Neuroinflammation induced because of chronic HIV-1 infection in the CNS is an intense topic of investigation in order to both expand the mechanistic framework of the underlying pathophysiology and to characterize potential targets for further therapeutic interventions. In this regard, the current investigations have characterized that the initial TIMP-1 upregulation in reactive astrocytes during HIV-1 CNS infection is a protective mechanism against HIV-1-associated neurotoxicity. Thus, loss of TIMP-1 during chronic inflammation may not only exacerbate ECM breakdown due to lack of MMP inhibition, but also lead to loss of direct neuroprotective effects mediated by TIMP-1.
Our studies, for the first time, identified the neuroprotective potential of TIMP-1 in cultured primary human neurons from toxicity induced by both HIV-1ADA and cytotoxins such as STS. Our investigations demonstrated that the anti-apoptotic property of TIMP-1 is direct, specific and independent of its MMP-inhibition abilities. The exogenous supply of recombinant human TIMP-1 not only increased the cell viability, but also preserved the cell morphology following cytotoxic treatments of STS or HIV-1ADA. Further, TIMP-1 modulates the expression of Bcl-2 family of proteins and inhibits mPTP opening, which promote neuronal cell survival pathways. It is noteworthy that neuroprotective effect of TIMP-1, T2G mutant or BDNF was not only quantitatively but also mechanistically comparable in the studied parameters.
The MMP activity is tightly regulated through gene expression and protein secretion, proenzyme activation, sequestration and inhibition by their endogenous inhibitors, TIMPs.23
An TIMP/MMP imbalance and ECM degradation are implicated in a variety of neuroinflammatory diseases including HAD.8
Together, TIMP-1 and -2 possess the potential to inhibit the activities of all known MMPs (although TIMP-1 is a poor inhibitor of MMP-14, 16, -19 and -24) and have an essential role in maintaining the balance between ECM formation and degradation.8
Interestingly, TIMP-1 and -2 levels increase in many neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis.8
We have previously reported that TIMP-1 is inducible in astrocytes after acute exposure to IL-1β
Further, TIMP-1 protein levels decrease and MMP-2 and pro-MMP-9 levels increase in the CSF of HAD patients as compared with non-demented HIV-1 seropositive or seronegative controls.9
The differential regulation of TIMP-1 by glia during neuroinflammation serves multiple purposes. For example, increased MMP expression can facilitate MP infiltration into the CNS in HAD involving degradation of the ECM, which can be countered by TIMP-1.24
Owing to MMP-inhibition activity, TIMP-1 has a variety of roles, viz. protecting neurons during severe cerebral ischemia,25
reducing neurite length, increasing the size of growth cones,26
generation and differentiation of oligodendrocytes27
and attenuation of demyelination in experimental autoimmune encephalomyelitis (EAE).4
The adenoviral delivery of TIMP-2 also had a protective effect in cerebral ischemia due to MMP-inhibition activity.28
TIMP-1 protects rat hippocampal neuronal death in culture against glutamate-induced excitotoxicity.11
Our results show the neuroprotective potential of TIMP-1 and demonstrate its role in protection from HIV-1-induced neurotoxicity for the first time. Additionally, TIMP-1 also protected human neurons from a broad-spectrum cytotoxin, STS. There are indications that TIMP-1 is implicated in diverse neuroprotection pathways during various neuronal injuries, viz. inflammatory, (EAE model),4
excitoxicity (glutamate dysregulation),11
and neuronal activity-dependent injuries (kainate-seizure mouse model).5
TIMP-1 has also been linked to ECM preservation through MMP-inhibition, non-inhibiting MMP-regulation and through the non-classical modulation of ionotropic glutamate receptors.5, 11, 29
Studies with potassium cyanide and TIMP-1 knockout also indicate that TIMP-1-mediated neuroprotection may not be evident in all types of neuronal injuries.5, 11
Thus, TIMP-1 has differential effects on neurons depending on the respective mechanisms of the specific insults and associated tissue contexts. Therefore, throughout this study, we included STS, an apoptosis induction model used extensively in a variety of cell types, including neurons to study neurodegeneration and neuroprotection.30
Our data support that STS induces apoptosis in cultured primary human neurons. Interestingly, cotreatment with TIMP-1 or T2G mutant reduced apoptosis, preserved cell morphology, and increased cell viability in STS-treated human neurons equivalent to a well-known neurotrophic factor (BDNF). Microglia is the predominant target of HIV-1 infection in the CNS. Infected microglia produce progeny virus and cytokines, thereby activating bystander cells via cytokines and viral toxins. Consequently, HIV-1-induced neuronal apoptosis may be mediated by both viral- and glial cell-derived soluble factors including proinflammatory cytokines. Various mechanisms have been demonstrated depending on the viral proteins, suggesting it is a complex process. Therefore, it is a novel finding that TIMP-1 protects neurons from a broad-spectrum cytotoxin. A better understanding of TIMP-1-mediated neuroprotection against HIV-1 will be helpful in developing new therapeutic strategies.
