HIV-1 enters the central nervous system early in the infection process and may ultimately result in an array of deficits, collectively known as HIV-1-associated neurocognitive disorders (HAND) (Antinori et al. 2007). Although the incidence of the most severe dementia has decreased as the development of combination anti-retroviral therapy (cART), neurocognitive deficits continue to persist in more than 40% of HIV-1-infected individuals (Lindl et al. 2010; Letendre 2011). The continued prevalence of HAND despite the success of cART (Ances and Ellis 2007; Lindl et al. 2010; Letendre 2011) indicates a need to understand the underlying mechanism(s) of HAND and identify effective treatments.
Neuronal cell death has been observed in post-mortem brain tissue of patients with HAND; however, cell death does not correlate well with neurocognitive status (Adle-Biassette et al. 1999; Kaul et al. 2001; Ellis et al. 2007). Dendritic pruning, decreases in spine density, and degradation of synaptic proteins all correlate more readily with HIV-1-induced neurocognitive decline than does cell death (Masliah et al. 1997; Adle-Biassette et al. 1999; Kaul et al. 2001). Moreover, loss of dendrites and decreased synaptic density in the frontal cortex from HIV+ patients has been reported with latent viral infection (Desplats et al. 2013).
During latent HIV-1 infection, the provirus is incorporated into cellular DNA, without the expression of viral RNA (Wu 2004). Current anti-retroviral drugs effectively suppress peripheral viral load, but do not prevent the continued latent production of HIV-1 neurotoxic proteins (i.e. Tat) once the proviral DNA is incorporated into the brain (Li et al. 2009). HIV-1 provirus containing cells can thereby produce and release Tat into the extracellular space (Pugliese et al. 2005; Bachani et al. 2013) and Tat can interact with the cell surface of other, non-infected cells, such as neurons. HIV-1 Tat protein has been found to decrease dendritic spine and synaptic density in vitro and in vivo (Everall et al. 1999; Kim et al. 2008; Fitting et al. 2010). Therefore, a more detailed understanding of how HIV-1 Tat interacts with the dendritic network may aid in preventing neuropathological dendritic pruning and synaptic loss during latent HIV-1 infection.
Filamentous actin (F-actin) is one of the major cytoskeletal proteins that make up pre- and post-synaptic structures. Polymerization of globular actin (G-actin) into F-actin, a more stable form of actin, occurs prior to spinogenesis (Johnson and Ouimet 2006). Although dendritic spines are rich in F-actin, where F-actin is found in the spine head and shaft (Sekino et al. 2007; Dent et al. 2011), dendritic spines are not the only F-actin rich structures found on the neurite (Halpain et al. 1998; Lau et al. 1999; Johnson and Ouimet 2006; Hotulainen et al. 2009). F-actin is associated with both pre- and post-synaptic structures (Johnson and Ouimet 2006), including non-spiny synapses; therefore, changes in the F-actin rich structures, i.e. dendritic F-actin puncta, suggest overall alterations in synaptic connectivity not limited to spines. Phalloidin, a form of phallotoxin isolated from the death cap mushroom (Amanita phalloides), selectively binds to F-actin, but not monomeric G-actin, and has been used to examine the dynamic activity of F-actin in modulating synaptic plasticity (Kaech et al. 1997; Halpain et al. 1998; Hotulainen et al. 2009; Korobova and Svitkina 2010).
Interestingly, estrogens modulate spine concentrations of F-actin (Kramar et al. 2009; Sanchez et al. 2009), suggesting estrogenic therapeutic approaches to correcting F-actin loss and restoration of neuroplasticity. Phytoestrogens are plant-derived diphenolic compounds found to partially mimic mammalian estrogen in structure and function (Glazier and Bowman 2001; Lephart et al. 2005). Daidzein (DAI) is a phytoestrogen isoflavone found in soybeans (Mortensen et al. 2009). DAI has been shown to be neuroprotective against glutamate excitotoxicity and Aβ25-35-induced apoptosis (Zhao et al. 2002). We have reported that DAI is protective against acute HIV-1 Tat-induced apoptosis (Adams et al. 2012).
The novel flavonoid, liquiritigenin (LQ), is one of the active compounds of MF101, a herbal remedy used to treat menopausal symptoms (Mersereau et al. 2008). LQ has been isolated from Chinese licorice root, glycyrrhiza uralensis, and identified as a selective estrogen receptor beta (ERβ) agonist (Paruthiyil et al. 2009). Previous studies have shown that LQ has anti-inflammatory effects, prevents neurocognitive deficits and neurotoxicity induced by the Aβ protein in animal models (Liu et al. 2009, 2010, 2011). LQ has been used in Traditional Chinese Medicine to treat senile dementia (Lin et al. 2012). Together, the ERβ specificity of LQ as well as the ability of LQ to prevent neurocognitive decline in animal models, suggest that LQ could be neuroprotective against HIV-1 Tat-mediated cell death and synaptodendritic damage.
