7,8-Dihydroxyflavone improves neuropathological changes in the brain of Tg26 mice, a model for HIV-associated neurocognitive disorder

The combined antiretroviral therapy era has significantly increased the lifespan of people with HIV (PWH), turning a fatal disease to a chronic one. However, this lower but persistent level of HIV infection increases the susceptibility of HIV-associated neurocognitive disorder (HAND). Therefore, research is currently seeking improved treatment for this complication of HIV. In PWH, low levels of brain derived neurotrophic factor (BDNF) has been associated with worse neurocognitive impairment. Hence, BDNF administration has been gaining relevance as a possible adjunct therapy for HAND. However, systemic administration of BDNF is impractical because of poor pharmacological profile. Therefore, we investigated the neuroprotective effects of BDNF-mimicking 7,8 dihydroxyflavone (DHF), a bioactive high-affinity TrkB agonist, in the memory-involved hippocampus and brain cortex of Tg26 mice, a murine model for HAND. In these brain regions, we observed astrogliosis, increased expression of chemokine HIV-1 coreceptors CXCR4 and CCR5, neuroinflammation, and mitochondrial damage. Hippocampi and cortices of DHF treated mice exhibited a reversal of these pathological changes, suggesting the therapeutic potential of DHF in HAND. Moreover, our data indicates that DHF increases the phosphorylation of TrkB, providing new insights about the role of the TrkB–Akt–NFkB signaling pathway in mediating these pathological hallmarks. These findings guide future research as DHF shows promise as a TrkB agonist treatment for HAND patients in adjunction to the current antiviral therapies.


Materials and methods
Animals. HIV-1 transgenic Tg26 mice FVB/N expressing high levels of 7 of the 9 HIV-1 proteins were established using the 7.4 kb transgene construct lacking the 3 kb sequence overlapping the gag/pol region of provirus pNL4-3 as described previously 29 . The model was originally obtained from the National Institute of Dental Research, and the colony has been maintained in the Institute of Human Virology (IHV) since 1995 by cross breeding heterozygous mice and continuously backcrossing to the wild-type FVB/N. To ensure little to no genetic drift, we also obtained colonies from Jackson Labs for comparison. Wild-type (WT) mice with an FVB/N genetic background generated from the same litter of Tg26 mice were used as controls for these studies. Female transgenic (Tg26) mice were housed under pathogen-free conditions at the animal facility of the Institute of Human Virology, University of Maryland School of Medicine, Baltimore. Female mice were used because Tg26 female mice show more cognitive deficits including short and long term spatial memory, loss in novel object location memory, and learning deficits, while male Tg26 mice don't express any such changes 30 . The study was carried out in compliance with the ARRIVE guidelines. All experimental procedures were conducted following NIH guidelines under an Institutional Animal Care and Use Committeeapproved protocol from the University of Maryland School Of Medicine, Baltimore.
Drug. We administered DHF (Tokyo Chemical Industry) intraperitoneally at a dose of 5 mg/kg in a 200 μl vehicle of 0.2% dimethyl sulfoxide (DMSO) in phosphate-buffered saline (PBS). Mice were divided into 3 groups untreated-Tg26 mice and DHF-treated Tg26 mice (Tg + DHF) and Wild type mice. Tg26 + DHF mice received a daily dose from day 90 to day 120. Vehicle-treated Tg26 mice received a daily dose of vehicle (200 μl of 0.2% DMSO in PBS) from day 90 to day 120.
Tissue pathology. Mice were euthanized on day 120 using the general anesthetic isoflurane. Brains were removed for analysis. Paraffin sections of the brain were prepared as previously described 25 . 7 μm thick sections were stained.
Immunohistochemistry. Immunohistochemistry was performed as previously described 25 using VEC-TASTAIN ABC kits (Vector Laboratories, Burlingame, CA, USA). Primary antibodies used in the experiment are listed in Table 1. Nuclei were counterstained with hematoxylin. Slides were examined using standard bright field microscopy.
