HIV and FIV glycoproteins increase cellular tau pathology via cGMP-dependent kinase II activation

Human immunodeficiency virus (HIV) continues to be a major public health issue because HIV infection has become a chronic disease with the advent of combination antiretroviral therapy (cART), which has allowed individuals infected at a younger age to survive into older age (Farhadian et al., 2017). Importantly, a large proportion of HIV patient population faces aging-associated brain disorders, including Alzheimer's disease (AD) (Miller et al., 2011). In fact, prevalence of AD is elevated among patients with HIV and is even higher among patients who are not being treated with cART (Alisky, 2007; Brew et al., 2005; Nebuloni et al., 2001). It has been shown that hyperphosphorylated tau, a known AD pathological characteristic, is prematurely increased in the brains of HIV-infected patients as early as in their 30s and that its levels increase with age (Anthony et al., 2006). This suggests that HIV might lead to accelerated AD-associated tau phenotypes. However, whether HIV infection can lead to accelerated AD development in the brain remains unclear (Jha et al., 2020).

One of the major limitations in searching for mechanisms and treatments for AD in HIV infection is the lack of reliable animal models for investigating the pathophysiology (Chambers et al., 2015). Rodent models are heavily used for the study of HIV-associated neuropathology (Jaeger and Nath, 2012). However, results obtained in these models are often not easily translated to human pathology, given that rodents are not naturally susceptible to HIV infection and thus do not reflect chronic in vivo nature of infection (Fox and Gendelman, 2012). Although non-human primates infected with simian immunodeficiency virus (SIV) or genetic chimeras of SIV and HIV have a number of important advantages over small-animal models, they have obvious disadvantages in terms of high maintenance costs and considerable genetic variation that greatly complicate studies, especially when the number of animals used is low (Hatziioannou and Evans, 2012). Moreover, SIV is unable to cause acquired immune deficiency syndrome (AIDS) in its natural hosts (sooty mangabeys and chimpanzees), whereas SIV shows a relatively high pathogenic potential in rhesus macaques and humans following cross-species transmission events (Apetrei et al., 2005; Gardner, 1996; Sharp et al., 2005), which have extremely unbalanced epidemiologic consequences (Apetrei et al., 2005; Gardner, 1996). Most importantly, these animal models are unable to naturally develop neurofibrillary tangles (NTFs), a known tau pathology in AD (Chambers et al., 2015). Thus, new animal models to examine HIV-associated tau pathology are an important current and future need.

Feline immunodeficiency virus (FIV) infection in domestic cats represents an animal model of immunodeficiency that shares similarities to the pathogenesis of HIV in humans (Meeker and Hudson, 2017). Additionally, certain strains of FIV can enter the central nervous system (CNS), and underlie neurological symptoms similar to those observed in some individuals infected with HIV (Elder et al., 2010). Several neurological deficits have been also found as early as 12 months post-infection in studies of experimentally infected specific-pathogen-free (SPF) cats (younger than 6 months) designed to mimic HIV infection in humans (Meeker and Hudson, 2017). β-amyloid-associated senile plaques are a major pathological hallmark in AD and are found in many animal species, including chimpanzees and dogs; however, many of these animals are unable to exhibit NFTs and subsequent neurodegeneration (Chambers et al., 2015). Notably, cats express multiple tau isoforms like humans, and cats older than 14 years are a unique animal species that naturally replicates NFTs like humans (Chambers et al., 2015; Head et al., 2005; Janke et al., 1999). In addition, application of a combination of HIV antiretroviral drugs on cats naturally infected with FIV in the late phase of the asymptomatic state of the disease significantly reduces viral loads (Gomez et al., 2012). Therefore, FIV infection in domestic cats can act as an important model to substantially improve our understanding of the HIV-induced tau pathophysiology relevant to an older population of HIV-infected patients that has undergone antiretroviral therapy.

