Marina V. Aksenova

Cell Culture Models of Oxidative Stress and Injury in the Central Nervous System

Constantly growing body of evidence suggests that hallmarks of oxidative stress are present in various central nervous system (CNS) disorders. Technological advantages in cell culturing made it possible to use neural cell/tissue cultures as experimental models for investigation of molecular mechanisms which underlie the development of oxidative stress condition, damage and adaptive responses to oxidative insults. This review is focused on the application of cell culture methodology for studies of oxidative stress condition in the brain. The review describes studies of biomarkers of oxidative stress-dependent cell damage and adaptive responses in various kinds of brain cell culture models. It discusses the use of cell/tissue culture models for elucidation of the role and pathogenesis of oxidative stress in neurodegenerative brain disorders, AIDS-associated brain pathology, drug abuse, and aging. The review underscores the importance of cell/tissue-based studies for testing of new antioxidants and development of therapeutic strategies for amelioration of oxidative damage in the CNS. The impact of new advances in gene and protein expression analysis on the cell/tissue culture-based research of oxidative stress in the CNS is also discussed.

HIV-1 Tat neurotoxicity in primary cultures of rat midbrain fetal neurons : Changes in dopamine transporter binding and immunoreactivity

HIV-1 neurotoxic proteins (Tat, gp120) are believed to play a major role in pathogenesis of dementia in a significant portion of the AIDS patient population. Dopaminergic systems appear to be particularly important in HIV-associated dementia. In the current studies, we determined that primary cell cultures prepared from the midbrain of 18-day-old rat fetuses are sensitive to Tat neurotoxicity and investigated the possible effects of Tat on DAT-specific ligand binding and DAT immunoreactivity in rat fetal midbrain cultures. We found that Tat neurotoxicity was associated with a significant decrease in [3H]WIN 35428 binding. Immunostaining of cell cultures with antibodies recognizing the C-end epitope of DAT did not reveal significant changes in DAT immunoreactivity. The results of this study implicate involvement of monoamine transmission systems in HIV-associated dementia.

Glutamine Synthetase-Induced Enhancement of β-Amyloid Peptide Aβ(1–40) Neurotoxicity Accompanied by Abrogation of Fibril Formation and Aβ Fragmentation

Abstract: β‐Amyloid peptide (Aβ) is the main constituent in both senile plaques and diffuse deposits in Alzheimers diseased brains. It was previously shown that synthetic Aβs were able to form free radical species in aqueous solution and cause both oxidative damage to cell proteins and inactivation of key metabolic enzymes. We also previously demonstrated that an interaction of Aβ(1–40) with the oxidatively sensitive enzyme glutamine synthetase (GS) resulted in both inactivation of GS and an increase of Aβ toxicity to hippocampal cell cultures. In the present study the enhancement of Aβ toxicity during interaction with GS was found to be accompanied by abrogation of fibril formation and partial fragmentation of Aβ(1–40). HPLC elution profiles demonstrated the production of several peptide fragments. Analysis of the amino acid sequence of the major fragments identified them as the first 15 and the last six amino acids of Aβ(1–40). The fragmentation of Aβ was inhibited by immunoprecipitation of GS.

Neuronal survival and resistance to HIV-1 Tat toxicity in the primary culture of rat fetal neurons.

In this study we report that primary cultures of rat fetal neurons contain subpopulations of cells that may be sensitive or resistant to HIV-1 Tat neurotoxicity. We demonstrate that rapid binding/uptake of Tat 1-86 for 2 h was sufficient to trigger caspase activation and neurodegeneration in rat fetal midbrain cell cultures. The uptake of Tat was followed by an increase in MCP1 (CCL2) immunoreactivity. Approximately 70% of neurons were able to survive transient or continuous (7 days) Tat exposure. The surviving neurons did not contain bound/internalized Tat, but were able to interact with Tat after medium replacement. These neurons were resistant to Tat toxicity. In neurons that resisted the toxic effects of continuous and repeated Tat treatment, levels of NR2A subunit of the NMDA receptor complex were significantly lower than in controls. We suggest that the subunit composition of NMDAR complexes may be important for the sensitivity of neurons to Tat toxicity.

Endogenous amyloidogenesis in long-term rat hippocampal cell cultures.

BackgroundLong-term primary neuronal cultures are a useful tool for the investigation of biochemical processes associated with neuronal senescence. Improvements in available technology make it possible to observe maturation of neural cells isolated from different regions of the rodent brain over a prolonged period in vitro. Existing experimental evidence suggests that cellular aging occurs in mature, long-term, primary neuronal cell cultures. However, detailed studies of neuronal development in vitro are needed to demonstrate the validity of long-term cell culture-based models for investigation of the biochemical mechanisms of in vitro neuronal development and senescence.ResultsIn the current study, neuron-enriched hippocampal cell cultures were used to analyze the differentiation and degeneration of hippocampal neurons over a two month time period. The expression of different neuronal and astroglial biomarkers was used to determine the cytochemical characteristics of hippocampal cells in long-term cultures of varying ages. It was observed that the expression of the intermediate filament nestin was absent from cultures older than 21 days in vitro (DIV), and the expression of neuronal or astrocytic markers appeared to replace nestin. Additionally, morphological evaluations of neuronal integrity and Hoescht staining were used to assess the cellular conditions in the process of hippocampal culture development and aging. It was found that there was an increase in endogenous production of Aβ1-42 and an increase in the accumulation of Congo Red-binding amyloidal aggregates associated with the aging of neurons in primary culture. In vitro changes in the morphology of co-existing astrocytes and cell culture age-dependent degeneration of neurodendritic network resemble features of in vivo brain aging at the cellular level.ConclusionIn conclusion, this study suggests that long-term primary CNS culture is a viable model for the study of basic mechanisms and effective methods to decelerate the process of neuronal senescence.