Analysis of histological images using ImageJ. Hippocampal and cortical regions of the brain sections were selected from all mice for pathology and immunohistochemistry. Histological quantification was performed by a blind observer using Image J. All cell labeling experiments (antibodies listed in Table 1) were quantified based on the number of positive cells/field. (Each field = 400 × magnification picture). All fields covering the hippocampus and cortex were analyzed from each brain. Antibodies are listed below. Statistical analysis. Statistical analyses were done using Prism software (GraphPad, San Diego, CA). Values are expressed as means ± SEM. Statistical analysis was performed with one-way ANOVA, and Bonferroni's

DHF treatment induces phosphorylation of TrkB and Akt in Tg26 hippocampus.
To determine the effect of DHF on the phosphorylation of TrkB and downstream signaling pathways in Tg26 mice, we immunohistochemically labeled phosphorylated TrkB (P-TrkB), AKT (P-AKT) were examined in the mouse brains. As shown in Fig. 1A-H, Tg26 mice exhibited significantly decreased phosphorylation of TrkB in comparison to normal mice (p < 0.001), and DHF treatment significantly increased the expression of P-TrkB in the hippocampus (p < 0.01) and cortex (p < 0.001) in comparison to those of the Tg26 mice without DHF treatment.  www.nature.com/scientificreports/ The expression of phospho-AKT (P-AKT) was also decreased significantly in the hippocampus (p < 0.001) and cortex (p < 0.001) in Tg26 mice, and increased significantly in both the hippocampus (p < 0.05) and in the cortex (p < 0.001) in the DHF group compared to the Tg26 mice without DHF treatment ( Fig. 1I-P).
DHF downregulates expression of HIV-1 chemokine co-receptors CXCR4 and CCR5. CXCR4 and CCR5 have been implicated in mediating HIV/gp120 neurotoxicity 31,32 . To determine whether DHF mimics the role of BDNF and modulates the availability of chemokine receptors CXCR4 and CCR5 implicated in HIV-1 infection, the levels of CXCR4 and CCR5 expression were examined in the mouse brains by immunohistochemical labeling. We found that in both the hippocampus and cortex of Tg26 mice, CXCR4 ( Fig. 2A-H) expression was significantly upregulated in the hippocampus (p < 0.05) and cortex (p < 0.001) in comparison to normal  www.nature.com/scientificreports/ mice, and DHF treatment significantly downregulated this co-receptor (p < 0.001). CCR5 ( Fig. 2I-P) expression was also significantly upregulated in the hippocampus (p < 0.001) and cortex (p < 0.001) in comparison to normal mice, and DHF treatment significantly downregulated CCR5 expression (p < 0.001). These results suggest that DHF modulates the expression of CXCR4 and CCR5 on the hippocampal and cortical regions of Tg26 mice.
DHF treatment downregulates activation of the TLR4 and NFkB. The TLR4-NFkB pathway plays a significant role in the activation of proinflammatory responses during infection 33 . Hippocampal and cortical sections were examined immunohistochemically for cells expressing TLR-4 and NF-kB to evaluate the effect of DHF on inflammatory signaling in the brain of Tg26 mice. It was found that the number of cells expressing positive for TLR4 ( Fig. 3A-H) and NF-kB ( Fig. 3I-P) in Tg26 mice was significantly increased in the hippocampus (p < 0.001) and the cortex (p < 0.05, p < 0.001 respectively). In DHF-treated mice, TLR4 expression was significantly reduced in both regions (p < 0.001). For NF-kB however, this effect was only observed in the hippocampus (p < 0.001) but not the cortex (p > 0.05). These results suggest that in the Tg26 model, the TLR4-NFkB pathway is activated in the hippocampus and brain cortex, and DHF treatment suppresses proinflammatory signaling in these brain regions.