HIV is believed to prematurely age the brains of those with the disease and lead to brain dysfunction, as can AD (Chakradhar, 2018). Given these commonalities, it is possible that HIV could create conditions ripe for the development of AD (Chakradhar, 2018). Although the molecular and cellular mechanisms underlying this connection have not been fully investigated (Chakradhar, 2018), one potential mechanism of a HIV–AD connection is excitotoxicity, the pathological process by which neurons are damaged by neuronal hyperexcitation (Hinkin et al., 1995; Rottenberg et al., 1996; von Giesen et al., 2000). However, HIV does not directly infect neurons but instead interacts with neurons via a non-infectious interaction between the viral envelope and the neuronal surface (Bragg et al., 1999; Brenneman et al., 1988). HIV gp120 indirectly and/or directly interacts with neurons, which enhances excitatory synaptic receptor activity, resulting in synaptic damages, although the mechanisms are not currently understood (Kettenmann et al., 2013; Kim et al., 2011; Xu et al., 2011). We have previously demonstrated that both HIV gp120 and FIV gp95 interact with the receptor for the α-chemokine stromal cell-derived factor 1, CXCR4, which promotes Ca²⁺ influx through NMDA receptors (NMDARs) and subsequently activates cGMP-dependent protein kinase II (cGKII; also known as PRKG2) via nitric oxide (NO) signaling (Sztukowski et al., 2018). Notably, cGKII can phosphorylate the AMPA receptor (AMPAR) subunit GluA1 (also known as GRIA1), which triggers their synaptic trafficking, a critical step for inducing synaptic plasticity (Kim et al., 2015a; Serulle et al., 2007). In fact, we have found that HIV gp120- and FIV gp95-induced cGKII activation induces AMPAR-mediated Ca²⁺ hyperactivity in cultured mouse and feline hippocampal neurons (Sztukowski et al., 2018). In addition, viral glycoprotein-induced Ca²⁺ hyperactivity in cultured neurons is mediated by GluA2 (GRIA2)-lacking and GluA1-containing Ca²⁺-permeable AMPARs (Sztukowski et al., 2018). We have further confirmed that Ca²⁺ from the ER and extracellular space is the source of elevated Ca²⁺ in cultured neurons treated with viral glycoproteins (Sztukowski et al., 2018). This suggests that viral glycoproteins induce neuronal hyperexcitation via cGKII activation. This hyperexcitation is also strongly associated with early AD pathogenesis (Bero et al., 2011; Cirrito et al., 2008; Gibbons et al., 2019; Pooler et al., 2013; Wu et al., 2016; Yamada et al., 2014). Therefore, neuronal cGKII activation induced by HIV gp120 and FIV gp95 might provide a molecular link between HIV and AD.

Here, our data using cultured mouse cortical neurons have demonstrated that HIV gp120 and FIV gp95 significantly increases cellular tau pathology, including intracellular hyperphosphorylated tau and extracellular tau release. In addition, we have found that this cellular tau pathology is dependent on cGKII activation. Together, existing data and our new findings have revealed that HIV infection accelerates AD-related neural hyperexcitation and tau pathology via cGKII activation.

Neuronal Ca²⁺ is the second messenger responsible for transmitting depolarization status and synaptic activity (Gleichmann and Mattson, 2011). Ca²⁺ regulation is a vital process in neurons because of these characteristics, and abnormal Ca²⁺ activity in neurons is one of the major contributors to many neurological diseases (Gleichmann and Mattson, 2011). By measuring neuronal Ca²⁺ activity, we have previously shown that HIV gp120 or FIV gp95 treatment significantly increases neuronal activity in cultured mouse and feline hippocampal neurons (Sztukowski et al., 2018). We thus examined whether HIV gp120 or FIV gp95 treatment affected Ca²⁺ activity in cultured wild-type (WT) mouse cortical neurons infected with adeno-associated virus (AAV) expressing GCaMP7s, a genetically encoded Ca²⁺ indicator (Dana et al., 2019). First, we acutely treated day in vitro (DIV) 12–14 neurons with 1 nM HIV gp120 or 1 nM FIV gp95, and determined Ca²⁺ activity immediately after treatment. We found active spontaneous Ca²⁺ transients in control cells (CTRL) and neurons treated with viral proteins (Fig. 1A; Fig. S1). However, total Ca²⁺ activity in HIV gp120 or FIV gp95-treated cells was significantly higher than in controls (CTRL, 1±0.41 ΔF/F_min; HIV gp120, 1.70±0.51 ΔF/F_min, P<0.0001, and FIV gp95, 1.55±0.67 ΔF/F_min, P=0.0002; mean±s.d.), confirming that viral glycoproteins were sufficient to increase neuronal Ca²⁺ activity in cultured mouse cortical neurons (Fig. 1A). Importantly, Ca²⁺ flux through the NMDAR–nNOS (nNos is also known as NOS1) pathway activates cGKII through the production of cGMP (Bredt, 2003). cGKII mediates phosphorylation of serine 845 of GluA1 (pGluA1), which is important for activity-dependent trafficking of GluA1-containing AMPARs and increases the level of extrasynaptic receptors (Kim et al., 2015a; Sathler et al., 2021; Serulle et al., 2007). We have also shown that HIV gp120 or FIV gp95 treatment activates cGKII, which subsequently phosphorylates the AMPAR subunit GluA1, leading to the elevation of surface GluA1 expression and AMPAR-mediated synaptic activity, which is the cellular basis of hyperexcitation in viral glycoprotein-treated cultured hippocampal neurons (Sztukowski et al., 2018). We thus tested the possibility that cGKII was a downstream effector of viral glycoprotein-induced Ca²⁺ hyperexcitation in cultured cortical neurons. We found that HIV gp120 or FIV gp95 treatment was unable to increase Ca²⁺ activity when cGKII activity was blocked by treating neurons with 1 μM Rp8-Br-PET-cGMPS (RP), a cGKII inhibitor (HIV gp120+RP, 1.19±0.51 ΔF/F_min, P=0.0005, and FIV gp95+RP, 1.18±0.64 ΔF/F_min, P=0.0388; Fig. 1A). However, RP treatment in control neurons had no effect on Ca²⁺ activity (CTRL+RP, 1.04±0.53 ΔF/F_min, P=0.9992; Fig. 1A). We additionally cultured cortical neurons from cGKII-knockout (KO) mice as described previously (Kim et al., 2015a; Sztukowski et al., 2018) and confirmed that 1 nM HIV gp120 or 1 nM FIV gp95 treatment had no effect on Ca²⁺ dynamics in KO neurons (CTRL, 1±0.54 ΔF/F_min, HIV gp120, 0.85±0.50 ΔF/F_min, P=0.2560, and FIV gp95, 1.00±0.50 ΔF/F_minP=0.9988; Fig. 1B). This suggests that the viral glycoproteins HIV gp120 and FIV gp95 both use the cGKII-mediated core cellular pathway to induce neuronal hyperexcitation in cultured mouse cortical neurons, which is consistent with the previous findings in cultured mouse and feline hippocampal neurons (Sztukowski et al., 2018).

