HIV infection and illicit drugs are known to induce oxidative stress and ROS production, which can ultimately impact cellular functions like oxidative metabolism and cause energy deficits [25, 39–41]. GSH plays an important cell-protectant role by exhibiting an antioxidant defense mechanism. It is closely linked to the energy source of ATP production. Low levels of GSH have been associated with impaired immune and neuronal dysfunction; its metabolites also interface with energetics and neurotransmitter syntheses through several prominent metabolic pathways [42, 43]. HIV infection affects redox expression in GSS, SOD and catalase, as evidenced in HIV-infected patients and cocaine users [44–46]. However, inhibition of redox imbalance GSH/GSSG  activates energy sensor AMPKs and subsequently impacts the energy-profile machinery of metabolic enzymes such as HK-1, HK-II PKM, LDH, MCT-1, and MCT-4, all of which play vital roles in neuronal dysfunction and in the disease progression of HIV-associated dementia (HAD) patients . In CNS, astrocytes are the major reservoirs of energy storage and maintain cellular energy metabolism. Astrocytes are modulated by HIV infection as well as drug use, both of which lead to neurodegeneration [12, 33, 49]. HIV-1 Tat impacts several cellular functions, but we do not yet understand the effects of HIV-1 Tat and cocaine-induced redox imbalance on energy deficits, oxidative metabolism, and ultimately, neurodegeneration. Our observations provide new insights into the functional role of redox imbalance, which adequately alters the energy profiles of glycolytic enzymes and mitochondrial biogenesis, which leads to Nrf transcription and is a potential sign of neurodegeneration by HIV-1 Tat with cocaine.
We have demonstrated for the first time that HIV-1 Tat, cocaine, and HIV-1 Tat with cocaine led to redox inhibition of GSS and catalase and subsequently increased SOD and activation of the energy sensor AMPK and mRNA expression (Fig-1 and 2), as compared with the control. It’s known that redox imbalance disturbs cellular homeostasis . Our study suggests that cocaine with HIV–1 Tat may play an enhanced role in oxidative metabolism compared to the control. Our results are consistent with earlier reports of HIV-gp120-induced microglia and HIV-Tat-induced astrocytes, where activation of the redox and oxidative pathways were observed [25, 26]. AMPK is a key regulator of metabolism and survival during energy stress. Dysregulation of AMPK is strongly associated with oxidative injury, which impacts redox modification. Defective oxidative metabolism and a reduced level of ATP are both known to activate AMPK . AMPK plays a critical role in controlling both cellular and whole-body metabolic responses. However, we still do not know how HIV-1 Tat and cocaine regulate AMPK, which can impact intracellular redox (GSS, CAT and SOD) status. These studies confirm that redox imbalance and energy dysfunction affect glycolytic enzymes and mitochondrial biogenesis. These factors, in turn, activate OXPHOS and impair Nrf transcriptions, which ultimately leads to astrocytic signaling mediated neurodegeneration.
Our results also show that in cocaine, HIV-1 Tat, and cocaine with HIV-1 Tat, induction of redox inhibition and activation of AMPK is associated with altered glycolytic enzymes and increased mitochondrial biogenesis. Importantly, HIV-1 Tat, cocaine, and cocaine with HIV-Tat all led to oxidative metabolism, which in turn impaired mitochondrial biogenesis and subsequently reduced the level of energy resource and transfer. However, HIV-positive cocaine users have higher energy demands which increase ATP utilization and subsequently affect glycolytic enzymes. This study suggests that HIV-1 Tat-induced redox inhibition and energy demands are accelerated by cocaine use when compared with either cocaine use or HIV infection. These results confirm previous reports of HIV-1 Tat-induced metabolic profiles .