DHF treatment reduced astrogliosis in the brain of Tg26 mice. Reactive astrogliosis is a pathological hallmark of HIV-1 and is apparent in mouse and human HIV + brain tissues, indicated by increased glial fibrillary acidic protein (GFAP) staining 34 . GFAP was significantly increased in the hippocampus (p < 0.001) and cortex (p < 0.01) in Tg26 mice, indicative of astrogliosis. Interestingly, we found a significant decrease of GFAP ( Fig. 4) in the hippocampus (p < 0.05) and cortex (p < 0.05) of DHF treated Tg26 mice compared to Tg26 mice. These results suggest that DHF reduces astrogliosis in the hippocampus and cortex of Tg26 mice.

DHF treatment shifts the cytokine profile from Th1 towards Th2 response. Interferon Gamma
(IFN-y) and Tumor Necrosis Factor alpha (TNF-a) are proinflammatory Th1/17 cytokines. Interleukin-10 (IL-10), on the other hand, is a Th2 cytokine with potent anti-inflammatory processes. Corresponding to the results described in Sect. 3.4, we observed the same pattern of expression was observed in pro-inflammatory cytokines TNF-a ( Fig. 5A-H) and IFN-y ( Fig. 5I-P) in both the hippocampus (p < 0.001) and cortex (p < 0.001). In the Tg26 + DHF group, expression of these cytokines was significantly decreased in both the hippocampus and the cortex (p < 0.001). In contrast, expression of anti-inflammatory cytokine IL-10 ( Fig. 5Q-X) was significantly reduced in the hippocampus (p < 0.001) and the cortex (p < 0.001) of Tg26 mice, and DHF significantly reversed this pattern in both the hippocampus (p < 0.05) and the cortex (p < 0.05). These results suggest that DHF treatment promotes a shift from the Th1/17 towards the Th2 cytokine response in the hippocampus and cortex of Tg26 mice.

DHF treatment ameliorated mitochondrial dysfunction and biogenesis.
HIV + brains exposed to cART present altered mitochondrial biogenesis 35 . Hippocampal and cortical regions of Tg26 mice were immunohistochemically stained for Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) and NAD-dependent deacetylase sirtuin-3 (SIRT-3) to examine changes in mitochondrial biogenesis. Co-transcriptional regulation factor PGC-1a is known to induce mitochondrial biogenesis 36 . We found that PGC1-α ( Fig. 6A-H) was significantly decreased (p < 0.001) in the hippocampus of Tg26 mice in comparison to wild type mice, and was significantly upregulated (p < 0.001) in DHF-treated Tg26 mice in comparison to vehicle-treated Tg26 mice. SIRT3 is localized in mitochondria and is involved in energy metabolism, mitochondrial biogenesis, and mitochondrial fission/fusion [37][38][39][40] . Similarly, SIRT3 ( Fig. 6I-P) expression was downregulated in the hippocampus of Tg26 mice compared to normal mice (p < 0.001) and upregulated in DHF-treated Tg26 mice compared to untreated Tg26 (p < 0.01). DHF induced no significant changes in expression of either PGC-1a or SIRT-3 in the cortex of Tg26 mice. These results suggest that DHF improves mitochondrial biogenesis in the hippocampus, but not in the cortex. Citrate synthase is an enzyme-marker for intact mitochondria and is a good indicator of mitochondrial mass 41,42 . We found a significant decrease in the expression of citrate synthase in the hippocampus (p < 0.001) of Tg26 mice in comparison to the brains of normal mice, and DHF treatment significantly increased its expression in the hippocampus (p < 0.001) (Fig. 6Q-X). However, we observed no significant changes in citrate synthase levels between all three groups in the cortex (p > 0.05).
