HTLV-1 Secreted Proteins Induce Axonal Degeneration Through Kinase and Phosphatase Activity Dysregulation

Abstract

HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) pathology has been associated with Tax protein secreted from HTLV-1 infected CD4⁺-T-lymphocytes, which interacts with soluble SEMA-4D (Semaphorin 4D) inducing growth cone collapse in neurons. We investigated HTLV-1-induced phenotypic and signaling changes during PC12 neuronal differentiation that may mediate growth cone collapse effects. We measured kinases and phosphatases associated with microtubule-associated proteins and molecular motor functions. Phosphorylation status of proteins that participate in the cytoskeleton and axonal transport such as Tau, microtubule-associated protein 1B (MAP1B), motor proteins (kinesin-1 and dynein) and collapsin response mediator protein (CRMP-2), all involved in neurite extension and branching, were measured. The phosphorylation/dephosphorylation of these proteins is catalyzed by Cyclin-dependent kinase-5 (CDK5), Glycogen synthase kinase-3β (GSK3β), and Protein phosphatase-2 (PP2A). Our results show that viral secreted proteins produced a reduction of neurite extension and branching in PC12 cells during neuronal differentiation. We observed that GSK3β activity increased, while CDK5 and PP2A activities decreased. In addition, we found reduced levels of Tau phosphorylated at Thr¹⁸¹ and increased levels of CRMP-2 phosphorylated at Ser⁵²². No changes in motor proteins or MAP1B phosphorylation were found. Neurotoxic effects of HTLV-1 secreted proteins on neuronal differentiation of PC12 cells include lower CDK5 activity, which could explain the reduced levels of Tau-(pThr¹⁸¹); this could induce conformational changes in Tau protein, altering microtubule dynamics. Increased CRMP-2-(pSer⁵²²) phosphorylation precedes further phosphorylation at Thr⁵⁰⁹/⁵¹⁴ residues by GSK3β. All these phosphorylations are associated with growth cone collapse. The increased CRMP-2-(pSer⁵²²) levels found here suggest that CDK5 activity, even when decreased, is sufficient for this priming phosphorylation. Reduction in PP2A activity could importantly contribute to maintaining the increased phosphorylation in CRMP-2. These results suggest the involvement of extracellular Tax/sSEMA-4D complex in the activation of Plexin1B receptor, activating downstream cascade involving PI3K/AKT/GSK3β/CRMP-2, inducing growth cone collapse.

Keywords

HTLV-1 GSK3β CDK5 PP2A tax tau MAPB1 CRMP-2 motor proteins

Introduction

HTLV-1 is a retrovirus known as the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive central neuropathy and Adult T-cell Leukemia (ATL), an aggressive neoplasia.^1–3 A global estimate indicates 10–20 million infected people, and approximately 0.25%–4% of them will develop HAM/TSP.³ Clinicopathological features of the bilateral pyramidal syndrome associated with HAM/TSP are gait alterations, chronic progressive lower extremity weakness with spasticity and hyperreflexia, back pain, neurogenic bladder disturbances, Babinski’s sign, uveitis and Sjögren syndrome.^2,4,5 Magnetic resonance imaging indicates brain lesions and spinal cord atrophy in the thoracic region.⁶

HTLV-1 infection of T lymphocytes in vivo occurs initially by cell-cell contact and later by clonal expansion, where most infected people remain as asymptomatic carriers (ACs), probably immune system controlled.⁷ HTLV-1 proviral load is an important biomarker for HAM/TSP, with highest load in CD4⁺-T-lymphocytes and lower in CD8⁺-T-lymphocytes.^3,7 Viral Tax protein is secreted from lymphocytes, and it is also a transcriptional activator of viral and host proteins, being used as marker of HAM/TSP pathology.^3,7,8 HAM/TSP patients compared with ACs show higher levels of CD4⁺-CD25⁺-Tax⁺ lymphocytes, proviral load, mRNA encoding Tax in cerebrospinal fluid (CSF) and plasma, and specific Tax antibody titers in CSF.^3,8–12 We also detected tax gene in CSF cells.⁸ Since the virus does not infect neurons, the pathogenic mechanism of dysregulation of neuronal function is through extracellular Tax.¹⁰ HTLV-1-infected CD4⁺-T cells in HAM/TSP transmigrate to the CNS (Central Nervous System) through activation of small GTPases (Cdc42, Rac, and RhoA), producing cytoskeleton reorganization facilitating migration and increased expression of adhesion molecules and extracellular MMPs (Matrix Metallo Proteinases); thus, infected lymphocytes can cross the endothelial barrier and migrate into the spinal cord.¹³

