostasis. Senataxin (SETX) mutations are linked to two different neurological disease: Amyotrophic Lateral Sclerosis (ALS4) and Ataxia Oculomotor Apraxia (AOA2) both defective in R-loop resolution. We show that loss of SETX promotes RPA translocation into nucleolus in an R-loop dependent manner where it associates with rDNA. The same nucleolar RPA phenotype is evident in SETX- deficient AOA2 patient cells. We further explored this phenotype under conditions of CPT- induced genotoxic stress, which is coupled with accumulation of nucleolar R loops. Additionally, we show that loss of RPA decreased 47S pre-rRNA levels, but increased “promoter and pre-rRNA antisense” RNA (PAPAS) and promoter RNA (pRNA). Meanwhile, we also showed loss of RPA disrupted nucleolar structure. Section 3: I describe the identification and characterization of nucleolar lncRNA (PAPAS) which encodes a short polypeptide RIEP that plays a role in combating genomic instability and mitochondrial stress. We show that this novel peptide encoded by PAPAS is localized to the nucleolus and mitochondria but is translocated to the peri-nucleolus and peri-nucleus region upon heat shock induced cellular stresses. We also showed that RIEP facilitates SETX protein stability and plays a role in restricting genomic instability possibly through its association with H3K9me3 which maintains a heterochromatin state. Finally, we show that RIEP interacts with CHCHD2 and C1QBP(P32) and may consequently function in mitochondrial stress responses.

Huntington disease (HD) is an inherited neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Metabolic dysfunction is a feature of HD that is recapitulated in HD mouse models. Our lab has shown that circadian feeding rhythms are disrupted in humanized HD mice and restored by suppression of brain HTT. Furthermore, when circadian feeding rhythm is artificially restored, in addition to normalization of metabolic function, liver and striatal HTT is temporarily reduced, demonstrating that HTT is involved in gut-brain feedback. The gut microbiome, which can regulate gut-brain feedback, has been implicated in the pathogenesis of other central nervous system disorders and we hypothesize it also plays a role in HD. The objective of this study is to investigate alterations in relative abundance of HD gut microbiota using existing plasma metabolomics data to identify candidate bacteria. If distinct microbiota profiles are demonstrated, this would provide the basis for future unbiased studies to investigate the complete HD microbiome.

No description available.

The aberrant accumulation of aggregated proteins is a pathologic hallmark across adult-onset neurodegenerative diseases, the majority of which have no effective treatment. Although the relative importance of these structures to pathogenesis has been proposed in several diseases, there is little understanding of how we might accelerate the turnover of aggregated proteins, and in turn, a lack of consensus about whether targeting them would provide any therapeutic benefit. The overarching goal of my dissertation is to address both of these questions by focusing on how the pathway macroautophagy might handle protein aggregates in the adult brain. Aggregation-prone proteins are preferentially degraded through the lysosome-mediated degradation pathway macroautophagy (referred to hereafter as autophagy) (Ravikumar 2002; Iwata 2005; Yamamoto 2006). Although studies suggest that aggregates are degraded in bulk by autophagy (Ravikumar 2002; Iwata 2005), studies show that they are more likely degraded in an adaptor-protein dependent manner (Lemasters, 2005; Kraft, 2008; Hanna, 2012; Isakon, 2012; Filimonenko, 2010). In the Yamamoto lab, we have found that the adaptor, the Autophagy-linked FYVE protein (Alfy/WDFY3), is required for the degradation of detergent-insoluble aggregated proteins through selective autophagy in cell-based systems and the adult brain (Simonsen, 2004; Eenjes, 2016; Filimonenko, 2010; Fox, 2020). Through immunohistochemical and loss-of-function studies, Alfy has been implicated in the turnover of disease-relevant protein aggregates including mHtt, α-synuclein, SOD1, and TDP-43, as well as protein complexes such as the midbody ring (Filimonenko, 2010; Clausen, 2010; Han, 2014; Hocking, 2010; Isakson, 2013; Kadir, 2016). Here, I present a potential strategy to suppress disease progression across neurodegenerative disorders by increasing the levels, and thereby the function, of Alfy. I hypothesized that genetically augmenting Alfy levels in the brain will be sufficient to alleviate aggregate burden and delay the onset of proteotoxic stress in different mouse models of neurodegeneration. Using biochemical and genetic approaches, I conducted an extensive in vivo study, demonstrating that augmenting Alfy expression levels in mice can be neuroprotective, and that Alfy may be an influential genetic modifier of neurodegenerative disease. Using two independent genetic approaches that upregulate Alfy expression, I found that they both dramatically delay the onset of disease phenotypes in mouse models of Huntington’s disease, synucleinopathy and TDP-43 proteinopathy. First, I found that ectopic overexpression of Alfy has a pronounced, neuroprotective effect on reducing aggregation, improving motor function, and extending survival in disease models. In parallel, I used mouse genetics to verify the potency of a rare Alfy variant identified in a large Venezuelan cohort of Huntington’s disease that correlated with delayed onset in Huntington’s disease by 10-20 years. Excitingly, in support of our hypothesis, I found that the presence of this single nucleic acid polymorphism led to an increase in steady state levels of Alfy in both humans and in mice, and it was sufficient to recapitulate the benefits of ectopic Alfy overexpression. Taken together, these studies demonstrate that increasing Alfy levels in the brain are sufficient to augment the turnover of aggregated proteins, and may be an effective therapeutic strategy that can be beneficial across neurodegenerative diseases.