Although tau is primarily a cytoplasmic protein that stabilizes microtubules, it can be released into the extracellular space, enter neighboring neurons, and spread tau pathology throughout the brain; this process is not dependent on neuronal death but can be stimulated by increased neuronal activity as AD progresses (Braak and Braak, 1991; Chai et al., 2012; Clavaguera et al., 2009; Frost et al., 2009; Guo and Lee, 2011; Iba et al., 2013; Karch et al., 2012; Kfoury et al., 2012; Pooler et al., 2013; Sanders et al., 2014; Wu et al., 2016; Yamada et al., 2014). Several recent studies have shown that tau can be physiologically released to the extracellular fluid both in vivo and in cultured cells, and such release appears to be regulated by neuronal activity (Pooler et al., 2013; Wu et al., 2016; Yamada et al., 2014). We thus determined whether an increase in neuronal activity was sufficient to induce extracellular tau release in cultured mouse cortical neurons. Extracellular tau levels were measured using a total tau solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) with a mouse anti-tau antibody (Yan et al., 2016). To directly activate neurons’ activity, we treated DIV 14 neurons with 1 mM glutamate, an excitatory neurotransmitter, for 10 min and collected the conditioned culture medium to measure extracellular tau concentration. As expected, glutamate treatment significantly increased extracellular tau levels compared to control cells (CTRL, 631.45±188.60 pg/ml and glutamate 1341.52±266.63 pg/ml, P=0.0286; mean±s.d.; Fig. 2A), confirming that elevated neuronal activity was sufficient to increase extracellular tau levels in cultured neurons. Given that HIV gp120 or FIV gp95 treatment is sufficient to increase neuronal activity in cultured mouse cortical neurons (Fig. 1), we next examined whether viral glycoproteins enhanced tau release to the extracellular space in cultured neurons. To test this, we treated DIV 14 neurons with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h and measured extracellular tau levels in the conditioned culture medium by ELISA as described above. We found that both HIV gp120 and FIV gp95 significantly increased extracellular tau concentration (CTRL, 661.10±169.09 pg/ml, HIV gp120, 1506.31±224.96 pg/ml, P=0.0055, and FIV gp95, 987.11±250.15 pg/ml, P=0.0325; Fig. 2B). This suggests that viral glycoprotein-induced neuronal hyperexcitation contributes to an increase in tau release to the extracellular space. Given that viral glycoprotein-induced neuronal hyperexcitation is dependent on cGKII activation (Fig. 1), we examined whether inhibition of cGKII activity reversed the viral glycoprotein effects on extracellular tau levels in cultured neurons. 1 μM RP was added to DIV 14 cultured neurons treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h, and conditioned culture medium was collected to determine extracellular tau levels by ELISA. We found that inhibition of cGKII activity significantly reversed a viral glycoprotein-induced increase in extracellular tau concentrations (HIV gp120+RP, 516.17±131.04 pg/ml, P=0.0003, and FIV gp95+RP, 526.82±219.69 pg/ml, P=0.0024; Fig. 2B). However, RP treatment in the absence of viral glycoproteins had no effect on tau release (CTRL+RP, 571.35±193.31 pg/ml, P=0.9634; Fig. 2B). To further address whether cGKII activation was crucial for an increase in extracellular tau levels by viral glycoproteins, we used cGKII KO cultured cortical neurons treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h, with the extracellular tau concentration measured by ELISA as described above. We found that HIV gp120 or FIV gp95 treatment was unable to increase extracellular tau levels in cGKII KO neurons (CTRL, 588.10±198.33 pg/ml, HIV gp120, 644.23±272.66 pg/ml, P=0.8633, and FIV gp95, 617.97±313.13 pg/ml P=0.9591; Fig. 2C). Taken together, these results show that the viral glycoproteins HIV gp120 and FIV gp95 are sufficient to increase extracellular tau concentration via cGKII-dependent neuronal hyperexcitation in cultured cortical neurons.