HIV-1 protein-mediated amyloidogenesis in rat hippocampal cell cultures.

Since the beginning of the highly active antiretroviral therapy (HAART) era, epidemiological evidence indicates an increasing incidence of Alzheimers (AD)-like brain pathology in aging HIV patients. Emerging evidence warns of potential convergent mechanisms underlying HIV- and Abeta-mediated neurodegeneration. We found that HIV-1 Tat B and gp120 promote the secretion of Abeta 1-42 in primary rat fetal hippocampal cell cultures. Our results demonstrate that the variant of Tat expressed by the neurotropic subtype of HIV-1 virus (HIV-1 clade B) specifically induces both the release of amyloidogenic Abeta 1-42 and the accumulation of cell-bound amyloid aggregates. The results of the research rationalize testing of the ability of beta-amyloid aggregation inhibitors to attenuate HIV protein-mediated cognitive deficits in animal models of NeuroAIDS. The long-term goal of the study is to evaluate the potential benefits of anti-amyloidogenic therapies for management of cognitive dysfunction in aging HIV-1 patients.

Enhancement of β‐Amyloid Peptide Aβ(1–40)‐Mediated Neurotoxicity by Glutamine Synthetase

Abstract: The β‐amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimers disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C‐end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation‐sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ‐derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ‐mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.

Synaptodendritic recovery following HIV Tat exposure: Neurorestoration by phytoestrogens

HIV‐1 infects the brain and, despite antiretroviral therapy, many infected individuals suffer from HIV‐1‐associated neurocognitive disorders (HAND). HAND is associated with dendritic simplification and synaptic loss. Prevention of synaptodendritic damage may ameliorate or forestall neurocognitive decline in latent HIV‐1 infections. The HIV‐1 transactivating protein (Tat) is produced during viral latency in the brain and may cause synaptodendritic damage. This study examined the integrity of the dendritic network after exposure to HIV‐1 Tat by labeling filamentous actin (F‐actin)‐rich structures (puncta) in primary neuronal cultures. After 24 h of treatment, HIV‐1 Tat was associated with the dendritic arbor and produced a significant reduction of F‐actin‐labeled dendritic puncta as well as loss of dendrites. Pre‐treatment with either of two plant‐derived phytoestrogen compounds (daidzein and liquiritigenin), significantly reduced synaptodendritic damage following HIV‐1 Tat treatment. In addition, 6 days after HIV‐1 Tat treatment, treatment with either daidzein, or liquiritigenin enhanced recovery, via the estrogen receptor, from HIV‐1 Tat‐induced synaptodendritic damage. These results suggest that either liquiritigenin or daidzein may not only attenuate acute synaptodendritic injury in HIV‐1 but may also promote recovery from synaptodendritic damage.

HIV-1 Tat protein variants: critical role for the cysteine region in synaptodendritic injury.

HIV-1 enters the central nervous system early in infection; although HIV-1 does not directly infect neurons, HIV-1 may cause a variety of neurological disorders. Neuronal loss has been found in HIV-1, but synaptodendritic injury is more closely associated with the neurocognitive disorders of HIV-1. The HIV-1 transactivator of transcription (Tat) protein causes direct and indirect damage to neurons. The cysteine rich domain (residues 22-37) of Tat is important for producing neuronal death; however, little is known about the effects of the Tat protein functional domains on the dendritic network. The ability of HIV-1 Tat 1-101 Clades B and C, Tat 1-86 and Tat 1-72 proteins, as well as novel peptides (truncated 47-57, 1-72δ31-61, and 1-86 with a mutation at Cys22) to produce early synaptodendritic injury (24h), relative to later cell death (48h), was examined using cell culture. Treatment of primary hippocampal neurons with Tat proteins 1-72, 1-86 and 1-101B produced a significant early reduction in F-actin labeled puncta, implicating that these peptides play a role in synaptodendritic injury. Variants with a mutation, deletion, or lack of a cysteine rich region (1-86[Cys22], 1-101C, 1-72δ31-61, or 47-57) did not cause a significant reduction in F-actin rich puncta. Tat 1-72, 1-86, and 1-101B proteins did not significantly differ from one another, indicating that the second exon (73-86 or 73-101) does not play a significant role in the reduction of F-actin puncta. Conversely, peptides with a mutation, deletion, or lack of the cysteine rich domain (22-37) failed to produce a loss of F-actin puncta, indicating that the cysteine rich domain plays a key role in synaptodendritic injury. Collectively, these results suggest that for Tat proteins, 1) synaptodendritic injury occurs early, relative to cell death, and 2) the cysteine rich domain of the first exon is key for synaptic loss. Preventing such early synaptic loss may attenuate HIV-1 associated neurocognitive disorders.

Soy Isoflavones Genistein and Daidzein Exert Anti-Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat1–86 Exposure

Background HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity. Methodology/Principal Findings We demonstrate in primary cortical cell cultures that 17β-estradiol or isoflavones (genistein or daidzein) attenuate Tat1–86-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERα or ERβ specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERβ selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling. Conclusions/Significance Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons.