The severity of cognitive impairment in HIV positive subjects correlates with synaptodendritic degeneration (Ellis et al., 2007
). The present work was undertaken to provide insight into novel molecular mechanisms by which HIV promotes synaptic simplification. Here we show that neurons exposed to gp120 exhibit lower concentrations of mBDNF and higher levels of proBDNF than controls. Most importantly, these data were reproduced in human subjects. In fact, proBDNF levels in HAD were higher than those in HIV negative as well as HIV positive subjects without dementia. mBDNF plays a key role in axonal branching whereas proBDNF reduces synaptic plasticity [reviewed in (Greenberg et al., 2009
)]. Thus, the altered mBDNF/proBDNF ratio in HIV subjects could compromise synaptic connections and neuronal survival. Overall, our data suggest that the neurotoxic effects of HIV may encompass a reduction of the neurotrophic factor environment. Understanding how HIV inhibits the availability of mBDNF is crucial for the development of new therapies.
Most of the neurotoxic properties of HIV have been attributed to the combined effect of the virus and viral proteins, and/or host immune responses (Kaul et al., 2001
). In this study we show that HIV-infected individuals with HAD exhibit lower BDNF levels than non-demented HIV in the CX, ST and HP. BDNF is made in the cerebral cortex and delivered to the striatal neurons where it is particularly important for their survival and for the activity of the cortico-striatal synapses (Zuccato and Cattaneo, 2007
). Conversely, loss of BDNF has been suggested to be a risk factor in chronic diseases of the basal ganglia such as Parkinson’s (Nagatsu et al., 2000
) and Hintington’s diseases (Zuccato et al., 2001
). BDNF is also abundant in the HP where it is important in maintaining dendritic morphology and synaptic function (Horch and Katz, 2002
), as well as the survival of neurons and their connections (Xu et al., 2000
). Indeed, evidence has shown a strong correlation between reduction of BDNF and decrease in hippocampal neuronal survival and memory (Erickson et al., 2010
). Pathological features consistent with lack of BDNF in these brain areas have also been described in HAD. In fact, cortical neurons of HAD subjects are characterized by axonal injury (Ellis et al., 2007
) as well as apoptosis (Garden et al., 2002
). Hippocampal dysfunction has also been described in HIV positive women (Maki et al., 2009
). In addition, HIV promotes pathological changes in the basal ganglia. Abnormalities include neuronal loss in the putamen (Everall et al., 1995
) and globus pallidus (Fox et al., 1997
), loss of nigro-striatal dopamine neurons (Reyes et al., 1991
; Itoh et al., 2000
), and dysfunctional dopaminergic transport (Wang et al., 2004
). These correlations allow us to speculate that the decrease in BDNF evoked by HIV contributes to the development of synaptic simplification and neuronal damage seen in HAD.
An important finding reported here is the effect of HIV and its soluble envelope protein gp120 on proBDNF. ProBDNF was detected in the medium of rodent neurons, consistent with the notion that mature neurons are capable of producing and releasing proBDNF (Peng et al., 2005
; Yang et al., 2009b
). Secreted proBDNF has an opposite effect of mBDNF on neuronal plasticity (Pang et al., 2004
; Woo et al., 2005
). In fact, BDNF promotes neuronal survival and maintenance of synaptic spines through the high affinity receptor TrkB (Dorsey et al., 2006
), whereas through sortilin, proBDNF binds to p75NTR and induces apoptosis (Teng et al., 2005
). We have observed that proBDNF reduces the survival of cortical neurons and CGC. In addition, proBDNF reduced the length of neuronal processes in these neuronal cultures. These toxic properties of proBDNF were inhibited by blocking p75NTR activity, either by TAT-Pep5 or by a p75ab. On the contrary, K252a, an inhibitor of Trk signaling (Berg et al., 1992
; Ohmichi et al., 1992
) failed to reverse proBDNF toxicity. In contrast, BDNF prevented gp120 activity confirming previous results (Bachis et al., 2003
). Thus, our data support the notion that mBDNF and proBDNF elicit opposite effects through the activation of two distinct receptors, Trk and p75NTR. Most importantly, a similar neurotoxic profile was elicited by gp120 (or HIV). In fact, TAT-Pep5 but not K252a reduced significantly the ability of gp120 to promote neuronal injury. Intriguingly, while TAT-Pep 5 inhibited the ability of gp120 to reduce the length of neuronal processes, this p75NTR antagonist could not reverse completely gp120-mediated cell death. This could be due a number of mechanisms. For instance, two studies have showed that the DR6 receptor, which like p75NTR is a member of the tumor necrosis factor receptor family, causes axonal degeneration (Nikolaev et al., 2009
; Park et al., 2010
). Thus, a blockade of p75NTR may be insufficient to fully protect against gp120 because it does not prevent DR6 activation. In addition, the loss of BDNF combined with an increase of pro-inflammatory cytokines (Medders et al., 2010
) might exacerbate the neurotoxic profile of gp120. Thus, we cannot exclude that gp120 promotes cell death via more than one mechanisms. Overall our data support the hypothesis that HIV, most likely through gp120 (Bachis et al., 2009
), causes a change in the ratio proBDNF to mBDNF that ultimately results in an increased release of proBDNF. This altered ratio promotes an environment that is conducive to activation of p75NTR. This could be a risk factor for the development of synaptic simplifications and neuronal apoptosis seen in HAD.
ProBDNF can be converted into mBDNF intracellularly in the trans
-Golgi by endoproteases such as furin or in the immature secretory granules by proprotein convertases (Mowla et al., 2001
). However, proBDNF can also be cleaved to mBDNF extracellularly by proteases including tPA. Reduced tPA release has been shown to enhance proBDNF signaling through p75NTR leading to loss of spines and synapses and subsequently neuronal loss (Head et al., 2009
). Therefore, we examined the levels of tPA and furin in order to reveal the molecular mechanisms underlying the effect of HIV on proBDNF processing. Our data show that gp120 reduces the release of tPA. However, this reduction does not temporally correlate with the gp120-mediated increase in proBDNF, which occurs as early as 15 min after exposure of neurons to the envelope protein. Contrary to tPA, furin levels were reduced as early as 15 min after gp120, suggesting that furin is a key enzyme in gp120-mediated effect on proBDNF. How gp120 reduces furin levels is still under investigation. Furin mRNA levels did not change up to 3hr (data not shown), suggesting that gp120 may not affect furin transcription. On the other hand, furin has been shown to localize in early endosomes to be recycled to cell surface or trafficked to the trans
-Golgi network (Molloy et al., 1998
). Gp120 is internalized within minutes by axons and transported in endosomes (Bachis et al., 2006
). Thus, gp120 might change furin trafficking and help promote furin degradation, perhaps through ubiquitination. This could be consistent with the rapid (15 min) effect of gp120 on furin and the consequent increase in the levels of unprocessed (within 1 hr) proBDNF. Regulation of furin activity/synthesis might therefore have a central role in determining which neurons die even if they are not productively infected. However, this hypothesis must be proven.
In conclusion, our data suggest that gp120 shed by the virus can inhibit the appropriate processing of proBDNF into mature BDNF by reducing furin levels. proBDNF, in turn, initiates neuronal damage which, in combination with other neurotoxins such as glutamate or TNFα, can lead to apoptosis and neuronal loss. Our data provide support for new pharmacological agents against HIV-mediated neuronal toxicity that are able to increase mBDNF as well as promote the processing of proBDNF or inhibit proBDNF activity.