Hyperphosphorylated tau has been identified in the brains of HIV-positive people with brain impairment as early as in their 30s, and its levels rise with age, which is known to contribute to neurodegeneration in HIV brains (Anthony et al., 2006; Patrick et al., 2011). HIV gp120 is known to induce tau hyperphosphorylation, which in turn contributes to NFT formation in rodent brains (Cho et al., 2017; Kang et al., 2010). We thus examined whether HIV gp120 or FIV gp95 treatment induced tau hyperphosphorylation in cultured mouse cortical neurons. DIV 14 cultured cortical neurons were treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h, and total cell lysates were collected to determine tau hyperphosphorylation using immunoblotting. We found that HIV gp120 or FIV gp95 treatment significantly increased the signal from AT8 antibody, which recognizes tau phosphorylation at serine 202 and threonine 205, and thus phosphorylated paired helical filament (PHF) tau (CTRL, 1.00, HIV gp120, 1.92±0.49, P<0.0001, and FIV gp95, 1.47±0.35, P=0.0012; mean±s.d.; Fig. 3A). As we have shown that cGKII activation is critical for viral glycoprotein-induced neuronal hyperexcitation (Fig. 1) and elevated extracellular tau levels (Fig. 2), we examined whether viral glycoprotein-induced cGKII stimulation was responsible for tau hyperphosphorylation. 1 μM RP was added to DIV 14 cultured neurons treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h, and hyperphosphorylated tau levels were examined using immunoblots. Notably, inhibition of cGKII activation was sufficient to abolish a HIV gp120- or FIV gp95-induced increase in phosphorylated PHF tau levels (HIV gp120+RP, 0.82±0.29, P<0.0001, and FIV gp95, 0.97±0.28, P=0.0026; Fig. 3A). In contrast, RP treatment in control cells was unable to affect tau hyperphosphorylation (CTRL+RP, 1.00±0.40, P>0.9999; Fig. 3A). To further address whether cGKII activation was important for viral glycoprotein-induced tau PHF formation, we employed cGKII KO cultured cortical neurons and treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h, hyperphosphorylated tau was determined by immunoblotting using the AT8 antibody as described above. We found that HIV gp120 or FIV gp95 treatment was unable to increase phosphorylated PHF tau levels in cGKII KO neurons (CTRL, 1.00, HIV gp120, 1.10±0.95, P=0.9841, and FIV gp95, 0.87±0.46, P=0.9513; Fig. 3B). Importantly, tau contains multiple phosphorylation sites (Hole and Williams, 2021). We thus examined an additional tau phosphorylation at threonine 217 (tau-pThr217) using immunoblots. DIV 14 cultured cortical neurons were treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h, and we determined tau-pThr217. We found that HIV gp120 or FIV gp95 treatment significantly increased tau-pThr217 (CTRL, 1.00, HIV gp120, 1.99±0.63, P=0.008, and FIV gp95, 1.96±0.58, P=0.011; Fig. S2A). To further test whether viral glycoprotein-induced cGKII stimulation was responsible for tau-pThr217, 1 μM RP was added to DIV 14 cultured neurons treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h, and tau-pThr217 levels were examined. Consistent with the findings in Fig. 3A, inhibition of cGKII activation was sufficient to abolish viral glycoprotein-induced elevated tau-pThr217 (HIV gp120+RP, 1.03±0.34, P=0.0106, and FIV gp95, 0.97±0.45, P=0.0079; Fig. S2A). In contrast, RP treatment in control cells was unable to affect tau-pThr217 (CTRL+RP, 0.97±0.44, P>0.9999; Fig. S2A). We further revealed that HIV gp120 or FIV gp95 treatment was unable to increase tau-pThr217 levels in cGKII KO neurons (CTRL, 1.00, HIV gp120, 1.10±0.55, P=0.9580, and FIV gp95, 1.10±0.64, P=0.9293; Fig. S2B). Taken together, these results show that the viral glycoproteins HIV gp120 and FIV gp95 are sufficient to induce tau hyperphosphorylation, including phosphorylation at serine 202, threonine 205 and threonine 217, via cGKII activation in cultured cortical neurons.

