Masters Degrees (Physiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/7046

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Effect of highly active antiretroviral therapy (HAART) on HIV-1 tat protein-induced neurotoxicity.
(2014) Zulu, Simo Siyanda.; Daniels, William Mark Uren.; Mabandla, Musa Vuyisile.
Background: HIV-1-trans-activating (Tat) protein has been associated with development of HIV associated neurocognitive disorder (HAND). Previous studies have demonstrated that Tat protein causes neurotoxicity through an increase in reactive oxygen species (ROS) leading to damage of proteins and other cellular components. Tat has also been shown to cause excessive production of pro-inflammatory cytokines. However the role of antiretroviral agents in the neuropathology of HIV is not known. The objective of this study was to investigate whether a combination of antiretroviral drugs (Zidovudine, Lamivudine and Efavirenz, a highly active antiretroviral therapy, HAART) is effective in reducing the toxic effects of Tat protein in the rat hippocampus. Materials and methods: Male Sprague-Dawley rats were divided into four groups (n=10 per group). Each rat received bilateral intrahippocampal injection of either Tat protein (5μg/10μL) or vehicle, followed 7 days later by a combination of antiretroviral drugs (Zidovudine 12mg/kg, Lamivudine 6mg/kg and Efavirenz 24mg/kg) or saline injected intraperitoneally, twice a day, for 7 days. After treatment, animals were sacrificed and hippocampal tissue was collected for analysis of cleaved caspase-3, 4- hydroxynonenal (NHE), tumor necrosis factor alpha (TNF-α), phosphorylated extracellular signal regulated kinase (pERK) and Synaptophysin. Results: Tat increased cleaved caspase-3 levels in the hippocampus. Antiretroviral treatment decreased the Tat-induced increase in cleaved caspase-3. Tat increased HNE, a marker of lipid peroxidation and reduced hippocampal synaptophysin. The latter Tat-induced effects were not reversed by antiretroviral treatment. The antiretroviral drug combination activated the pERK pathway and increased TNF-α levels in hippocampal tissue, independent of Tat infusion. Discussion: Our findings showed that antiretroviral drugs reversed Tat-induced cleaved caspase-3, reducing apoptosis but did not reverse Tat-induced increase in lipid peroxidation and the synaptic marker, synaptophysin. The evidence suggests that the combination of antiretroviral drugs may be toxic, elevating hippocampal pERK and TNF-α levels. However, these effects could also be beneficial to the individual, since TNF-α has been shown to inhibit viral replication. The present results provide novel insight into the mechanism of antiretroviral action.
The effects of vasopressin and oxytocin on methamphetamine : induced place preference behaviour in rats.
(2012) Subiah, Cassandra.; Daniels, William Mark Uren.; Mabandla, Musa Vuyisile.
Methamphetamine is a highly addictive stimulant drug whose illicit use and resultant addiction has become an alarming global phenomenon. The mesolimbic dopaminergic system in the brain, originating in the ventral tegmental area and terminating in the nucleus accumbens, has been shown to be central to the neurobiology of addiction and the establishment of addictive behaviour. This pathway, as part of the reward system of the brain, has also been shown to be important in classical conditioning, which is a learnt response. This common pathway has supported theories suggesting addiction as a case of maladaptive associative learning. Within the modulation of learning and memory, the neurohypophyseal hormones vasopressin and oxytocin have been seen to play a vital role. Vasopressin exerts a long- term facilitatory effect on learning and memory processes. Studies have shown that the stress responsive AVP V1b receptor systems are a critical component of the neural circuitry underlying emotional consequences of drug reward. Oxytocin, on the other hand, has an effect on learning and memory opposite to that of vasopressin. Previous studies have shown that oxytocin caused a decrease in heroin self-administration, as well as attenuated the appearance of cocaine-induced hyperactivity and stereotyped behaviour. Therefore, we adopted a reinstatement conditioned place preference model to investigate whether a V1b antagonist or oxytocin treatment would cause a decrease in methamphetamine seeking behaviour. Behavioural findings indicated that methamphetamine induced a change in the place preference in the majority of our animals. This change in preference was not seen after vasopressin administration in the extinction phase. On the other hand, the change in place preference was enhanced during the reinstatement phase in the animals treated with oxytocin. Striatal dopamine levels were determined, as methamphetamine is known to increase dopamine transmission in this area. Results showed that rats that received both methamphetamine and oxytocin had significantly higher striatal dopamine than those that received oxytocin alone. Western blot analysis for hippocampal cyclic AMP response element binding protein (CREB) was also conducted as a possible indicator of glutamatergic NMDA receptor activity, a pathway that is important for learning and memory. The Western blot analysis showed no changes in hippocampal pCREB expression. Overall our data led us to conclude that methamphetamine treatment can change place preference behaviour in rats and that this change may be partially restored by vasopressin antagonism, but exaggerated by oxytocin.
Evaluating a new drug to combat Alzheimer's disease.
(2014) Sibiya, Siyabonga G.; Daniels, William Mark Uren.; Mabandla, Musa Vuyisile.