Neurocognitive deficits associated with latent HIV-1 infection continue to persist despite successful suppression of peripheral viral load by anti-retroviral therapy (Heaton et al. 2010; Desplats et al. 2013). Synaptodendritic injury is closely tied to cognitive decline in HAND (Ellis et al. 2007). However, there are currently no treatments to prevent or attenuate synaptodendritic injury that occurs during HIV-1 infection. In this study, the neurorestorative potential of DAI and LQ was evaluated by monitoring their ability to prevent, and enhance recovery from, synaptodendritic injury produced in vitro by the HIV-1 protein, Tat 1-86B. The effects of DAI and LQ on restoration of the dendritic network were determined following withdrawal of Tat, and the role of estrogen receptors in the phytoestrogen-induced recovery process was determined through the use of tamoxifen. Collectively, these studies determined the neuroprotective and neurorestorative potential of DAI and LQ treatment in HIV-1 Tat-induced synaptodendritic injury.
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- Materials and methods
The major findings of this study are (i) acute pre-treatment with either LQ or DAI, two plant-derived phytoestrogenic compounds, prevents the loss of F-actin puncta induced by HIV-1 Tat. Synaptodendritic damage was quantified by the significant loss of F-actin puncta after a short exposure to Tat, and Tat was found to be acutely associated with the dendtric arbor. Moreover, (ii) these phytoestrogens were able to promote restoration of F-actin dendritic puncta and dendrites following damage from HIV-1 Tat in hippocampal cells. This recovery occurred via an estrogen receptor-mediated mechanism, as restoration of F-actin puncta was blocked by tamoxifen. These results suggest that DAI, LQ, and compounds of similar chemical structures, could be beneficial in HIV-1 therapeutics because of their ability to either prevent, and/or enhance recovery, from synaptodendritic injury.
The brain serves as a reservoir for HIV-1, as cART effectively suppresses viral replication in the periphery, but does not eradicate the virus from the brain (Heaton et al. 2010). Synaptodendritic injury has been found in latent HIV-1 infection, in which active viral replication is not present in the brain, despite the presence of proviral DNA. Latent HIV-1 infection in humans produces dendritic loss (decreased MAP2 staining) and synaptic loss (decreased synaptophysin staining) in the frontal cortex without viral replication (Desplats et al. 2013). HIV-1 proviral DNA may produce early proteins, such as Tat, without productive viral infection (Wu 2004). In this study, the co-localization of Tat within the F-actin rich dendritic network and the concomitant loss of puncta suggests a mechanistic interaction between Tat and synaptodendritic injury; however, it is currently unknown whether the synaptodendritic loss in human latent HIV-1 infections is caused by Tat expression per se. Nevertheless, our in vitro model of HIV-1 Tat exposure demonstrates dendritic loss (decreased MAP2 staining) and synaptic loss (decreased F-actin puncta), similar to that observed in post-mortem tissue from humans brains with latent HIV-1 infection.
F-actin puncta encompass a variety of neuronal structures, the most well understood being dendritic spines. Dendritic spines are F-actin rich protrusions from the dendritic arbor, are the sites of post-synaptic excitatory synapses, and change shape rapidly in response to extracellular signaling (Kaech et al. 1997; Calabrese et al. 2006; Hotulainen et al. 2009; Dent et al. 2011). Non-spiny synapses, typically GABA-ergic (Craig et al. 1994), have also been found to contain a significant concentration of F-actin and appear as patches or ‘hot spots’ on the dendritic arbor (Halpain et al. 1998; Lau et al. 1999). We found that intensity profiling, a semi-automatic computer-based method, and manual counting produced similar puncta densities in vitro. As a result of the varying morphology of spines, the ability of F-actin staining to detect non-spiny synapses, and the early filamentous stages of spinogenesis, the quantification of all F-actin rich structures, or puncta, provides a measurement of overall synaptic integrity, health of the dendritic network, and potential to recover from Tat-induced injury.
Although F-actin is known to play a role in spinogenesis and synaptic plasticity, the staining of F-actin is a relatively new technique used to image dendritic spines and monitor synaptic integrity (Matus et al. 1982; Allison et al. 1998; Sekino et al. 2007). In comparison, the Golgi method has been used for well over a century to identify changes in spine morphology and density, and is still in use. The Golgi method has been favored over the years because of the particular ability of Golgi to produce images with little to no background and complete neuronal filling. However, Golgi randomly and unpredictably stains neurons and has been found to significantly underestimate spine number and, moreover, fails to identify non-spiny thin synaptic structures and patch morphology (Mancuso et al. 2012). Non-spiny inhibitory synapses, which can be detected as patches by F-actin staining, but are not detected by the Golgi method, are important for normal communication between neurons (Heller et al. 2012).
