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.

Tax secretion from peripheral blood mononuclear cells and Tax detection in plasma of patients with human T-lymphotropic virus-type 1-associated myelopathy/tropical spastic paraparesis and asymptomatic carriers.

Human T-lymphotropic virus-type 1 (HTLV-1) is the etiologic agent of the neurologic disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Tax viral protein plays a critical role in viral pathogenesis. Previous studies suggested that extracellular Tax might involve cytokine-like extracellular effects. We evaluated Tax secretion in 18 h-ex vivo peripheral blood mononuclear cells (PBMCs) cultures from 15 HAM/TSP patients and 15 asymptomatic carriers. Futhermore, Tax plasma level was evaluated from other 12 HAM/TSP patients and 10 asymptomatic carriers. Proviral load and mRNA encoding Tax were quantified by PCR and real-time RT-PCR, respectively. Intracellular Tax in CD4(+)CD25(+) cells occurred in 100% and 86.7% of HAM/TSP patients and asymptomatic carriers, respectively. Percentage of CD4(+)CD25(+) Tax+, proviral load and mRNA encoding Tax were significantly higher in HAM/TSP patients. Western blot analyses showed higher secretion levels of ubiquitinated Tax in HAM/TSP patients than in asymptomatic carriers. In HTLV-1-infected subjects, Western blot of plasma Tax showed higher levels in HAM/TSP patients than in asymptomatic carriers, whereas no Tax was found in non-infected subjects. Immunoprecipitated plasma Tax resolved on SDS-PAGE gave two major bands of 57 and 48 kDa allowing identification of Tax and Ubiquitin peptides by mass spectrometry. Relative percentage of either CD4(+)CD25(+) Tax+ cells, or Tax protein released from PBMCs, or plasma Tax, correlates neither with tax mRNA nor with proviral load. This fact could be explained by a complex regulation of Tax expression. Tax secreted from PBMCs or present in plasma could potentially become a biomarker to distinguish between HAM/TSP patients and asymptomatic carriers.

Tax posttranslational modifications and interaction with calreticulin in MT-2 cells and human peripheral blood mononuclear cells of human T cell lymphotropic virus type-I-associated myelopathy/tropical spastic paraparesis patients.

The human retrovirus human T cell lymphotropic virus type-I (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Axonal degeneration in HAM/TSP patients occurs without neuron infection, with the secreted viral Tax protein proposed to be involved. We previously found that Tax secreted into the culture medium of MT-2 cells (HTLV-1-infected cell line) produced neurite retraction in neuroblastoma cells differentiated to neuronal type. To assess the relevance of Tax posttranslational modifications on this effect, we addressed the question of whether Tax secreted by MT-2 cells and peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected subjects is modified. The interaction of Tax with calreticulin (CRT) that modulates intracellular Tax localization and secretion has been described. We studied Tax localization and modifications in MT-2 cells and its interaction with CRT. Intracellular Tax in MT-2 cells was assessed by flow cytometry, corresponding mainly to a 71-kDa protein followed by western blot. This protein reported as a chimera with gp21 viral protein-confirmed by mass spectrometry-showed no ubiquitination or SUMOylation. The Tax-CRT interaction was determined by confocal microscopy and coimmunoprecipitation. Extracellular Tax from HAM/TSP PBMCs is ubiquitinated according to western blot, and its interaction with CRT was shown by coimmunoprecipitation. A positive correlation between Tax and CRT secretion was observed in HAM/TSP PBMCs and asymptomatic carriers. For both proteins inhibitors and activators of secretion showed secretion through the endoplasmic reticulum-Golgi complex. Tax, present in PBMC culture medium, produced neurite retraction in differentiated neuroblastoma cells. These results suggest that Tax, whether ubiquitinated or not, is active for neurite retraction.

Molecular and clinical effects of betamethasone in human T-cell lymphotropic virus type-I-associated myelopathy/tropical spastic paraparesis patients.

There is no effective therapy for human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Glucocorticoids are effective to reduce the motor disability in these patients, but its role as anti-spastic drugs is unknown. Here it is reported the use of corticosteroids in HAM/TSP. The goal was to find reliable molecular markers linked to treatment effectiveness. The clinical efficacy of corticosteroids was studied in 22 HAM/TSP. The treatment was a single dose of 7.0 mg of systemic betamethasone. Pre-treatment samples were obtained immediately before steroid administration and post-treatment samples were collected after 5 days. Neurological disability was evaluated by the Osame's Motor Disability Scales. Relative levels of Tax, Foxp3, IL-10, TGF-ß, CTLA-4, and GITR mRNA were measured and the percentage of CD4(+) Foxp3(+) and CD4(+) Tax(+) populations was quantified in PBMCs by real-time PCR and flow cytometry, respectively. The same parameters were studied in eight untreated carriers. Betamethasone treatment showed neurological improvement in 21 HAM/TSP patients, with one patient without response to treatment. This therapy was associated with a decrease in Tax mRNA load and CD4(+) Tax(+) T cells in HAM/TSP. Simultaneously, an increase in Foxp3 mRNA and CD4(+) Foxp3(+) T cell was detected in these patients. The other markers studied had no significant changes after treatment. Clinical improvement in betamethasone-treated HAM/TSP was associated with an inverse relationship between a decrease in Tax and an increase in Foxp3 at the mRNA and protein levels. These results suggest that both Tax and Foxp3 may represent potential biomarkers for drug treatment assessments in HAM/TSP.

Comparative study of CSF neurofilaments in HTLV-1-associated myelopathy/tropical spastic paraparesis and other neurological disorders.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive CNS disease leading to corticospinal tract degeneration. Various degenerative diseases have increased neurofilament subunit concentration in cerebrospinal fluid (CSF), frequently showing hyperphosphorylation in neurofilaments. The aim of this study was to determine if there were elevated concentrations of neurofilament light subunit (NFL) and phosphorylated forms of neurofilament heavy subunit (PNFH) in HAM/TSP CSF. NF concentrations were compared with those of controls and patients with neurodegenerative diseases associated with other retroviruses (HIV-associated dementia, HAD) and a form of prion disease (familiar Creutzfeldt-Jakob, FCJD). Western blotting of CSF with antibodies against NFL showed two immunoreactive bands of 66 and 59 kDa, the latter probably corresponding to a partially degraded NFL form. The concentration of the 59-kDa form was not different in HAM/TSP compared with controls, but it was significantly increased in HAD and FCJD groups. ELISA assay for PNFH did not show differences among HAM/TSP, HAD, and control groups, while PNFH concentration was significantly elevated in FCJD. Our results show that CSF NFL and PNFH are not molecular markers of axonal damage for HAM/TSP probably due to the slow progression of this disease. NFL phosphorylation studies required previous immunoprecipitation from CSF for mass spectrometric analysis. This preliminary analysis indicated phosphorylation at S472 and at some other residues.

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.