DHF treatment improved mitochondrial fission, but not fusion. Mitochondrial fission/fusion are simultaneous actions that occur in mitochondrial structures to regulate their morphology 43 . To test if DHF had an effect on mitochondrial fission/fusion, sections of the hippocampus and cortex of Tg26 mice were immunohistochemically labeled for mitochondrial fusion-2 (MFN-2) and mitochondrial fission (FIS-1). MFN-2 plays a role in the regulation of fusion processes 44 . While MFN-2 ( Fig. 7A-H) was significantly downregulated in both the hippocampus and cortex of Tg26 mice (p < 0.001), no significant increase was observed in the DHF treated group (p > 0.05). On the other hand, we found a significant increase in the expression of FIS-1, which is involved in the fragmentation of mitochondrial networks, (Fig. 7I-P) in both the hippocampus (p < 0.001) and cortex (p < 0.001) of Tg26 mice in comparison to wild type mice. Interestingly, DHF significantly lowered the expression of FIS-1 in Tg26 mice close to normal levels in the hippocampus (p < 0.001) and the cortex (p < 0.01). These results suggest that DHF may play a role in restoring normal mitochondrial fusion and fission in the hippocampus and cortex of Tg26 mice, but more research is necessary to reach a firm conclusion.  8I-P) by using immunohistochemical labeling. PACS-2 is a multifunctional sorting protein that regulates communication between the mitochondria and the endoplasmic reticulum 45 . In Tg26 mice, it was found that PACS-2 was significantly downregulated in the hippocampus (p < 0.001) and the cortex (p < 0.001). In the DHF treated group, no significant changes were found in either the hippocampus (p > 0.05) or the cortex (p > 0.05). VDAC-1 is an outer mitochondrial membrane and plasma membrane channel that regulates the release of extracellular ATP 46 . VDAC-1 was also significantly decreased in the hippocampus (p < 0.001) and cortex (p < 0.001) of Tg26 mice, but DHF only had a positive effect in the

Discussion
In this study, we demonstrated for the first time that administration of DHF resulted in reduced expression of HIV-1 chemokine co-receptors CXCR4 and CCR5, decreased astrogliosis, suppressed inflammatory activity, and improved mitochondrial function/biogenesis in the hippocampal and brain cortical regions of Tg26 mice. DHF also enhanced the phosphorylation of TrkB and its downstream signaling pathway Akt, which was correlated with a downregulation in downstream NFkB signaling, indicating that enhancing TrkB activation is a potential therapeutic mechanism in HAND.
TrkB is a receptor of BDNF that plays a role in stimulating neuronal survival, morphogenesis, and plasticity 47 , and DHF upregulates TrkB phosphorylation via the Akt pathway 48 . The PI3K/Akt signaling pathway protects against apoptosis and promotes neuronal survival 48 . DHF activates the TrkB receptor and the PI3K/AKT and MAPK pathway in hippocampal neurons 49 . In the Tg26 model, immunohistochemical staining for phosphorylated TrkB and Akt (Fig. 1) in the hippocampus and cortex revealed upregulated phosphorylation of the TrkB/ Akt pathway in DHF-treated Tg26 in comparison to Tg26 mice without treatment. These results indicate that DHF successfully crossed the BBB and sufficiently binded to the TrkB receptor. NFkB, which is downstream of Akt 50 , was upregulated in the same brain regions of Tg26 mice, and DHF reversed this change (Fig. 3A-H). These findings suggest that the antiinflammatory neuroprotective effects of DHF may be via activation of the TrkB/Akt pathway and downregulation of downstream NF-kB.