In this study, we used F-actin to assess the synaptodendritic injury induced by HIV-1 Tat, whereas other studies have used Golgi (Sa et al. 2004; Fitting et al. 2010), green fluorescent protein coupled to post-synaptic density 95 (GFP-PSD95) labeling (Kim et al. 2008; Shin et al. 2012), or MAP-2 staining (Maragos et al. 2003) to evaluate the damage to the dendritic arbor. Golgi detects changes in spine morphology, but not changes in non-spiny synapses or thin filopodia. GFP-PSD95 staining can detect only post-synaptic excitatory structures, as PSD-95 has not been found in non-spiny inhibitory synapses (Heller et al. 2012) and is not found in pre-synaptic structures. Although pre- and post-synaptic terminals generally correspond to one another (Craig et al. 1994), PSD-95 can still be located after F-actin has disappeared from the spine (Halpain et al. 1998), suggesting that F-actin may be a more sensitive marker. MAP-2 staining can detect changes in overall structure of the neuronal arbor, but cannot reliably detect subtle changes in spine or patch morphology as microtubules have been observed to enter dendritic spines selectively and microtubule entry is activity dependent (Dent et al. 2011). Although the aforementioned techniques vary, and the studies determined the effects of HIV-1 Tat both in vivo (Maragos et al. 2003; Fitting et al. 2010) and in vitro (Kim et al. 2008; Shin et al. 2012), collectively these studies indicate that HIV-1 Tat produces dendritic damage. Therefore, identifying mechanisms or compounds that either prevent or reverse synaptodendritic damage inflicted by HIV-1 Tat may be useful approaches for providing neuroprotection during HIV-1 infection.
As a potential therapeutic approach for recovery of synaptodendritic damage, estrogen has long been known to rapidly increase dendritic spine density in vivo (Gould et al. 1990; Woolley and McEwen 1992), specifically through regulation of the actin cytoskeleton in spines (Sanchez et al. 2009). Phytoestrogens are non-steroidal, diphenolic structures found in plants that have similar chemical and structural properties to 17-β-estradiol (Glazier and Bowman 2001; Lephart et al. 2005). We found that phytoestrogens promote recovery from the synaptodendritic injuries produced by HIV-1 Tat. Although synaptodendritic injury is correlated with the symptoms of HAND (Ellis et al. 2007; Desplats et al. 2013), the extent to which synaptic restoration is possible remains unknown. Also unknown is whether there might be a critical therapeutic window for promoting effective recovery wherein neurorestoration remains possible. Unfortunately, studies of therapeutic pathways for enhancing dendritic recovery are few (Kim et al. 2008; Shin et al. 2012); however, estrogen has been shown to promote spine formation via modulation of F-actin in spines (Kramar et al. 2009; Sanchez et al. 2009). The current data suggest that phytoestrogens, possibly acting via F-actin, may provide a novel intervention for promoting neurorestoration. The activation of estrogen receptors has been shown to play a key role in modulation of dendritic spine dynamics (Liu et al. 2008; Kramar et al. 2009; Sanchez et al. 2009; Srivastava et al. 2010; Phan et al. 2011), suggesting the estrogen receptor may be a useful target in ameliorating synaptodendritic injury, such as that seen in HAND.
Previous studies have shown that LQ is a highly specific ERβ agonist, with a 75-fold binding preference to ERβ over ERα (Mersereau et al. 2008) and only activates ERβ (Kupfer et al. 2008; Paruthiyil et al. 2009). DAI preferentially binds to ERβ, with a 14-fold selectivity for ERβ (Zhao et al. 2009). The selectivity of LQ for ERβ suggests that LQ acts through an ERβ dependent mechanism to prevent synaptodendritic damage induced by HIV-1 Tat; however, more experimentation is needed to determine the receptor-dependent mechanism of LQ.
Our results illustrate that HIV-1 Tat causes an early reduction in F-actin positive puncta. Interventions aimed at promoting synaptodendritic integrity following HIV-1 infection of the nervous system would therefore appear to be an effective approach for preventing HAND. Moreover, our results indicate that damage by HIV-1 Tat may be repaired by the phytoestrogens, liquiritigenin and daidzein, and estrogen receptor actions mediate this neurorestoration. Although it is presently unknown if such reversals can improve neurocognitive outcomes, phytoestrogenic compounds, like DAI and LQ, may prevent cumulative injury to the dendritic network, and ultimately, aid recovery from HIV-associated neurocognitive disorders.