In addition to the well-described MMP-dependent actions, recently several studies have demonstrated that TIMPs perform a number of MMP-independent actions. Importantly, TIMP-2 promotes neuronal differentiation by inhibiting cell proliferation in an MMP-independent manner.31
Using the T2G mutant of N-TIMP-1, we demonstrate directly that inhibition of apoptosis of human neurons by TIMP-1 is in fact mediated independently of its effects on MMP activity. The dose of T2G used in these experiments (28
nM) is slightly less than its Ki
a concentration at which it would be expected to only partially (<50%) inhibit MMP-9. Our data show that TIMP-1-mediated neuroprotection is, at least in part, MMP-independent and an addition into its non-classical functions because the T2G mutant was effective as wild type TIMP-1; but other MMP-inhibitors, TIMP-2 or -3, did not protect neurons from STS-induced apoptosis. In contrast to Tan et al.
where they demonstrated that TIMP-1-mediated neuroprotection against glutamate is MMP-dependent; in our STS model, neuroprotection was independent of MMP-inhibition activity. This seeming disagreement with our results may be due to different paradigms, toxic mechanisms and species used in the two studies. There are significant sequence differences between the rat and human TIMP-1 most notably in the crucial N-terminal region,15
human is CTCVPPH and rat is CSCAPTH, which has large effects on specificity and might reflect differences in targets between the two species. Further, in our study we supplied TIMP-1 exogenously to neurons, which is similar to the natural method of TIMP-1 delivery, as compared with transfection of neurons.11
We utilized STS and HIV-1ADA
to cause cytotoxicity. Exactly how MMP-independent effects are mediated at the molecular level is still to be determined. However, studies suggest that TIMPs can bind to a variety of cell-surface receptors, including CD63 for TIMP-1, α
1 integrin for TIMP-2 and TIMP-3 to the vascular endothelial growth factor receptor-2, raising the possibility that TIMPs can directly signal through these specific receptors.6, 32, 33
Our study indicates a possible neurotrophic pathway by TIMP-1, independent of binding, sequestration, or inhibition of MMPs. It is possible that pro-survival pathways are activated in cells directly by TIMP-1 without involvement of MMP-binding.
Outer mitochondrial membrane permeability is regulated by Bcl-2 family protein expression and the ratio of Bax to Bcl-2.34
Our results demonstrate that TIMP-1 attenuates STS-induced neurotoxicity through upregulation of anti-apoptotic proteins, Bcl-2 and Bcl-xL
, and downregulation of the pro-apoptotic protein, Bax. Data demonstrate that TIMP-1 preserves the Bax/Bcl-2 and Bax/Bcl-xL
ratios at basal control levels. Further, this effect was direct, independent of MMP-inhibitory activity and equal to BDNF as the T2G mutant and BDNF had equivalent effects. Our data demonstrate that STS-induced neurotoxicity in human neurons involves changes in Bax/Bcl-2 or Bax/Bcl-xL
ratios and opening of mPTP. Our data show that TIMP-1 inhibits the opening of mPTP induced by either STS or HIV-1, and thus, indicates direct trophic signaling by TIMP-1 in human neurons.
As TIMP-1 is a small extracellular protein, any potential signaling is likely to be mediated through a cell-surface receptor. To activate pro-survival or anti-apoptotic pathways, TIMP-1 modulates Bcl-2, Bax, and Bcl-xL
protein levels during neurotrophic factor deprivation and through phosphotidylinositol-3 kinase (PI3K), c-Jun N-terminal kinase, JAK2/PI3K/Akt pathways as shown in a variety of cell types.8, 20
Interestingly, TIMP-1 binds to cell surface targets and translocates into the nucleus.35, 36
Recently, CD63, a tetraspanin, which modulates signaling by integrin complexes, has been identified as a cell-surface interacting partner for TIMP-1 in the non-malignant breast epithelial cell line, MCF10A.6
Interestingly, this study characterized the C-terminal domain of TIMP-1 as the interaction partner for CD63, whereas the N-TIMP-1 mutant T2G that lacks the C-terminal domain, was sufficient for neuroprotection in our study.6, 37
Moreover, modulation of anti-apoptotic proteins by TIMP-1 in human neurons suggests that different domains of the TIMP-1 protein may mediate several separate signaling pathways. This appears to indicate that TIMP-1 neuroprotective signaling can be mediated through other receptors and/or interaction partners besides CD63 and the net outcome of these signaling pathways confers neuroprotection. This finding, along with the diverse anti-apoptotic or growth promoting pathways activated or modulated by TIMP-1 indicates mechanisms not classically mediated through MMPs.
In summary, TIMP-1 expression by glial cells is neuroprotective against general neurotoxicity, exogenous HIV-1 and possibly from neuroinflammation or neurodegenerative diseases. Moreover, our results indicate that TIMP-1-neuroprotection could be mediated through a neurotrophic pathway without the involvement of MMPs. We also demonstrate that TIMP-1 modulates the anti-apoptotic/pro-survival pathways in human neurons. Therefore, we propose that the TIMP-1 protein is a neuroprotective signal to neurons. In this regard, the measurement of TIMP-1 level in the CSF of CNS disease patients could be a novel prognostic tool to predict the pathological outcome, while careful immune-modulation to manipulate TIMP-1 levels may slow or reverse neuronal damage in CNS diseases and injuries. Furthermore, interventions to restore or supplement TIMP-1 during the early stages of neuronal inflammation may significantly decrease the severity of neurotoxicity, tissue damage and associated degenerative symptoms. Bearing in mind the broad range of signaling activities of TIMP-1 in other cell types, it would be important to dissect the precise signaling pathways modulated by TIMP-1 that affect neuron viability before implementation of TIMP-1-delivery therapy for CNS neurodegeneration.