Several studies have demonstrated that astrocytes, neurons, and microglia express chemokine receptors CXCR4 and CCR5 which facilitate HIV-1 entry, making them highly susceptible to cytokine signaling and HIV-1 induced neurotoxicity 32,34,[51][52][53] . We demonstrate that CXCR4 and CCR5 immunoreactivity were significantly upregulated in Tg26 hippocampi and cortices as expected in a HAND model. BDNF, by activating TrkB, has been shown to be neuroprotective in vivo 10 and in vitro 11 against two strains of gp120, which binds to CXCR4 or CCR5 11,53,54 . In the hippocampus and brain cortex of BDNF (+ /-) mice, it was found previously that CXCR4 is highly expressed particularly in neurons rather than astrocytes 53 , perhaps because neurons are known to express full length TrkB receptors 56 . This suggests that BDNF may not regulate CXCR4 expression in nonneuronal cells. Nevertheless, the BDNF-TrkB pathway's ability to modulate CXCR4 expression underlies BDNF's neuroprotective properties against gp120, and our findings show that DHF successfully downregulates CXCR4 expression. The cognitive deficits induced by HIV-1 may also be due in part to the overexpression of CCR5; neuronal overexpression of CCR5 causes memory deficits, and decreasing the function of CCR5 improved long-term potentiation and neuroplasticity 56 . In a transgenic model of HIV-associated brain injury induced by a CXCR4-utilizing viral envelope gp120, CCR5 knockout prevented neuronal injury and behavioral impairment 57 . PWH carry a homozygous deletion of 32 base pairs in the CCR5 gene, which prevents CCR5 cell surface expression and protects against  (61,74,107). The total numbers of cortex fields analyzed (WT, Tg26, TG + DHF) were (15,15,20 [58][59][60] . Furthermore, CCR5 antagonist Maraviroc improves neurocognitive status in PWH when administered as a supplement to cART regimens 61 . Our findings suggest that DHF enhances neuroprotection and mediates neurotoxicity due to HIV-1 associated genes  www.nature.com/scientificreports/ by reducing CXCR4 and CCR5 expression in the hippocampus and brain cortex (Fig. 2), perhaps by increasing BDNF-TrkB signaling mechanisms, in the Tg26 model. Downregulating NF-kB may also have attenuated the expression of CXCR4 and CCR5, as these chemokine receptors play a role during neuroinflammation in HAND and previous studies suggest that NFkB mediates their expression [62][63][64] . Given that TrkB activation modulates CXCR4 and CCR5 expression, our findings suggest that this effect may be mediated by NF-kB 54,65 . Viral proteins such as gp120, Tat, and Vpr are indirectly neurotoxic by binding to CXCR4 and CCR5 and activating macrophages, microglia, and astrocytes 66 . This indirect neurotoxicity fosters an environment of chronic inflammation that is characteristic of HAND 66 . HAND is characterized by glial activation, cytokine/ chemokine dysregulation, and neuronal damage and loss 9,67 . Even in the cART era, microgliosis, microglial www.nature.com/scientificreports/ nodules, astrocytosis, and other neuroinflammation indicators have been found in the postmortem brains of PWH 9,68,69 , especially in the memory-associated areas of the brain including the hippocampus and entorhinal and temporal cortices 69 . A major barrier in the treatment of HIV-1 is the reduced activity of cART in the brain, where long-lived macrophages/microglia and astrocytes serve as viral reservoirs 6,7 . HIV-1 Tg26 mice exhibited an increase of GFAP + astrocytes in the cerebral cortex and hippocampus compared to WT mice, and DHF reversed these changes (Fig. 4). Various mouse models of HAND and human HIV + brain tissues have indicated that reactive astrogliosis is a pathological hallmark of HIV-1 34,70 . It is suggested that additional treatment targeting astrocytosis may be necessary to further reduce the effects of HIV-1 in the CNS 71 . Vartak-Sharma et al. demonstrated that astrogliosis in HIV-associated neuroinflammation may be mediated by NFkB signaling via  64, 119, 124). The total numbers of cortex fields analyzed (WT, Tg26, TG + DHF) for each antibody were PACS-2 (15,10,20), VDAC-1 (15,15,20 www.nature.com/scientificreports/ astrocyte elevated gene-1 72,73 . Our findings suggest that DHF protects against HIV-mediated astrogliosis, which may be through down-regulating NF-kB activation 74,75 . This is consistent with a study that examined the effect of DHF in mice exposed to perinatal hypoxia and ischemia 76 . In our findings, we show that DHF treatment mitigates neuroinflammation in the hippocampus and cortex of TG26 mice (Figs. [3][4][5]. Previously, it has been reported that TNF, IL-1β and IFN-γ are produced by activated monocytes/macrophages, microglia and T cells of PWH presenting signs of dementia, and the cerebrospinal fluid of PWH contain higher levels of proinflammatory cytokines such as IFN-γ, TNF-α, IL-2, IL-6, IL-7, and IL-8 77 . TNF-α is recognized to be an inducer of neuronal injury as it increases the permeability of the BBB, resulting in the migration of HIV-infected monocytes into the CNS 78 . IFN-γ correlated with the severity of neurologic impairment in PWH 79 . Prolonged abnormal presence of IFN-γ reduced heme oxygenase-1 expression in human astrocytes, which contributes to oxidative stress, another pathogenic characteristic of HAND 80 . Several previous studies have found that HIV viral genes induce the expression of IL-10; however, in the Tg26 mouse model [81][82][83][84][85] , we found the opposite. DHF upregulated IL-10, similar to our previous findings in a murine model for multiple sclerosis, a neurodegenerative disease 25 . DHF likely downregulated TNF-a indirectly by activating the TrkB signaling cascade, which in turn down-regulated NFkB and suppressed the neuroinflammatory response. The reduced levels of TNF-a may lead to increased levels of BDNF because TNF-a is known to prevent the activity of the glucocorticoid receptor, forming a positive-feedback type mechanism 84 . TLR-4 is an upstream mediator of NF-kB and is upregulated in astrocytes during HIV-1 infection 85 . The HIV-1 Tat protein binds to the TLR4-MD2-CD14 complex, activating the NF-kB pathway, and, in turn, inducing the production of pro-inflammatory cytokines 86,87 . In our findings with Tg26 mice, TLR4-NFkB activation possibly triggered the secretion of proinflammatory cytokines TNF-a and IFN-y, and inhibited secretion of antiinflammatory IL-10. It is widely accepted that NFkB is activated during HIV-induced neuroinflammation 88,89 . The activation of NFkB often acts as an initiating signal for the transcription of HIV-1, and targeting this signaling pathway could prevent ongoing low-level neurodegeneration by disturbing HIV-1's ability to sense immune cell activation 90 . Inhibition of NF-κB activity can reverse neuronal autophagy induced by Tat 91 . We recently reported that HIV-1 Vpr-induced proinflammatory response and apoptotic cell death are mediated through the NF-kB activation in astrocytes 92 . Targeting NFkB may ameliorate HIV-associated neuroinflammation. We report for the first time that DHF downregulates TLR4-NFkB signaling during HIV-associated neuroinflammation, resulting in an anti-inflammatory shift in cytokine release. Previously Park et al. 93 reported that DHF reduces LPS-induced NF-kB activity via the suppression of the nuclear translocation of NF-kB p65 and the degradation of inhibitor kB and reduced inflammation (70). Our data sheds light on DHF's ability to reduce neuroinflammation by attenuating the proinflammatory responses.
Persistent inflammation can be attributed to the metabolic abnormalities in PWH 94,97 . HIV-1 transgenic mice exhibit mitochondrial abnormalities associated with impaired energy homeostasis 96 . Impaired mitochondrial metabolism, altered mitochondrial biogenesis, and abnormal mitochondrial morphology are prominent in postmortem brains of PWH 97-100 . Our findings with SIRT3 and citrate synthase (Fig. 6I-X) suggest that DHF improves mitochondrial biogenesis and function. SIRT3 mediated mitochondrial biogenesis is regulated by PGC-1α 101 . Just as we found in the Tg26 model, PGC-1a levels are reduced in HIV + brains exposed to ART 35 . In a TBI model, it was previously established that DHF restored levels of PGC-1a 102 . This mechanism, similar to that of what we present in our animal model of HAND (Fig. 6 A-H), is likely TrkB dependent, as the BDNF receptor activates the cAMP-response-element-binding protein (CREB), a transcription factor that regulates PGC-1a 102 . Low PGC-1α levels are associated with stimulating NF-kB activation, as seen in our Tg26 model, suggesting a link between metabolic disturbances and the inflammatory response. DHF appears to ameliorate these changes. DHF has also been shown to exert neuroprotection independently through its antioxidant properties 103,104 , and SIRT3 regulates antioxidant activity 105 , so DHF may perhaps exert antioxidant effects in Tg26 mice. Mitochondrial dynamics also play an important role in HAND 106 .