Tau can be phosphorylated by various kinases, including p38 mitogen-activated protein kinases (p38Ks) and glycogen synthase kinase 3β (GSK3β) (Billingsley and Kincaid, 1997; Han et al., 2005; Pei et al., 1999; Wang et al., 2013). Among these kinases, p38Ks are responsible for HIV gp120-induced neuronal death, microglial and macrophage activation, and proinflammatory cytokine production (Kaul and Lipton, 1999; Kaul et al., 2007). Moreover, pharmacological inactivation of p38Ks in mixed neuronal-glial cultures prevents neuronal death triggered by HIV gp120 (Kaul and Lipton, 1999; Kaul et al., 2007). Importantly, HIV gp120 directly interacts with neurons in the absence of macrophages, and that interaction also results in activation of neuronal p38Ks (Medders et al., 2010). However, the exact mechanism linking HIV gp120 activation of p38Ks in both macrophages and neurons currently remains unknown. Previous studies report that activation of p38Ks requires cGMP-dependent protein kinases in various cell types, including cardiomyocytes, thrombocytes, and fibroblasts (Browning et al., 2000; Kim et al., 2000; Li et al., 2006). We thus hypothesized that HIV gp120 and FIV gp95-induced cGKII stimulation activated p38Ks and, in turn, induced tau hyperphosphorylation in neurons. In fact, p38Ks are known to phosphorylate tau at several sites, including serine 202 and threonine 205 (recognized by the AT8 antibody) and threonine 217 (Hole and Williams, 2021). We thus treated DIV 14 mouse cortical neurons with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h and determined p38K activation using immunoblots. Notably, HIV gp120 or FIV gp95 treatment significantly increased p38K phosphorylation at threonine 180 and tyrosine 182, an active form of p38K (CTRL, 1.00, HIV gp120, 1.49±0.32, P<0.0001, and FIV gp95, 1.52±0.24, P=0.0012; mean±s.d.; Fig. 4A). We next examined whether viral glycoprotein-induced p38K activation was dependent on cGKII activation. We added 1 μM RP to DIV 14 cultured neurons treated with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h and determined p38K activation using immunoblots. Notably, pharmacological inhibition of cGKII was sufficient to abolish viral glycoprotein-induced p38K activation (HIV gp120+RP, 0.87±0.29, P<0.0001, and FIV gp95+RP, 0.92±0.18, P=0.0001; Fig. 4A). However, RP treatment in control neurons had no effect on p38K phosphorylation levels (CTRL+RP, 0.94±0.24, P=0.6229; Fig. 4A). We further used cGKII KO cultured cortical neurons and treated them with 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h to see if cGKII activation was crucial for viral glycoprotein-induced p38K activation. HIV gp120 or FIV gp95 treatment in KO cells was unable to activate p38Ks (CTRL, 1.00, HIV gp120, 1.04±0.18, P=0.8773, and FIV gp95, 1.03±0.28, P=0.9462; Fig. 4B), suggesting that neuronal p38K activation by HIV gp120 and FIV gp95 requires cGKII activity.

We next examined whether viral glycoprotein-induced p38K activation was crucial for tau hyperphosphorylation. DIV 14 cultured cortical neurons were exposed to 1 nM HIV gp120 or 1 nM FIV gp95 with 10 μM SB203580 (SB), a p38K inhibitor, for 24 h. As seen previously (Fig. 3A), viral glycoprotein treatment was sufficient to increase phosphorylated PHF tau levels, which was significantly reversed by pharmacological inhibition of p38K activity (CTRL, 1.00, HIV gp120, 1.65±0.27, P<0.0001, FIV gp95, 1.48±0.19, P=0.0028, HIV gp120+SB, 1.00±0.28, P<0.0001, FIV gp95+SB, 0.89±0.278, P=0.0001; Fig. 4C). However, SB treatment in control cells had no effect on tau hyperphosphorylation (CTRL+SB, 0.90±0.24, P=0.9526; Fig. 4C). Taken together, we demonstrate that the viral glycoproteins HIV gp120 and FIV gp95 induce tau hyperphosphorylation via cGKII-mediated p38K activation.

It has been shown that glutamate treatment causes a rapid rise in hyperphosphorylated tau protein immunoreactivity in primary neuronal cells (Sindou et al., 1994). It has been further suggested that glutamate acts on AMPARs, NMDARs and metabotropic receptors, and can trigger activations of intracellular second messengers leading to tau hyperphosphorylation in cultured cortical neurons (Sindou et al., 1994). The mechanism underlying the glutamate-induced increase in neuronal activity via induction of tau hyperphosphorylation has not been elucidated. Given that viral glycoprotein-induced neuronal hyperexcitation was sufficient to increase tau hyperphosphorylation via cGKII activation in cultured cortical neurons (Figs 1 and 3), it is possible that cGKII activity was required for this phenomenon. We first treated DIV 14 neurons with 1 mM glutamate for 10 min and determined tau hyperphosphorylation using immunoblots with AT8 antibody and found that glutamate treatment significantly increased hyperphosphorylated tau (CTRL, 1.00 and glutamate, 1.44±0.41, P=0.0300; mean±s.e.m.; Fig. 5A), which is consistent with the previous findings (Sindou et al., 1994). To determine whether such an increase in tau hyperphosphorylation was mediated by cGKII activation, 1 μM RP was added to DIV 14 cultured neurons treated with 1 mM glutamate for 10 min, and we evaluated tau phosphorylation as described above. Interestingly, RP treatment was unable to reverse glutamate-induced tau hyperphosphorylation (glutamate+RP, 1.83±0.87, P=0.0019; Fig. 5A). To further confirm whether cGKII activation was distinct from glutamate-induced tau hyperphosphorylation, DIV 14 cultured cGKII KO neurons were treated with 1 mM glutamate for 10 min, and hyperphosphorylated tau levels were determined by immunoblotting. We found that glutamate treatment in KO cells significantly increased tau hyperphosphorylation (CTRL, 1.00 and glutamate, 1.68±0.68, P=0.0312) (Fig. 5B). Taken together, these results show that the glutamate-induced increase in neuronal activity significantly elevated hyperphosphorylated tau levels in cultured cortical neurons is independent from cGKII activation.

