HIV-induced redox changes and inflammation in the central nervous system and modulatory factors

Ph.D. University of Hawaii at Manoa 2013.Includes bibliographical references.Currently 34 million people live with human immunodeficiency virus (HIV) infection. Although the lifespan of HIV patients has been prolonged by highly active antiretroviral treatment, the prevalence of HIV-associated neurocognitive disorders (HANDs) is increasing. Oxidative stress and inflammation in the central nervous system (CNS) are major causes of HANDs, and concurrent drug abuse may accelerate the progression of symptoms. This dissertation aims to study HIV-induced changes in antioxidants, oxidative stress, and inflammation in the CNS, assess the effects of concurrent methamphetamine (Meth) exposure, and evaluate the protective effect of dietary supplementation of bamboo Phyllostachys edulis extract (BEX). This study centered on the most abundant antioxidant in the brain-glutathione (GSH). The changes of GSH metabolism and GSH-dependent antioxidant enzymes were measured in cerebrospinal fluid (CSF) of human cohorts with HIV infection and/or uses of Meth, and in the brain tissues of HIV-1 model transgenic rats (HIV-1Tg expressing 7 HIV viral proteins) with or without Meth exposure. HIV infection resulted in elevated gamma-glutamyl transpeptidase (GGT) activity, GSH depletion and a several-fold increase in lipid peroxidation in human CSF. Compared to HIV infection, use of Meth resulted in less severe oxidative stress, which may be partially explained by the upregulation of glutathione peroxidase (GPx) and GSH in the CSF. Meth did not interact with HIV in modulating the redox changes in the CSF. In HIV-1Tg rats, redox and inflammatory changes were observed in the brain in a region-specific manner, with little synergy from Meth exposure. The thalamus was highlighted by its high GSH content and systematic upregulation of GSH biosynthesis and GSH-dependent antioxidant enzymes in the HIV-1Tg rats. On the other hand, neuroinflammation markers, such as glial fibrillary acidic protein (GFAP) and p65, were increased in the hippocampus of the transgenic rats, and such changes were effectively normalized by dietary supplementation of BEX. In summary, this dissertation documents the significance of GSH and GSH-dependent antioxidant enzymes in regulating the redox status of CNS, and the potential anti-neuroinflammatory effects of BEX in the context of HIV infection

Inhibition of Vascular Endothelial Growth Factor Receptor 2 Exacerbates Loss of Lower Motor Neurons and Axons during Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are inflammatory demyelinating and neurodegenerative diseases in the central nervous system (CNS). It is believed that MS and EAE are initiated by autoreactive T lymphocytes that recognize myelin antigens; however, the mechanisms responsible for neurodegeneration in these diseases remain elusive. Data indicate that vascular endothelial growth factor A (VEGF-A) plays a role in the development of MS and EAE. Interestingly, VEGF-A is regarded as a neurotrophic factor in the CNS that promotes neuron survival and neurogenesis in various neurodegenerative diseases by activating VEGF receptor 2 (VEGFR2). In this study, we sought to explore the role of the VEGF-A/VEGFR2 signaling in neurodegeneration in MS and EAE. We showed that the expression of VEGF-A was decreased in the spinal cord during EAE and that VEGFR2 was activated in lower motor neurons in the spinal cord of EAE mice. Interestingly, we found that treatment with SU5416, a selective VEGFR2 inhibitor, starting after the onset of EAE clinical symptoms exacerbated lower motor neuron loss and axon loss in the lumbar spinal cord of mice undergoing EAE, but did not alter Purkinje neuron loss in the cerebellum or upper motor neuron loss in the cerebral cortex. Moreover, SU5416 treatment had a minimal effect on EAE clinical symptoms as well as inflammation, demyelination, and oligodendrocyte loss in the lumbar spinal cord. These results imply the protective effects of the VEGF-A/VEGFR2 signaling on lower motor neurons and axons in the spinal cord in MS and EAE

Treatment with the low dose of SU5416 after EAE onset did not alter the disease severity.

<p><b>A.</b> Mean clinical score. <b>B.</b> Peak clinical score for individual mice. <b>C, D.</b> H&E staining revealed typical EAE pathology in the lumbar spinal of mice treated with vehicle and SU5416. N = 5 animals. Error bars represent SD. Scale bar: C, D, 200 μm.</p

Treatment with the low dose of SU5416 after EAE onset exacerbated lower motor neuron loss and axon loss in the lumbar spinal cord.

<p><b>A, B, C, G.</b> NeuN IHC showed that the number of lower motor neurons was significantly reduced in the lumbar spinal cord of vehicle-treated EAE mice at PID 21 as compared to naïve mice, and that SU5416 treatment further reduced the motor neuron numbers in SU5416-treated EAE mice. N = 5–7 animals. <b>D, E, F, H.</b> SMI31 IHC showed that the number of axons was significantly reduced in the lumbar spinal cord of vehicle-treated EAE mice at PID 21 as compared to naïve mice, and that SU5416 treatment further reduced the axon numbers in SU5416-treated EAE mice. N = 5 animals. Error bars represent SD, *<i>P</i> < 0.05, **<i>P</i> < 0.001,***<i>P</i> < 0.0001. Scale bar: A–C, 100 μm; D–F, 25 μm.</p

Treatment with the low dose of SU5416 after EAE onset did not affect Purkinje neuron loss or upper motor neuron loss.

<p><b>A, B, C, G.</b> Calbindin 2 IHC showed the number of Purkinje neurons was significantly reduced in the cerebellum of vehicle-treated EAE mice at PID 21 as compared to naïve mice, and that SU5416 treatment did not significantly change the number of Purkinje neurons in the cerebellum of EAE mice. N = 5 animals. <b>D, E, F, H.</b> NeuN IHC showed the number of neurons in the layer V of the primary motor cortex was significantly reduced in vehicle-treated EAE mice at PID 21 as compared to naïve mice, and that SU5416 treatment did not change the number of neurons in the layer V of the primary motor cortex of EAE mice. N = 5 animals. ***<i>P</i> < 0.0001. Scale bar: A–C, 100 μm; D–F, 50 μm.</p

Activation of the VEGF-A/VEGFR2 signaling in lower motor neurons during EAE.

<p><b>A</b>. VEGF-A ELISA showed that the protein level of VEGF-A was not altered in the spinal cord of EAE mice at PID14, but was significantly reduced at PID 19 and PID 50, as compared to naïve mice. N = 5 animals. <b>B—J</b>. VEGFR2 and pVEGFR2 double immunostaining revealed activation of VEGFR2 in lower motor neurons in the lumbar spinal cord of both naïve mice and EAE mice. Importantly, treatment with SU5416 noticeably reduced the levels of pVEGFR2 in the lower motor neurons of mice with EAE at PID 21. N = 5 animals. Error bars represent SD, **<i>P</i> < 0.01, ***<i>P</i> < 0.0001. Scale bar: B–J, 50 μm.</p