In current literature, there are mixed results as to how the HIV alters mitochondrial fusion/fission. While one study determined that HIV-1 Vpr post-transcriptionally reduces the expression of MFN-2 and causes a loss in mitochondrial membrane potential 107 , another study claims that MFN-2 levels remained unchanged after exposure to HIV-1 tat in neurons. Interestingly, however, another study presented that in the brains of HIV + donors, mitochondrial fusion protein MFN-1 expression was increased in neurons, suggesting a shift towards mitochondrial fusion. For the first time, we show that MFN-2 levels decrease in the Tg26 model, and DHF did not affect MFN-2 levels in this model (Fig. 7A-H). As for mitochondrial fission, one study presented that HIV proteins induce the translocation of fission protein DRP-1 to promote mitochondrial fission 108 . Another study, however, determined that FIS-1 immunoreactivity was decreased in gp120 Tg mice 99 . In the Tg26 model, it was seen that mitochondrial fission was abnormally increased in the hippocampus and cortex regions by analysis of FIS-1, and unlike MFN-2, DHF did have a significant restoring effect on FIS-1 ( Fig. 7I-P), suggesting that DHF may have an ameliorative effect on mitochondrial fission. Mitochondrial fission/fusion is also regulated by NF-kB, suggesting an intricate relationship between mitochondrial dysfunction and chronic inflammation in HAND 109 . Together, our data suggests that HIV-1 viral proteins (expressed in Tg26 mice) produce chronically dysfunctional mitochondria, possibly contributing to the HAND pathology. DHF treatment may increase mitochondrial integrity, function by perhaps downregulating mitochondrial fission. Additionally, mitochondrial dysfunction and ER stress are pathological characteristics of HAND, which results in the disruption of ER-mitochondria communication [110][111][112][113] . In turn, this may lead to disturbances in mitochondrial bioenergetics/dynamics. For the first time, we show that PACS-2 ( Fig. 8A-H) and VDAC-1 ( Fig. 8I-P), both of which are involved in ER-mitochondria communication, mitophagy, and calcium influx 114,115 , are dysregulated in the Tg26 hippocampus and cortex. However, we did not observe a significant effect of DHF treatment on PACS-2 and VDAC-1 indicating need for further research to determine the effect of DHF on ER-mitochondria communication. www.nature.com/scientificreports/ While the study lays a strong foundation for the potential therapeutic efficacy of DHF, we also highlight several limitations. Our study only used in-vivo studies and IHC methods, so further research is needed to confirm the mechanisms underlying DHF's neuroprotective effect in HAND. Future studies should also use various techniques such as western blots and RT-PCR to further analyze protein levels and interactions with and without administration of DHF. Future research should also specifically explore if the shift towards anti-inflammation and metabolic homeostasis is independent or dependent of the TrkB-Akt signaling cascade. Behavioral testing and neuronal functional assays will also better evaluate the role of DHF in mediating the cognitive deficits of HAND. Furthermore, while the Tg26 mouse model has been used to study various HIV-associated comorbidities, its potential use for HAND has not been extensively investigated. Future studies should investigate DHF in other animal models such as HIV-1 humanized mice. By addressing these limitations with future research, we can gain better insight into DHF's potential as an adjunct therapeutic agent to current antiviral therapy.

Conclusion
We investigated the neuroprotective effects of BDNF using 7,8-dihydroxyflavone, a small molecule that is a bioactive high-affinity TrkB agonist utilizing the HIV-1 transgenic mouse model (Tg26). Our in vivo studies identified that HIV-1 Tg26 mice have neurologic deficits, associated with hippocampal and brain cortical changes in astrogliosis, CXCR4/CCR5 expression, inflammatory activity, and mitochondrial changes, all of which are characteristic of HAND. Following treatment with DHF in Tg26 mice, the mice exhibited a reversal of the pathological changes, suggesting the therapeutic potential of DHF in HAND. We provide an overview of how targeting BDNF-TrkB signaling in the pathophysiology of HAND may be relevant for future therapies, and how 7,8 Dihydroxyflavone may be a potential adjunct therapeutic agent to current antiviral therapy.

Data availability
"The datasets supporting the conclusions of this article are available in the National Addiction and HIV Data Archive Program repository, NAHDAP-122302.