The focus of this study was to assess the cardiovascular effects in hens of a single 2.5 mg/kg intramuscular injection of phenyl saligenin phosphate (PSP) into the breast muscle. Parameters were measured at 1, 3, 7, and 20 days post treatment. All hens developed clinical signs of delayed neuropathy by day 10 and these signs were maximal by day 20. Alterations of measured parameters were observed prior to the onset of clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) (days 1, 3, and 7) as well as when maximal clinical signs were evident (days 15-21). Significant decreases in the activities of brain NTE and plasma cholinesterase as well as decreases in weight and the level of pcO2 and an increase in peripheral resistance were observed prior to evidence of clinical signs of OPIDN. When maximal signs of OPIDN were present, brain NTE and plasma cholinesterase were at control levels but brain cholinesterase was significantly increased. Significant decreases in body weight and arterial pCO2 and significant increases in limb venous flow, arterial blood pressure, and hematocrit were seen at this time. / Master of Science

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 17, 2009). Includes bibliographical references.

Sprague-Dawley rats were administered daily, subcutaneous injections of methylmercuric chloride at a dose of 5 mg/Hg/kg beginning on postnatal day 5. By their fourth postnatal week, animals exhibited a constellation of neurological signs of motor impairment which resembled the cerebral palsy syndrome of humans perinatally exposed to methylmercury. Routine histological examination of the brain revealed no gross differences between methylmercury-treated (MeHg), normal control (NC) or weight-matched littermates. The histochemical localization of the mitochondrial enzyme cytochrome oxidase (CO) was utilized in Experiment I to examine possible alterations in the metabolic activity of motor nuclei which might contribute to the observed movement and postural disorders. A population of intensely-staining cytochrome oxidase neurons (ICONs) in the magnocellular portion of the red nucleus (RMC) and interrubral mesencephalon (IRM) were conspicuously present in all MeHg animals at the onset of motor impairment. These morphologically, histochemically, and anatomically distinct neurons did not exhibit intense CO staining in control animals. Conversely, a significant decrease was demonstrated in the oxidative metabolic activity of many neurons in the substantia nigra, zona reticulata of MeHg animals. In Experiment II, the postnatal appearance of ICONs was morphometrically quantified in MeHg animals sacrificed at PND 14, 16, 18, 20, 22, or 25. The histochemically-defined onset of increased metabolic activity in ICONs was first observed on PND 16, at least one week before the onset of clinical signs of neurological impairment. This was the earliest manifestation of methylmercury neurotoxicity yet described in this animal model. A subsequent four-fold increase in the total number of ICONs at PND 18 was followed by a gradual decrease in number to PND 25. Significantly more of the ICONs were found in the IRM than in the RMC at PND 18 & 20. The possibility that the increased activity of ICONs may result from disinhibition of specific afferents to the red nucleus was addressed by introducing either hemidecortication or hemicerebellectomy on PND 10 and then morphometrically determining the deviation from symmetry in the bilateral distribution of the total number of ICONs in the RMC and IRM at PND 22. The distribution of ICONs was symmetrical and not different in either hemidecorticate or unoperated controls. A significant (36%) decrease in the total number of ICONs was observed in both the RMC and IRM contralateral to hemicerebellectomy. The identical ipsilateral regions did not differ from control or hemidecorticate MeHg animals. In Experiment III, the anatomical distribution of major histocompatability complex antigens (MHC) in the brain of MeHg animals was examined using immunohistochemical methods. MHC immunoreactivity was widely distributed throughout the brain of MeHg animals. Areas with low immunoreactivity, or lack of it, stand out and include all of the hippocampus, thalamus, pyriform and entorhinal cortex, and lateral cerebellar hemispheres. Moderate staining intensity was observed in neocortical areas, basal forebrain, caudate-putamen and cerebellar vermis. Strong immunoreactivity was found in red nucleus, substantia nigra, cingulate cortex, retrosplenial cortex, presubiculum, parasubiculum and vestibular nuclei. It was suggested that the increased activity of ICONs likely contributes to the movement and postural disorders resulting from methylmercury intoxication. The increased activity in ICONs was determined to be, at least partially, dependent upon cerebellar input. The results are discussed with reference to the toxic effects of methylmercury and specifically to the susceptibility of GABAergic interneurons in perinatal trauma. Possible analogies are drawn between the mechanisms of methylmercury-induced cerebral palsy syndrome and those of other developmental movement and postural disorders. / Medicine, Faculty of / Graduate

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.