Introduction of cART for HIV has dramatically reduced the number of HIV-related deaths, resulting in more HIV patients reaching ages when AD-like symptoms appear (Farhadian et al., 2017). Tau pathology has been seen in the brains of HIV-infected individuals as early as their 30s and further develops as the individuals get older (Anthony et al., 2006). HIV can prematurely age the brains of those with the disease and lead to brain dysfunction, as can AD (Chakradhar, 2018). Given these commonalities, it is possible that HIV could create suitable conditions for the development of AD (Chakradhar, 2018), although the molecular and cellular mechanisms underlying this connection have not been fully investigated (Chakradhar, 2018). Neuronal overexcitation is a putative biological mechanism of HIV-associated AD (Bero et al., 2011; Cirrito et al., 2008; Gibbons et al., 2019; Hinkin et al., 1995; Pooler et al., 2013; Rottenberg et al., 1996; von Giesen et al., 2000; Wu et al., 2016; Yamada et al., 2014). In fact, we have discovered that the viral glycoproteins HIV gp120 and FIV gp95, known neurotoxic viral antigens, stimulate cGKII, which controls AMPA receptor trafficking and hence induces neuronal hyperexcitation (Sztukowski et al., 2018) (Fig. 6). Here, our new data using cultured mouse cortical neurons further demonstrate that HIV gp120 and FIV gp95 significantly increase cellular tau pathology, including intracellular hyperphosphorylated tau and extracellular tau release (Fig. 6). Additionally, we find that this cellular tau pathology is dependent on cGKII activation (Fig. 6). Thus, our current work identifies a novel mechanism underlying the link between HIV and AD-related tau pathology relevant to an older population of HIV-infected patients.

It has been previously shown that viral glycoproteins induce neuronal cell death via an increase in intracellular Ca²⁺ levels. However, viral glycoprotein-induced excitotoxicity is still controversial because viral glycoprotein-induced neuronal death is dependent on a concentration of the viral proteins, incubation time, and maturity of cultured cells. For example, there is a study showing that long-term treatment (longer than 4 days) with a low concentration of HIV gp120 (20 pM) in cultured hippocampal neurons induces Ca²⁺-dependent cell death (Meucci and Miller, 1996). Another study demonstrates that a 12-h treatment of 8 nM HIV gp120 induces apoptosis of DIV 7 cultured cortical neurons (Guo et al., 2013). Furthermore, 1 nM HIV gp120 treatment for 24 h can induce apoptosis of DIV 10 cultured hippocampal neurons (Zhu et al., 2015), whereas in DIV 6–7 feline neurons, 200 pM FIV envelope protein treatment for 24 h significantly increases neuronal death (Bragg et al., 1999). Taken together, excitotoxicity by viral glycoproteins is likely enhanced when neurons are less mature, the concentration of the protein is higher and the incubation time is longer. Our experimental condition, 1 nM HIV gp120 or 1 nM FIV gp95 for 24 h in DIV 14 neurons, is unlikely to induce neuronal death. It is important to note that the increased Ca²⁺ activity in cultured neurons treated with viral glycoproteins is modest in our conditions, suggesting that a small increase in Ca²⁺ activity likely makes important contributions to pathogenesis without reaching a threshold that can cause neuronal death.

As of now, neither cure nor therapeutic approaches for HIV-associated AD-like pathology are available. Despite the massive investment in AD drugs, there have been more failures than treatment successes (Huang et al., 2019). One of the challenges that the research community has faced is the lack of a viable target for treatment (Saylor et al., 2016). In recent years, a hypothesis implicating the role of neuronal hyperexcitability, hypersynchronous network activity and aberrant hippocampal network rewiring in memory loss at the early stage of AD has emerged (Busche et al., 2012, 2008; Hall et al., 2015; Kazim et al., 2017; Minkeviciene et al., 2009; Šišková et al., 2014). Importantly, brain imaging studies reveal that hypermetabolic signatures of glucose abnormalities, an indication of elevated cerebral neural plasticity, also appear to be an early event in HIV-associated neurocognitive disorder pathogenesis (Hinkin et al., 1995; Rottenberg et al., 1996; von Giesen et al., 2000). However, the underlying cellular mechanisms of AD- and HIV-related neuronal hyperexcitability remain unclear. We have shown that HIV and FIV glycoprotein-induced neuronal hyperexcitation and cellular tau pathology are mediated by a cGKII-dependent cellular mechanism (Fig. 6). This thus suggests that aberrant cGKII stimulation might be a cellular basis for AD- and HIV-related neuronal hyperexcitability. Importantly, tau pathology trans-synaptically spreads throughout the brain, which can be stimulated by increased neuronal activity as AD progresses (Braak and Braak, 1991; Kfoury et al., 2012; Sanders et al., 2014; Wu et al., 2016). Indeed, increasing neuronal activity rapidly elevates the steady-state levels of extracellular tau in vivo (Sanders et al., 2014; Wu et al., 2016). Moreover, stimulation of excitatory glutamatergic synapses is sufficient to drive tau release (Yamada et al., 2014). Although the mechanism by which tau can be released from neurons is unknown, hyperexcitable neurons in AD- and HIV-infected brains might contribute to tau spread. Taken together, abnormal cGKII activation by viral infection can increase neuronal activity via enhancing surface expression of AMPARs, which promotes cellular tau pathology in HIV-positive brains (Fig. 6). Therefore, by identifying cGKII as a target of HIV-associated AD pathology, our study completes the pathological pathway and implicates cGKII as a new therapeutic target for limiting HIV-induced AD pathology. Thus, use of cGKII inhibition as a means for neuroprotection might be a novel and innovative approach to this therapeutically challenging disease pathway.

