Frank Denaro

Altered expression pattern of Nrf2/HO-1 axis during accelerated-senescence in HIV-1 transgenic rat

Chronic oxidative stress plays a central role in the pathogenesis of many diseases, including HIV-1 associated disorders. Concomitantly with the decline of endogenous antioxidant systems, it was reported that HIV-1-related proteins increase the production of radical species in cells and tissues that are not directly infected by the virus. In the context of HIV-1 infection, the role of Nrf2, a key transcription factor that contributes to the maintenance of cellular redox homeostasis, remains largely uncharacterized. One of the major stress-responsive player regulated by Nrf2 is the antioxidant enzyme HO-1. The Nrf2/HO-1 axis constitutes a crucial cell survival mechanism to counteract oxidative stress and inflammation. The present study aims to investigate the age-related patterns of Nrf2 and HO-1 in different brain regions and tissues of HIV-1 transgenic rat. Since HIV-1 induces an accelerated aging and the redox imbalance may actively promote senescence, we also evaluated the senescence phenotype-switching by quantifying levels of β-galactosidase activity. Our results showed changes in gene expression, with different trends depending on the brain regions and tissues examined. However, compared to age-matched controls, we observed in HIV-1 transgenic rats a significant reduction in the protein levels of Nrf2 and HO-1, suggesting a weakening in the protection exerted by Nrf2/HO-1 system. Moreover, we show that senescence occurs more rapidly in HIV-1 transgenic rats than in control animals. To our knowledge this is the first in vivo report showing the involvement of Nrf2/HO-1 pathway in a rat model of HIV-1.

Characterization of neuropathology in the HIV-1 transgenic rat at different ages

The transgenic HIV-1 rat (Tg) is a commonly used neuroHIV model with documented neurologic/behavioral deficits. Using immunofluorescent staining of the Tg brain, we found astrocytic dysfunction/damage, as well as dopaminergic neuronal loss/dysfunction, both of which worsening significantly in the striatum with age. We saw mild microglial activation in young Tg brains, but this decreased with age. There were no differences in neurogenesis potential suggesting a neurodegenerative rather than a neurodevelopmental process. Gp120 CSF levels exceeded serum gp120 levels in some animals, suggesting local viral protein production in the brain. Further probing of the pathophysiology underlying astrocytic injury in this model is warranted.

Topographical distribution and morphology of NADPH-diaphorase-stained neurons in the human claustrum

We studied the topographical distribution and morphological characteristics of NADPH-diaphorase-positive neurons and fibers in the human claustrum. These neurons were seen to be heterogeneously distributed throughout the claustrum. Taking into account the size and shape of stained perikarya as well as dendritic and axonal characteristics, Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd)-positive neurons were categorized by diameter into three types: large, medium and small. Large neurons ranged from 25 to 35 μm in diameter and typically displayed elliptical or multipolar cell bodies. Medium neurons ranged from 20 to 25 μm in diameter and displayed multipolar, bipolar and irregular cell bodies. Small neurons ranged from 14 to 20 μm in diameter and most often displayed oval or elliptical cell bodies. Based on dendritic characteristics, these neurons were divided into spiny and aspiny subtypes. Our findings reveal two populations of NADPHd-positive neurons in the human claustrum—one comprised of large and medium cells consistent with a projection neuron phenotype, the other represented by small cells resembling the interneuron phenotype as defined by previous Golgi impregnation studies.

Muscular Abnormalities in the HIV-1 Transgenic Rat

INTRODUCTION. Special attention is focused on the central nervous system (CNS) in AIDS because of the debilitating symptoms and unique neuropathology. Anti- viral therapy has been effective in treating many of the symptoms of AIDS. But the CNS and PNS can continue to display debilitating symptoms even when the patient is on antiviral therapy (1). Motor abnormalities and atrophy of skeletal muscle are two PNS problems that continue to resist treatment. In order to understand the disease process and to develop new therapies, animal model systems are needed (2). The recently developed HIV-1 Transgenic Rat (HIV-1 TgR) displays many neurological symptoms of AIDS (3). These symptoms include muscular wasting, weakness, difficulties in gait, and paralysis. Examination of the peripheral nerve has revealed degeneration of the axons and focal demyelination. The muscular changes may be secondary to this nerve pathology or myopathic mechanisms may be operational. The muscle in these animals was examined by histologic means. By identifying the type of degeneration it may be possible to eventual study the effect of growth factors or therapeutics. METHODS. HIV-1TGRs were constructed using transgenic protocols. The pronuclei were microinjected with an HIV-1-gag-pol-pEVd 1443 clone in which the SphI and Bal1 fragment that contained the 3’ gag region and 5’ pol region of the infectious proviral DNA pNL4.3 clone was removed. This produced a non-infectious model. Gp-120 is found in their serum. Immunocytochemistry to Gp-120, TAT and Nef localized the viral products to the lymphoid tissue. Animals were observed for neurologic abnormalities. Their clinical symptoms were recorded over time. Both nerve and muscle were removed at necropsy for histologic analysis. Tissues were prepared for light and electron microscopy. The special stains used were H&E, NADH, ATPase, lymphocyte markers, and ApopTag. Thick sections stained with Toluidine Blue. RESULTS. Depending on the muscle studied one could observe mild changes (inflammatory changes, and apoptosis to endothelial cells) to severe end stage changes (fatty replacement of muscle, phagocytosis of myocytes and necrosis). The hind limb muscle typically displayed the most marked changes. Intermediate changes include atrophic, angulated fibers (Fig.1), hypertropic fibers surrounded fat, increased connective tissue and internal nuclei (Fig. 2).

Peripheral Nerve Degeneration in the HIV-1 Transgenic Rat

INTRODUCTION. Neurological abnormalities of the peripheral (PNS) and central nervous system continue to cause debilitating symptoms in AIDS patients despite advances in antiviral therapy (1). How HIV-1 mediates PNS damage is unknown. Our understanding is limited by the lack of model systems in which HIV/PNS pathology can be studied. Studies on the recently developed HIV-1 transgenic rat (HIV-1TgR) reveal that a subset of these animals displays severe neurological signs. These symptoms include complete or partial paralysis, difficulties in walking and sensitivity to touch. Progressive skeletal muscle mass loss with increasing severity of motor symptoms occurs over time. These clinical observations suggest that morphological changes to the nerve might be found. Peripheral nerve was examined in a group of HIV-1 TGRs which displayed neurologic symptoms. By characterizing the abnormalities in this model, it may be possible to develop theories on pathogenesis relevant to treatment.

APOPTOSIS OF SPLEENOCYTES AND EXPRESSION OF HIV GENE PRODUCTS IN THE HIV-1 TRANSGENIC RAT

INTRODUCTION. Apoptosis is believed to play a major role in the progressive loss of CD4 + T cells, and other cell types in AIDS. Several studies support the theory that HIV-1 gene products may be the causative factor in the initiation of cell death (1-3). Moreover, post mortem studies reveal apoptotic cells in many tissues. Identification of mechanisms for the production of apoptosis in animal models of AIDS may clarify AIDS related cell death. A new model, developed at IHV for the study of AIDS related pathology is the HIV-1 Transgenic Rat. This model is unique among transgenic models in that measurable levels of Gp-120 have been identified in the serum and in spleenocyte lysates (4). In this study, we have characterized the location of HIV-1 transgene (GP120, NEF, TAT) expression in the spleen by means of immunocytochemistry. Moreover, identification of cells undergoing apoptosis was made in adjacent sections. The localization of HIV-1 gene products and the identification of apoptosis in this new animal model of AIDS could give insight in the mechanisms of HIV related cell lose.