Alzheimer’s disease (AD), a progressive neurodegenerative disorder that affects mostly the limbic system and the neocortical areas of the brain, is the most prevalent form of dementia affecting the elderly population. Twenty-nine million people live with the disease worldwide, 10% of the population > 65 years of age and 50 % of the population > 85 years of age. These figures are expected to increase exponentially over the next few decades and reach 81.1 million by the year 2040. Hallmark lesions include extracellular deposition of β-amyloid protein (Aβ) fibrillar plaques and intraneuronal neurofibrillary tangles (NFTs), which impair synaptic plasticity in the target regions of the brain thereby producing a progressive decline in cognitive function, with the earliest signs observed in learning and memory. Current therapies of AD are merely palliative and only slow down cognitive decline. In a recent study a novel compound, poly-N-methylated amyloid beta (Aβ)-peptide C-terminal fragments (MEPTIDES) was shown to reduce Aβ toxicity in vitro and in Drosophila melanogaster, however whether this novel drug is equally effective in mammals to inhibit Aβ-induced toxicity remains unclear. Accordingly in the present study we investigated the effects of MEPTIDES on the neurotoxicity induced by a single intracerebral (i.c.) injection of Aβ42 into the dorsal hippocampus of adult male Sprague-Dawley (SD) rats, a model of AD-like impaired learning and memory, and explored the implications of these findings for possible future management therapies or AD.
HIV-1 transactivator of transcription (TAT) protein causes neurotoxicity via astrocyte activation.
(2015) Ganga, Yashica.; Daniels, William Mark Uren.; Ramsuran, Duran.
HIV is most well-known for its negative effects on the immune system and the resulting development of AIDS, however it also has severe damaging effects on the central nervous system. Many infected individuals exhibit neuropsychological and behavioral dysfunctions which are collectively referred to as HIV-associated dementia (HAD). One of the worrying aspects of HAD is the fact that current anti-retroviral therapy, while being effective in managing the onslaughts of HIV on the immune system, is less efficient in addressing the impact of HIV on the CNS. The HIV-1 regulatory protein, transactivator of transcription (Tat), is responsible for the transactivation of viral transcription, and has been identified as a possible etiological factor of HAD. Neurotoxicity caused by HIV-1 is an indirect effect since the virus is unable to infect neurons directly. We subsequently hypothesized that HIV-1 infects non-neuronal cells in the CNS which leads to their activation, resulting in the release of cytokines that are detrimental to neurons. The aims of this study was therefore to (i) determine whether Tat activates astrocytes, (ii) establish whether astrocytes exposed to Tat result in the release of IL-6 and TNF-α, and to (iii) assess whether these cytokines can induce apoptosis of neuronal cells. Our study has shown that Tat does activate astrocytes and that activated astrocytes do indeed release cytokines IL-6 and TNF-α into their growth medium. Tat treated cells release more than double the amount of IL-6 than the control group of untreated astrocytes. We also observed that exogenous administration of these cytokines (individually or collectively) to neurons has the ability to cause neuronal apoptosis. Interestingly in combination, these cytokines show no cooperative effect. Our data also showed that neurons, when exposed to the culture medium of astrocytes that were subjected to Tat, exhibit hallmarks of apoptosis similar to that induced by IL-6 and TNF-α. Our findings led us to conclude that in individuals with HIV-infection, the virus activates astrocytes possibly via the production and release of Tat. This causes the astrocytes to secrete pro-inflammatory cytokines (e.g. TNF- α and IL-6) that may induce apoptotic cell death of neurons. This mechanism may explain the development of HAD.
Tat protein induced neurocognitive dysfunction.
(2013) Makhathini, Khayelihle Brian.; Daniels, William Mark Uren.; Mabandla, Musa Vuyisile.
Human immunodeficiency virus type 1 (HIV-1) is a major health threat in South Africa. Studies have shown that HIV affects the central nervous system (CNS), and can initiate a progressive neurodegenerative process culminating in HIV associated dementia. Tat protein and gp120 are two viral proteins that have been linked to the neurotoxic effects of HIV. The present study used an animal model to study the effects of tat protein on the brain. The study also investigated the ability of picolinic acid to block the damaging effects of Tat protein. Sprague-Dawley rats were divided into four groups. The control group received a bilateral injection of saline (100μl), and the experimental group received a bilateral injection of tat protein (5μg/100μl) into the hippocampus. The other two groups of rats were treated similarly, but also received picolinic acid at a concentration of 3.36mg/3ml, injected intraperitoneally (i.p) before the intrahippocampal injections. Two tests were performed to assess the cognitive behaviour of all the animals, namely the light/dark box and Morris water maze. After the behavioural tests, the brains of animals were collected for evaluating the activity of the enzyme caspase 3 by various means. Some brains were used to determine caspase 3 mRNA transcriptions by polymerase chain reaction; others were used to measure the expression of caspase 3 protein by Western blotting techniques, while the rest of the brains were used for flow cytometry analysis where a caspase 3 specific staining kit was used. Our behavioural results indicated that tat protein caused impairment in learning and memory in the experimental group, when compared to controls. The group that received both tat protein and picolinic acid showed a significant improvement in learning and memory in Morris water maze test, in comparison to the tat protein treated group. Our gene expression data showed a significant up regulation of caspase 3 gene in the tat protein treated group, compared to controls. This result was supported by the Western blot data that showed significantly increased caspase 3 protein expression in the tat protein treated group. While these increases in caspase 3 expression strongly point to an apoptotic mode of cell death in the hippocampus of animals that were treated with tat protein, our flow cytometry results were less convincing with marginal levels of caspase 3 staining being observed. Both increases in gene and protein expression were inhibited in animals that were pretreated with picolinic acid. Our data led us to conclude that tat protein can cause cognitive abnormalities through toxic sequelae that may include apoptosis. It is therefore likely that this viral protein may be one of the etiological factors of HIV associated neurocognitive impairment. Our results further suggest that picolinic acid may be considered as an adjunct therapy for HAND.

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Browsing Masters Degrees (Physiology) by Author "Daniels, William Mark Uren."