Tax and Semaphorin 4D Released from Lymphocytes Infected with Human Lymphotropic Virus Type 1 and Their Effect on Neurite Growth.

Human lymphotropic virus type 1 (HTLV-1) is a retrovirus causing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a neurodegenerative central nervous system (CNS) axonopathy. This virus mainly infects CD4(+) T lymphocytes without evidence of neuronal infection. Viral Tax, secreted from infected lymphocytes infiltrated in the CNS, is proposed to alter intracellular pathways related to axonal cytoskeleton dynamics, producing neurological damage. Previous reports showed a higher proteolytic release of soluble Semaphorin 4D (sSEMA-4D) from CD4(+) T cells infected with HTLV-1. Soluble SEMA-4D binds to its receptor Plexin-B1, activating axonal growth collapse pathways in the CNS. In the current study, an increase was found in both SEMA-4D in CD4(+) T cells and sSEMA-4D released to the culture medium of peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients compared to asymptomatic carriers and healthy donors. After a 16-h culture, infected PBMCs showed significantly higher levels of CRMP-2 phosphorylated at Ser(522). The effect was blocked either with anti-Tax or anti-SEMA-4D antibodies. The interaction of Tax and sSEMA-4D was found in secreted medium of PBMCs in patients, which might be associated with a leading role of Tax with the SEMA-4D-Plexin-B1 signaling pathway. In infected PBMCs, the migratory response after transwell assay showed that sSEMA-4D responding cells were CD4(+)Tax(+) T cells with a high CRMP-2 pSer(522) content. In the present study, the participation of Tax-sSEMA-4D in the reduction in neurite growth in PC12 cells produced by MT2 (HTLV-1-infected cell line) culture medium was observed. These results lead to the participation of plexins in the reported effects of infected lymphocytes on neuronal cells.

Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: an in vitro model of HTLV-I-induced disease.

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could be involved in this disease, configuring an axonal transport disease. We measured tubulin and its post-translational modified forms (acetylated and tyrosinated) in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulin between patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levels were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes the viral Tax protein) we observed neurite retraction and an increase in tau phosphorylation at T181. A disruption of normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: An in vitro model of HTLV-I-induced disease

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could beinvolved in this disease, configuring an axonal transport disease. We measured tubulin and its posttranslational modified forms (acetylated and tyrosinated) in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulinbetween patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levels were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes theviral Tax protein) we observed neurite retraction and an increase in tau phosphorylation at T181. A disruptionof normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

Trophic factors in cerebrospinal fluid and spinal cord of patients with tropical spastic paraparesis, HIV, and Creutzfeldt-Jakob disease.

HTLV-1-associated myelopathy/tropical spastic paraparesis (TSP/HAM) is a chronic CNS disease characterized by axomyelinic degeneration of the long axons of corticospinal tracts. Levels of NGF, NT-3, NT-4/5, BDNF, GDNF, CNTF, and FGF-2 were measured in the cerebrospinal fluid (CSF) of 21 TSP/HAM patients and 20 controls. NGF, BDNF, and FGF-2 levels were also determined in 19 patients with HIV motor cognitive motor syndrome, and in 21 subjects diagnosed with Creutzfeldt Jakob disease (CJD). No significant differences were detected in the concentrations of NGF, BDNF, NT-3, NT-4/5, GDNF, and CNTF in the CSF between TSP/HAM patients and controls. FGF-2 was significantly lower in the CSF of the three groups of patients compared with controls; the HIV group exhibited the lowest values. HIV patients differed from TSP/HAM in their significantly higher levels of NGF and lower levels of BDNF and FGF-2, whereas CJD patients differed only in their higher levels of NGF. Immunohistochemical studies were done of trophic factors (NGF and FGF-2) and neurotrophin receptors (trkA and p75) in spinal cord and motor cortical areas from anatomopathological cases of TSP/HAM. Results indicated that NGF is expressed in motoneurons and oligodendrocytes of the posterior column of the spinal cord. FGF-2 was detected in motoneurons and spinal cord vessels. p75 receptor was detected in cortical neurons. The absence of a significant change in the trophic factor levels in TSP/HAM may be attributed to a selective axonal lesion in a slow process.

TIMPs and MMPs expression in CSF from patients with TSP/HAM.

The tropical spastic paraparesis or human T-cell lymphotropic virus associated myelopathy (TSP/HAM), has been related with an overexpression of matrix metalloproteinases (MMPs), especially MMP-9. Initial studies of reverse zymography with cerebrospinal fluid (CSF) from TSP/HAM patients, and controls showed the presence of TIMPs, endogenous MMP inhibitors. We determined in CSF the levels of TIMPs by immunoanalysis in 25 patients with TSP/HAM, and compared with two groups: controls and patients with acute and subacute inflammatory neurological diseases. We found that TIMP-2, TIMP-3 and TIMP-4 levels were significantly higher than in controls in both TSP/HAM and inflammatory patients, while TIMP-1 was increased only in the inflammatory group. Levels of MMP-3 and MMP-9 from the two groups of patients showed a significant upregulation in CSF. In the CSF of around the 70% of TSP-HAM and inflammatory patients the presence MMP-9 was detected by zymography, but not in controls. MMP-2 was only overexpressed in the acute inflammatory group. The active form of MMP-2 was observed in both groups of patients with a similar high frequency (60%). MMPs overexpressions are independent of the evolution time of the disease in TSP/HAM. The chronic overexpression of these extracelullar matrix proteins detected in CSF of TSP/HAM should be indirectly produced by secreted viral proteins being responsible for the progression of this disease, accounting for the observed differences with acute inflammatory patients. Our results support the existence of an imbalance between MMPs and their endogenous tissue inhibitors, which could be a pathogenic factor in the chronicity of TSP/HAM.