Tau phosphorylation has significant physiological and pathological consequences (Šimić et al., 2016; Wang and Mandelkow, 2016; Yamada, 2017). Nonetheless, the phosphorylation state of released tau is still controversial (Busche et al., 2008; Šimić et al., 2016; Wang and Mandelkow, 2016). For example, phosphorylated tau can be released under normal conditions; however, the physiological function of released tau is unknown (Ismael et al., 2021; Pooler et al., 2013; Yamada, 2017; Yamada et al., 2011). Conversely, several studies have shown that released tau is unphosphorylated (Croft et al., 2017; Mohamed et al., 2014; Pooler et al., 2013). Differences between studies on phosphorylated- or unphosphorylated-released tau can be explained by several factors, including rodent versus human cells, endogenous versus overexpressed tau, physiological versus pathological tau, membrane bound versus vesicle bound, and others (Ismael et al., 2021). More research thus is needed to resolve the differences.

Interestingly, we reveal that glutamate treatment can induce tau hyperphosphorylation that is not dependent on cGKII activation (Fig. 5). Indeed, our results are in accordance with the literature showing that glutamate treatment is sufficient to induce tau phosphorylation in cultured cortical neurons (Sindou et al., 1994). Importantly, many mechanisms underlying glutamate treatment-induced tau hyperphosphorylation have been suggested, including modulation of protein kinases [GSK3β and cyclin dependent kinase 5 (CDK5)] and/or tau dephosphorylation phosphatases (Adamec et al., 1997; Fleming and Johnson, 1995; Revett et al., 2013). Therefore, it is possible that glutamate could modulate tau phosphorylation by a variety of mechanisms that might be independent of cGKII.

Neuroinflammation plays an important role in AD and possible links to viruses have been proposed (Canet et al., 2018). Microglia are the resident immune cells of the CNS and the major cell types infected by HIV in the brain (Dheen et al., 2007). Neuroinflammatory processes mediated by the activated microglia have been strongly implicated in a number of neurodegenerative diseases, including HIV-associated neurocognitive disorders (Gonzalez-Scarano and Baltuch, 1999). Similar to neuronal mechanisms, HIV gp120 interacts with microglial CXCR4 to increase Ca²⁺ activity via enhancing IP3R function, leading to stimulation of inducible NO synthase (iNOS; also known as NOS2) and subsequent production of NO in microglia (Persichini et al., 2014). Importantly, NO activates cAMP response element-binding proteins (CREBs) via cGMP-dependent kinase stimulation, leading to upregulation of microglial CD11b expression, an indication of microglial activation during neurodegenerative inflammation (Roy et al., 2006). This suggests that HIV gp120-induced cGKII stimulation in microglia can be critical for their activation. Moreover, HIV gp120 elevates synaptic receptor activity by enhancing the release of proinflammatory cytokines from activated microglia (Kaul et al., 2005; Sillman et al., 2018). This suggests that microglial cGKII stimulation by viral glycoproteins can be important for activation in microglia, which results in elevation of excitatory synaptic activity, leading to excitotoxicity and tau pathology.

Our cultured neuron system provides cGKII-dependent mechanisms underlying viral glycoprotein-induced neuronal hyperexcitation and cellular tau pathology, including an increase in intracellular hyperphosphorylated tau and extracellular tau release, but our in vitro system is unable to produce NFTs. Therefore, further in vivo studies are needed to better understand pathophysiology and to develop more effective treatments for AD in HIV infection, such as those alluded to above.

CD-1 (Charles River) and cGKII KO postnatal day 0 male and female mouse pups were used to produce mouse cortical neuron cultures as shown previously (Sathler et al., 2021; Sztukowski et al., 2018). cGKII KO animals were maintained as previously described (Kim et al., 2015a; Sztukowski et al., 2018; Tran et al., 2021). We confirmed no cGKII protein expression in cultured KO cortical neurons by immunoblotting (Fig. S3). Colorado State University's Institutional Animal Care and Use Committee reviewed and approved the animal care and protocol (978).

Primary mouse cortical neuron cultures were prepared by a previously described protocol (Kim and Ziff, 2014). Cortices were isolated from postnatal day 0 (P0) CD-1 or cGKII KO mouse brain tissues and digested with 10 U/ml papain (Worthington Biochemical Corp. Lakewood, NJ). Mouse cortical neurons were plated on following poly lysine-coated dishes for each experiment – 24 wells (200,000 cells) for ELISA, glass-bottom dishes (500,000 cells) for Ca²⁺ imaging, and 6-cm dishes (2,000,000 cells) for immunoblotting. Cells were grown in Neurobasal Medium with B27 supplement, 0.5 mM Glutamax, and 1% penicillin and streptomycin (all Thermo Fisher Scientific).

DIV 4 neurons were infected with adeno-associated virus (AAV) expressing GCaMP7s (pGP-AAV-syn-jGCaMP7s-WPRE) from Addgene (plasmid #104487, deposited by Douglas Kim, GENIE Project; Dana et al., 2019) for imaging cortical neurons. Neurons were grown in Neurobasal Medium without Phenol Red and with B27 supplement, 0.5 mM Glutamax, and 1% penicillin and streptomycin (all Thermo Fisher Scientific). Glass-bottom dishes were mounted on a temperature-controlled stage on an Olympus IX73 microscope and maintained at 37°C and 5% CO₂ using a Tokai-Hit heating stage and digital temperature and humidity controller. For GCaMP7s, the images were captured with a 10 ms exposure time and a total of 100 images were obtained with a 1 s interval. F_min was determined as the minimum fluorescence value during the imaging. Total Ca²⁺ activity was obtained by calculating 100 values of ΔF/F_min=(F_t−F_min)/F_min in each image, and values of ΔF/F_min<0.1 were rejected due to potential photobleaching. The average total Ca²⁺ activity in the control group was used to normalize the total Ca²⁺ activity in each cell. The average total Ca²⁺ activity for the control group was compared to the average for the experimental groups as described previously (Kim et al., 2015a,b; Kim and Ziff, 2014; Roberts et al., 2021; Sun et al., 2019; Sztukowski et al., 2018).

Expression, amplification and purification of the FIV envelope glycoprotein gp95 were performed using previously described methods (de Parseval and Elder, 2001; Sztukowski et al., 2018; Wood et al., 2013). HIV CXCR4-tropic gp120 (IIIB) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 IIIB gp120 Recombinant Protein from ImmunoDX, LLC. The following inhibitors were used in this study: 1 μM Rp8-Br-PET-cGMPS (RP) (Thermo Fisher Scientific), and 10 μM SB203580 (Thermo Fisher Scientific) for cultured mouse neurons. 1 mM glutamate (Thermo Fisher Scientific) was used to activate glutamate receptors.

Total mouse tau ELISA measurements were carried out using a total mouse tau ELISA kit (Thermo Fisher Scientific, catalog #KMB7011) according to the manufacturer's instruction. DIV 14 cultured mouse cortical neurons were treated with reagents in each experiment, and the conditioned culture medium was collected and centrifuged at 1000 g for 10 min. Extracellular tau levels (phosphorylated or unphosphorylated tau) were determined in a total tau solid-phase sandwich ELISA with an anti-mouse tau antibody-coated plate and mouse tau (total) biotin conjugate. Streptavidin conjugated to horseradish peroxidase was used for assays. All assays were developed using tetramethylbenzidine (TMB) and read on a plate reader at 450 nm. We used recombinant mouse tau as a standard.

Immunoblotting was performed as described previously (Farooq et al., 2016; Kim et al., 2016, 2018; Shou et al., 2018). The protein concentration in total cell lysates was determined by a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein samples were loaded on 10% glycine-SDS-PAGE gels. The separated proteins were transferred onto nitrocellulose membranes. The membranes were blocked (5% powdered milk or 5% bovine serum albumin) for 1 h at room temperature, followed by overnight incubation with the primary antibodies at 4°C. The primary antibodies used were: anti-phosphorylated tau (AT8) (Thermo Fisher Scientific, catalog #MN1020, 1:1000), anti-phosphorylated tau (phospho-Thr217) (GenScript, catalog #A00896, 1:1000), anti-Tau-1 (total), clone PC1C6 (MilliporeSigma, catalog #MAB3420, 1:1000), anti-phosphorylated p38K (Cell Signaling Technology, catalog #4511, 1:200), anti-p38K (Bioss, catalog #bs-0637R, 1:1000), anti-cGKII (Kim et al., 2016; Serulle et al., 2007; Tran et al., 2021; Covance, 1:1000), and anti-actin (Abcam, catalog #ab3280, 1:2000) antibodies. Membranes were subsequently incubated by secondary antibodies for 1 h at room temperature and developed with enhanced chemiluminescence (ECL) (Thermo Fisher Scientific). Protein bands were quantified using ImageJ (https://imagej.nih.gov/ij/).

All statistical comparisons were analyzed with the GraphPad Prism 9. A non-parametric Mann–Whitney test, or Wilcoxon signed rank test were used in single comparisons. For multiple comparisons, we used one-way or two-way ANOVA followed by Tukey test to determine statistical significance. Results are represented as mean±s.d., and a P value<0.05 was considered statistically significant.

We thank members of the Kim laboratory for their generous support. We also thank Mary Nehring in Dr Sue VandeWoude's laboratory for helping with the ELISA experiments. We thank Dr James Bamburg and Laurie Minamide for providing the phosphorylated tau antibody (tau-pThr217).