In infected peripheral blood mononuclear cells (PBMCs) and HTLV-1 infected cell lines, we demonstrated that the Tax–Calreticulin interaction participates in Tax intracellular localization and secretion.¹⁴ Extracellularly we detected a complex between Tax and sSEMA-4D, a soluble form of the transmembrane glycoprotein SEMA-4D upon shedding by MMP1, suggesting that Tax-SEMA-4D complex could be involved in Tax neurotoxic actions.¹⁰ Measuring PBMC transmigration in patients, we detected a chemotactic effect by sSEMA-4D in CD4⁺-Tax⁺-cells with a high content of CRMP-2-(pSer⁵²²) facilitating lymphocyte cytoskeleton reorganization and migration to the CNS, condition that induces axonal growth cone collapse.^10,13,15,16 Therefore, extracellular Tax-sSEMA-4D complex may bind to the neuronal semaphorin receptor Plexin-B1 triggering the neuropathological effects.^17–19

Anatomopathological studies of corticospinal-spinal tracts of HAM/TSP subjects showed abnormal accumulation of the amyloid precursor protein (APP), a marker of anterograde axonal transport deficiency, found in patients with Hereditary Spastic Paraparesis (HSP) with similar clinical characteristics to HAM/TSP but associated with mutations of motor proteins.^4,20–23 Therefore, we were interested in studying alterations of proteins participating in axonal transport comprising microtubules (MTs), MT-associated proteins (MAPs), and molecular motor proteins (ATP-dependent kinesin and dynein) that move cargoes along MT tracks.^23,24 Among these proteins we studied Tau, which stabilizes MTs and mediates the association between F-actin and stable/dynamic MTs,^25–27 MAP1B that enhances axonal growth cone by regulation of actin filament (F-actin) dynamics^28,29 and CRMP-2 that induces axonal MT formation through binding to tubulin dimers.^15,16,30 Phosphorylation status of Tau, CRMP-2, MAP1B, KLC (Kinesin Light Chain), KHC (Kinesin Heavy Chain), and DIC (Dynein Intermediate Chain) are controlled by an equilibrium between kinases (such as CDK5 and GSK3β) and protein phosphatases (importantly PP2A).^19,21–35 Upregulated kinase activities and reduced phosphatase activity have been reported in some neurodegenerative diseases including Alzheimer’s disease (AD),^{22,24,25,30,33,36–46} Parkinson’s Disease (PD),^{33,37,39,45,46} Huntington’s Disease (HD),^{37,39,40,45,46} Amyotrophic Lateral Sclerosis (ALS),^44–46 HIV-Encephalitis (HIVE),^30,45 Multiple Sclerosis (MS)^44,46 and Dementia with Lewy Bodies.³⁷ Several reports of neurogenerative diseases of diverse origins show alterations in Tau phosphorylation associated to dysregulation in axonal MT assembly, MT dynamics, and interactions with F-actin cytoskeleton.^{24–26,33,36,39–42,45–51}

Our group carried out studies on the neurotoxic effects of secreted proteins from MT2 cells (HTLV-I infected, normal human cord leukocytes) and with PBMCs from HAM/TSP patients on PC12 cells (rat pheochromocytoma) during their neuronal-like differentiation process.¹⁰ We observed a delay in the length/extension of neurites that was blocked in the presence of anti-Tax antibodies, strongly suggesting that Tax is the main viral protein responsible for the neurotoxic effects.¹⁰

In this paper, our aims include finding the association between the effects of secreted viral proteins on PC12 cells phenotype and alterations in posttranslational modifications of proteins related to neurite outgrowth and defining the participation of kinases (CDK5 and GSK3β) and PP2A phosphatase. We found that during PC12 cell differentiation secreted viral proteins (including Tax) produced: a) reduction of neurite growth and branching number; b) reduction of CDK5 and PP2A activities and increase in GSK3β activity; c) decrease of Tau phosphorylation at Thr¹⁸¹, hyperphosphorylation of CRMP-2 at Ser⁵²² and no changes in phosphorylation of CRMP-2 at Thr⁵⁵⁵, MAP1B, Kinesin and Dynein. These results further support our hypothesis that the neurotoxic role of HTLV-1 secreted proteins is mediated by Plexin-B1 activation (by Tax-sEMA4D), triggering the neuropathological effects associated with this receptor signaling pathway.

Materials and Methods

MT2 and K562 cell cultures

MT2 (HTLV-I infected, normal human cord leukocytes) and K562 cells (noninfected, myelogenous leukemia with T-cell properties) were used. Cell cultures were kept in RPMI 1640 with GlutaMAX™-I (Thermofischer-Scientific, cat. 72400) and 10% iFBS (inactivated fetal bovine serum, Thermofischer-Scientific, cat. 10438026) at 37°C and 5% CO₂. To obtain the culture medium, 10⁶ cells were cultured per ml of RPMI medium with 0.2% iFBS for 7 days, then centrifuged at 400×g (5 minutes) at 4°C. Supernatant corresponds to culture medium used with PC12 cell cultures during their differentiation.

Differentiation of PC12 cells and treatment with MT2 or K562 culture medium

Undifferentiated PC12 cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium-HEPES modification, Sigma-Aldrich, cat. 1152) with 6% iFBS and 6% HS (horse serum, Thermofischer-Scientific, cat. 16050122) at 37°C and 5% CO₂. PC12 cell differentiation to neuronal type was initiated by adding 50 ng/mL of NGF (Mouse Nerve Growth Factor 2.5S, N-100, Alomone-Labs, cat. N-240) to cells seeded at 45,000 cm². Cells were differentiated up to 3 days by gradually decreasing the serum concentration as follows: culture with DMEM containing 4% iFBS, 4% HS, and 50 ng/mL NGF on the first day; with DMEM containing 2% iFBS, 2% HS, and 50 ng/mL NGF on the second day; and with DMEM with 1% iFBS, 1% HS, and 50 ng/mL NGF on the third day. During differentiation, PC12 cells were cultured with 1/8 of the MT2 or K562 culture medium.

Neurite outgrowth evaluation

Cells were observed by inverted phase microscopy using a 20 × objective. Images were directly captured as black/white digital micrographs. Eight areas of PC12 cells were randomly acquired, measuring between 20 and 70 neurites/field. Neurite length was measured using the NIH ImageJ-1.38d-program plug-in NeuronJ. The neurite length in primary neurites corresponds to the net extension away from the cell body (from the margin of the cell body to its terminus). When secondary neurites were present (two possible end points), the largest possible extension from the cell body was assigned as the primary neurite, and the secondary neurite was measured as the remaining extension from the bifurcation point to its terminus.

CDK5 activity assay

CDK5 was immunoprecipitated from 200 µg of PC12 cell lysates (quantified using “Bio-Rad Protein Assay,” Bio-Rad cat. 500-0006), using 12 µL of anti-CDK5 (C-8) antibody (Santa Cruz Biotechnology, cat. sc-173) and 35 µL of Protein A/G PLUS-Agarose (Santa Cruz-Biotechnology, cat. sc-2003). Immunoprecipitated protein was washed 3 times with 20 mM Tris-HCl pH 7.4 buffer containing 10 mM MgCl₂ and 1 mM EDTA and centrifuged 5 minutes at 2,500×g (4°C); the pellet was recovered and resuspended in 10 µL of buffer. For the enzymatic assay, 4 µL of 100 mM Tris-HCl pH 7.4 buffer with 50 mM MgCl₂, 5 mM EDTA, 5 µL of H1-histone substrate (1 mg/mL), and 5 µCi of [γ-³²P]-ATP (0.5 mM) were added to the immunoprecipitated sample to final volume of 20 µL. Reaction was stopped by adding Laemmli buffer.⁵² After boiling at 100°C, samples were submitted to SDS-PAGE using 12% polyacrylamide gels. After electrophoresis, gels were exposed in “phosphoscreen” plaques.

Indirect measurements of GSK3β activity

GSK3β phosphorylates β-catenin, producing destabilization by activating its proteasomal breakdown,⁴⁷ thereby we measured the degradation of β-catenin as indirect measurement of activity by Western blot. In addition, the ratio of inactivated GSK3β phosphorylated on Ser⁹ versus total GSK3β was also determined by Western blot.

