Kathryn A. Lindl

Expression of Nrf2 in Neurodegenerative Diseases

In response to oxidative stress, the nuclear factor E2-related factor 2 (Nrf2) transcription factor translocates from the cytoplasm into the nucleus and transactivates expression of genes with antioxidant activity. Despite this cellular mechanism, oxidative damage is abundant in Alzheimer and Parkinson disease (AD and PD). To investigate mechanisms by which Nrf2 activity may be aberrant or insufficient in neurodegenerative conditions, we assessed Nrf2 localization in affected brain regions of AD, Lewy body variant of AD (LBVAD), and PD. By immunohistochemistry, Nrf2 is expressed in both the nucleus and the cytoplasm of neurons in normal hippocampi with predominant expression in the nucleus. In AD and LBVAD, Nrf2 was predominantly cytoplasmic in hippocampal neurons and was not a major component of beta amyloid plaques or neurofibrillary tangles. By immunoblotting, we observed a significant decrease in nuclear Nrf2 levels in AD cases. In contrast, Nrf2 was strongly nuclear in PD nigral neurons but cytoplasmic in substantia nigra of normal, AD, and LBVAD cases. These findings suggest that Nrf2-mediated transcription is not induced in neurons in AD despite the presence of oxidative stress. In PD, nuclear localization of Nrf2 is strongly induced, but this response may be insufficient to protect neurons from degeneration.

HIV-Associated Neurocognitive Disorder: Pathogenesis and Therapeutic Opportunities

Human immunodeficiency virus type 1 (HIV) infection presently affects more that 40 million people worldwide, and is associated with central nervous system (CNS) disruption in at least 30% of infected individuals. The use of highly active antiretroviral therapy has lessened the incidence, but not the prevalence of mild impairment of higher cognitive and cortical functions (HIV-associated neurocognitive disorders) as well as substantially reduced a more severe form dementia (HIV-associated dementia). Furthermore, improving neurological outcomes will require novel, adjunctive therapies that are targeted towards mechanisms of HIV-induced neurodegeneration. Identifying such molecular and pharmacological targets requires an understanding of the events preceding irreversible neuronal damage in the CNS, such as actions of neurotoxins (HIV proteins and cellular factors), disruption of ion channel properties, synaptic damage, and loss of adult neurogenesis. By considering the specific mechanisms and consequences of HIV neuropathogenesis, unified approaches for neuroprotection will likely emerge using a tailored, combined, and non-invasive approach.

Activation of cyclin‐dependent kinase 5 by calpains contributes to human immunodeficiency virus‐induced neurotoxicity

Although the specific mechanism of neuronal damage in human immunodeficiency virus (HIV) ‐associated dementia is not known, a prominent role for NMDA receptor (NMDAR)‐induced excitotoxicity has been demonstrated in neurons exposed to HIV‐infected/activated macrophages. We hypothesized NMDAR‐mediated activation of the calcium‐dependent protease, calpain, would contribute to cell death by induction of cyclin‐dependent kinase 5 (CDK5) activity. Using an in vitro model of HIV neurotoxicity, in which primary rat cortical cultures are exposed to supernatants from primary human HIV‐infected macrophages, we have observed increased calpain‐dependent cleavage of the CDK5 regulatory subunit, p35, to the constitutively active isoform, p25. Formation of p25 is dependent upon NMDAR activation and calpain activity and is coincident with increased CDK5 activity in this model. Further, inhibition of CDK5 by roscovitine provided neuroprotection in our in vitro model. Consistent with our observations in vitro, we have observed a significant increase in calpain activity and p25 levels in midfrontal cortex of patients infected with HIV, particularly those with HIV‐associated cognitive impairment. Taken together, our data suggest calpain activation of CDK5, a pathway activated in HIV‐infected individuals, can mediate neuronal damage and death in a model of HIV‐induced neurotoxicity.

Expression of the endoplasmic reticulum stress response marker, BiP, in the central nervous system of HIV‐positive individuals

The prevalence of HIV‐associated neurocognitive impairment (NCI), which includes HIV‐associated dementia (HAD) and minor cognitive and motor disorder (MCMD), has been increasing. HIV‐infected and/or activated macrophages/microglia in the brain initiate the neurodegeneration seen in HIV‐associated NCI via soluble neurotoxic mediators, including reactive oxygen species, viral proteins and excitotoxins. Neurotoxic factors released by macrophages/microglia injure neurones directly and alter astrocytic homeostatic functions, which can lead to excitotoxicity and oxidative stress‐mediated neuronal injury. Often, cells respond to oxidative stress by initiating the endoplasmic reticulum (ER) stress response. Thus, we hypothesize that ER stress response is activated in HIV‐infected cortex. We used immunofluorescence and immunoblotting to assess expression patterns of the ER stress proteins, BiP and ATF6, in HIV‐positive cortical autopsy tissue. Additionally, we performed immunofluorescence using cell type‐specific markers to examine BiP staining in different cell types, including neurones, astrocytes and macrophages/microglia. We observed a significant increase in BiP expression by both immunoblotting and immunofluorescence in HIV‐positive cortex compared with control tissue. Additionally, phenotypic analysis of immunofluorescence showed cell type‐specific increases in BiP levels in neurones and astrocytes. Further, ATF‐6β, an ER stress response initiator, is up‐regulated in the same patient group, as assessed by immunoblotting. These results suggest that ER stress response is activated in HIV‐infected cortex. Moreover, data presented here indicate for the first time that numbers of macrophages/microglia increase in brains of MCMD patients, as has been observed in HAD.

Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.

Activation status of integrated stress response pathways in neurones and astrocytes of HIV-associated neurocognitive disorders (HAND) cortex

C. Akay, K. A. Lindl, N. Shyam, B. Nabet, Y. Goenaga‐Vazquez, J. Ruzbarsky, Y. Wang, D. L. Kolson and K. L. Jordan‐Sciutto (2012) Neuropathology and Applied Neurobiology38, 175–200

Site-specific hyperphosphorylation of pRb in HIV-induced neurotoxicity ☆

HIV-Associated Neurocognitive Disorder (HAND) remains a serious complication of HIV infection, despite combined Anti-Retroviral Therapy (cART). Neuronal dysfunction and death are attributed to soluble factors released from activated and/or HIV-infected macrophages. Most of these factors affect the cell cycle machinery, determining cellular outcomes even in the absence of cell division. One of the earliest events in cell cycle activation is hyperphosphorylation of the retinoblastoma protein, pRb (ppRb). We and others have previously shown increased ppRb expression in the CNS of patients with HIV encephalitis (HIVE) and in neurons in an in vitro model of HIV-induced neurodegeneration. However, trophic factors also lead to an increase in neuronal ppRb with an absence of cell death, suggesting that, depending on the stimulus, hyperphosphorylation of pRb can have different outcomes on neuronal fate. pRb has multiple serines and threonines targeted for phosphorylation by distinct kinases, and we hypothesized that different stimuli may target separate sites for phosphorylation. Thus, to determine whether pRb is differentially phosphorylated in response to different stimuli and whether any of these sites is preferentially phosphorylated in association with HIV-induced neurotoxicity, we treated primary rat mixed cortical cultures with trophic factors, BDNF or RANTES, or with the neurotoxic factor, N-methyl-d-aspartate (NMDA), or with supernatants containing factors secreted by HIV-infected monocyte-derived macrophages (HIV-MDM), our in vitro model of HIV-induced neurodegeneration. We found that, while BDNF and RANTES phosphorylated serine807/811 and serine608 in vitro, treatment with HIV-MDM did not, even though these trophic factors are components of HIV-MDM. Rather, HIV-MDM targets a specific phosphorylation site, serine795, of pRb for phosphorylation in vitro and this ppRb isoform is also increased in HIV-infected brains in vivo. Further, overexpression of a nonphosphorylatable pRb (ppRb S795A) attenuated HIV-MDM-induced neurotoxicity. These findings indicate that HIV-infection in the brain is associated with site-specific hyperphosphorylation of pRb at serine795, which is not induced by other tested stimuli, and that this phosphorylation contributes to neuronal death in this disease, demonstrating that specific pRb sites are differentially targeted and may have diverse impacts on the viability of post-mitotic neurons.

E2F1 localizes predominantly to neuronal cytoplasm and fails to induce expression of its transcriptional targets in human immunodeficiency virus-induced neuronal damage

As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV-associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary postmitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By Q-PCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, Cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19(ARF)) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19(ARF), Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity.

Targeted gene mutation of E2F1 evokes age‐dependent synaptic disruption and behavioral deficits

Aberrant expression and activation of the cell cycle protein E2F1 in neurons has been implicated in many neurodegenerative diseases. As a transcription factor regulating G1 to S phase progression in proliferative cells, E2F1 is often up‐regulated and activated in models of neuronal death. However, despite its well‐studied functions in neuronal death, little is known regarding the role of E2F1 in the mature brain. In this study, we used a combined approach to study the effect of E2F1 gene disruption on mouse behavior and brain biochemistry. We identified significant age‐dependent olfactory and memory‐related deficits in E2f1 mutant mice. In addition, we found that E2F1 exhibits punctated staining and localizes closely to the synapse. Furthermore, we found a mirroring age‐dependent loss of post‐synaptic protein‐95 in the hippocampus and olfactory bulb as well as a global loss of several other synaptic proteins. Coincidently, E2F1 expression is significantly elevated at the ages, in which behavioral and synaptic perturbations were observed. Finally, we show that deficits in adult neurogenesis persist late in aged E2f1 mutant mice which may partially contribute to the behavior phenotypes. Taken together, our data suggest that the disruption of E2F1 function leads to specific age‐dependent behavioral deficits and synaptic perturbations.

Examining the endogenous antioxidant response through immunofluorescent analysis of Nrf2 in tissue.

As organisms designed to depend upon oxygen to sustain life, humans are necessarily and continually exposed to damaging oxidizing agents. As a vital protective measure, oxygen-dependent organisms have developed a highly evolutionarily conserved mechanism for preventing oxidative stress. NF-E2 (nuclear factor (erythroid-derived 2))-related factor-2 (Nrf2) is the primary regulator of this endogenous antioxidant response. Many diseases that plague human society, ranging from various cancers to neurodegenerative diseases, have oxidative stress as a component of their etiology, and thus, much disease research has focused on Nrf2, both as a potential point of biological failure and as a promising therapeutic target. As a transcription factor, Nrf2 is active only when it is nuclear, and is regulated largely by its subcellular distribution. Thus, Nrf2 protein levels and subcellular localization are both key pieces of information when studying the endogenous antioxidant response. Immunofluorescent analysis (IFA) of Nrf2 in human tissue is a particularly powerful tool in the study of Nrf2 in disease, because it allows examination of both of these regulatory mechanisms that modulate Nrf2 activity.