Previous studies have also demonstrated that HIV infection and HIV proteins affect oxidative metabolism and glycolytic enzyme regulation, which may impact mitochondrial biogenesis . The first step in the breakdown of glucose is to extract energy for cellular metabolism. Glucose phosphorylation is catalyzed by the enzyme HK, which is the predominant isoform of HK I and HK II. Importantly, HK I and HK II contain a hydrophobic terminal mitochondrial binding motif. The energy metabolism in glycolysis is mainly thought of as a cytosolic process and HK I is predominantly associated with mitochondria in ATP production. In contrast, HK II is located in either the cytosol or at the mitochondrial outer membrane . Interestingly, our results demonstrated that HIV–1 Tat and cocaine exposure significantly down-regulated HK I and upregulated HK II expression; moreover, these effects were accelerated by cocaine combined with HIV-1 Tat. These results confirm that increased HK II expression and its binding to mitochondria facilitate and increase the level of aerobic glycolysis and lactate production. However, the increased level of HK- II targets mitochondria-mediated cell death, which is known to activate LDH. Furthermore, monocarboxylic acid transport (MCT) is one of the metabolic targets wherein the flux of small ketone bodies such as lactic acid and pyruvic acid occurs to support metabolic demands. The predominant role of MCTs 1–4 is the transport of L-lactate, pyruvate, and ketone bodies in and out of cells; L-lactate is quantitatively the most important substrate . MCT1 is responsible for the efflux of lactic acid when oxygen supply is compromised, and glycolysis is stimulated. However, glycolytic profiles in astrocytes treated HIV-Tat and cocaine significantly upregulated MCT1 and MCT4, which may increase lactic acid and initiate neurodegeneration. Recent studies conclude that in the brain, MCT1 and MCT4 export astrocyte-produced lactic acid, which is then taken up by MCT1 or MCT2 into the neurons for oxidation as an important respiratory fuel .
The energy sensor AMPK is key regulator that activates PGC-1α, a member of the peroxisome proliferator-activated receptor-gamma (PGC) family of transcriptional coactivators and is the master regulator of mitochondrial biogenesis . AMPK activates different transcriptional factors, which promote the expression of TFAM, including Nrf-1 and Nrf-2. The oxidative stress response is increased by antioxidant defenses through the activation of Nrf, an important transcription factor [56, 57]. Nrf is the main player in the controlled antioxidant-response element (ARE) found in the promoter regions of many genes that encode antioxidants and detoxification enzymes (e.g., SOD1, CAT, and GPx1) . In addition, Nrf-1 and Nrf-2 are important contributors to the sequence of events that increase the transcription of key mitochondrial enzymes. Nrf-1 and − 2 can interact with TFAM, which drives the transcription and replication of mtDNA . TFAM is a downstream target gene of PGC-1α and controls the transcription of mitochondrial DNA-encoded genes as well as DNA replication during biogenesis .
In the present study, exposure to HIV-Tat with cocaine led to the activation of PGC-α1 (unlike the control group). The analysis of the level of TFAM protein in total and mitochondrial fraction was increased by exposure of HIV-1 Tat or cocaine alone. Compared to control, these effects were accelerated by HIV-Tat with cocaine co-morbidity in total cell lysates as well as a mitochondrial fraction. Other studies have confirmed that mitochondrial damage induced by increasing mitochondrial biogenesis depends on upregulated PGC-1α expression [60, 61]. Also, increased oxidative damage and the level of PGC-1α and TFAM in astrocytes could enhance the susceptibility of mtDNA, which might cause neuronal dysfunction and result in neurodegeneration. Therefore, it is possible that the impact of energy consumption during HIV infection inhibits redox expression and dysregulates AMPK-mediated metabolic function and mitochondrial impairments, thus leading to Nrf transcription. Several studies show that interrupting glycolysis can contain oxidative metabolism but has an adverse effect on CNS function that is out of proportion with any change in total-energy status [62, 63]. In addition, clinically significant energy imbalance may also occur as a result of an important energy-generating enzyme system in mitochondria. In support to our study a recent report demonstrated that mitochondrial biogenesis is differentially regulated in neurons and astroglia in HIV associated neurocognitive disorders (HAND) brains and that targeting astroglial bioenergetics processes . Taken together, these data indicate that PGC-1α plays a crucial role in linking stimuli such as HIV infection and cocaine abuse to an internal metabolic response like mitochondrial biogenesis via, among others, the NRF transcription factors.
Overall, the data suggest a connection between the redox gene and protein enhancing the activation of AMPKs and altered intracellular signaling mechanisms in cocaine and HIV- Tat treated cells. The upregulation of redox-gene inhibition and altered oxidative metabolism by cocaine and HIV-Tat protein may lead to increased energy fuel utilization and ultimately affect the storage of energy resources and transfer metabolic fuel which can lead to cell death. The present study supports the idea that oxidative stress and redox-dependent gene expression are associated with energy dysfunction, particularly with the reduction of glutathione (GSH) and the activation of energy sensor AMPK, glycolytic enzymes, and mitochondrial biogenesis. The contained medium exposed studies show that in SK-N-MC neuroblastoma cells there was a loss in the number of spines, and a decrease in dendrite diameter, dendrite area, and spine area compared to control. These results suggest that either cocaine or HIV-infected astrocytes play a vital role in energy storage, metabolic dysfunction, and neurodegeneration.