PP2A activity assay

PP2A activity determination was performed using the “PP2A Immunoprecipitation Phosphatase Assay Kit” (Millipore, cat. Seventeen–313), following the manufacturer’s instructions. In brief, PP2A was immunoprecipitated using anti-PP2A antibody from 300 µg of PC12 cell lysates. Diluted phosphopeptide was added to the immunoprecipitated PP2A, and the phosphate released as product was measured using Malachite Green Phosphate Detection assay (Sigma-Aldrich, cat. HT8028).

Cell lysis, protein determination, SDS-PAGE and Western-blot analysis

Cell lysis

PC12 cells were washed with PBS (phosphate-buffered saline) and then resuspended in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P40, 1% sodium deoxycholate, 0.1% SDS, and 0.2 mM Na₃VO₄) with 2 µL/mL protease inhibitor cocktail (Sigma-Aldrich, cat. P8340), followed by mechanical disruption. Cells were sonicated for 5 minutes, and soluble fractions of PC12 lysates were obtained by centrifugation at 14,000×g (15 minutes) at 4°C.

Protein determination of cell lysate

Pierce Micro BCA Protein Assay Kit™ (Thermofischer Scientific, cat. 23235) was used according to the manufacturer’s instructions.

Western-blot analysis

SDS/PAGE was performed as previously described⁵² and transfer to nitrocellulose membrane was carried out with the following specifications: 50 µg protein of cell lysate or 50 µL of MT2 and K562 culture medium were loaded on 10% polyacrylamide gels, except for MAP1B and β-catenin where 4%–15% sucrose gradient gels were used. The transfer to nitrocellulose membranes (0.45 μm from Bio-Rad Laboratories, cat. 162-0115) was done with 25 mM Tris-HCl, 192 mM glycine, and 20% (v/v) methanol for a total of 600 mA at 4°C. In the case of MAP1B visualization, Bjerrum Shaeffer-Nielsen transfer buffer was used (Tris-HCl 48 mM, glycine 39 mM, 20% methanol, and 0.0375% SDS) for a total of 4,000 mA at 4°C. Transferred membranes were blocked for 20 minutes at room temperature with 6% p/v of non-fat milk dissolved in TBS-T (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.1% [v/v] Tween-20), then incubated overnight at 4°C or 2 h at room temperature with the different primary antibodies at the appropriate dilution in TBS-T. The following antibodies at the specified dilutions in TBS-T were used: from Abcam: anti-Tau-(pThr¹⁸¹) (cat. ab38505, diluted 1:1,000), anti-Tau (cat. ab64193, 1:5,000), anti-GAPDH antibody (cat. 8245, 1:20,000), anti-GSK3β (cat. ab2602, 1:1,000), anti-β-catenin (cat. ab6302, 1:500), anti-PP2A (cat. ab59255, 1:2,000); from Santa Cruz-Biotechnology: anti-MAP1B (cat. sc8970, 1:1,000), anti-DIC (cat. sc-13524, 1:2,500); from ECM-Biosciences: anti-CRMP-2-(pSer⁵²²) (cat. CP2191, 1:1,000), anti-CRMP-2-(pThr⁵⁵⁵) (cat. CP2251, 1:1,000); from Covance: anti-pMAP1B corresponds to anti-phosphorylated neurofilament antibody with documented cross-reactivity to phosphorylated MAP1B⁵³ (cat. SMI-31P, 1:5,000); from Sigma-Aldrich: anti-CRMP-2 (cat. 2993, 1:30,000); from Thermofischer-Scientific: anti-GSK3β (cat. 39-9500, 1:2,000); from Millipore: anti-GSK3β-(pSer⁹) (cat. 05-643, 1:1,000); antibodies anti-KHC (1:2,500) and anti-KLC (1:2,500) were generously donated by Dr. Scott Brady. Secondary antibodies were used from Thermofischer-Scientific: ImmunoPure Goat® Anti-Rabbit IgG (H + L), Peroxidase Conjugated (cat. 31460, 1:500,000), or ImmunoPure Goat® Anti-Mouse IgG (H + L), Peroxidase Conjugated (cat. 31430, 1:500,000). Five washes with TBS-T (5 minutes each) were done after each antibody incubation. Detection used Super Signal West Femto® Maximum Sensitivity Substrate (Thermofischer-Scientific, cat. 34095) and CL-Xposure film (Thermofischer-Scientific, cat. 34090). Quantification of blots was carried out by scanning films using Uni-Scan-It Automated Digitizing System.

Two-dimensional (2D) electrophoresis

Portions of 50 µg of PC12 cell lysates were isolated using 2-D Clean-Up kit (GE-Healthcare, cat. 80648451) following the manufacturer’s instructions. The pellet was incorporated into UTAH rehydration buffer (3.2 mM ASB-14, 69 mM HED, 9.7 M urea, and 2.7 M thiourea) in contact with immobilized pH gradient gels Immobiline® Drystrip pH 3–11 NL, 7 cm (GE-Healthcare, cat. 17-6003-73) according to the manufacturer’s instructions. For the first dimension (isoelectric focusing), the strips were submitted to a five-step procedure using the Ettan 3 IPGphor system (GE-Healthcare) as follows: 10 V for 30 minutes; 300 V until reaching 200 V/h; 1,000 V until reaching 300 V/h; 5,000 V until reaching 4,000 V/h; 5,000 V until reaching 2,000 V/h. Then strips were incubated at room temperature for 15 minutes at pH 8.8 in buffer containing 75 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, and 65 mM DTT, and then in buffer with 75 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, and 135 mM iodoacetamide. For the second dimension, the strips were submitted to SDS-PAGE (10% acrylamide gel without stacking gel), and finally the proteins were transferred to nitrocellulose membrane for Western blot.

Statistical analysis

The statistical analysis used R-software (Version 1.0.136-©2009–2016 RStudio, Inc.). The values were expressed as mean ± SEM of at least three independent experiments. Neurites of at least 150 cells per condition were analyzed. Komolgorov–Smirnov test was used to test normality. Since data did not show normal distribution, comparison between groups was done using the Mann–Whitney nonparametric test. Data were significantly different if p < .05 in all cases.

Results

MT2 cell culture medium decreases branching in differentiated PC12 cells

We studied in our neuronal in vitro model of PC12 cells co-cultured with MT2 (HTLV-1 infected cell line) or K562 (as control, noninfected) cell medium the phenotypic effects of viral products on neurite length and branching (number of secondary neurites). Figure 1A and B shows significant phenotypic changes with significant reductions of 15% in the length of primary neurites in PC12 cells after the 3^rd day of differentiation to neuronal type (Fig. 1C). No significant changes in neurite length were found in the previous days (data not shown). Branching quantification shows a significant decrease of 25% in the number of secondary neurites (Fig. 1D) without changes in their length (Fig. 1E). In the next experiments we tested alterations in proteins that could be explaining these phenotypic changes.

FIG. 1.

Effect of MT2 culture medium on PC12 cell model. Representative microphotographs of PC12 cells after 3 days of differentiation incubated with MT2 culture medium are shown in (A) and K562 culture medium in (B). Black arrow shows a secondary neurite in panel B. Data analysis of primary neurite length (C), number of secondary neurites with respect to the total number of neurites (D), and secondary neurite length (E) expressed as mean values (μm) ±standard deviation (SD) are shown. Statistical significance with *p < .05.

MT2 cell culture medium affects kinase and phosphatase activities in differentiated PC12 cells

Considering the important role of the phosphorylation in proteins related to the cytoskeleton and axonal transport, we measured activities of two relevant kinases and one phosphatase in PC12 cell lysates after incubation with HTLV-1 secreted proteins. We found that both CDK5 and PP2A activities in PC12 cells were significantly reduced during differentiation (Fig. 2A,C, 2F). Since GSK3β phosphorylates β-catenin, producing destabilization and activating its proteasomal breakdown, GSK3β activity was indirectly measured by quantifying β-catenin degradation by western blot.⁴⁷ While the GSK3β activity was significantly increased as shown in Figure 2B,D that could be associated with the significant reduction of its inactive form, GSK3β-(pSer⁹) (Fig. 2B,E).

FIG. 2.

Enzymatic activity of kinases and phosphatases in PC12 model. (A) Representative autoradiography showing phosphorylation of histone H1 by CDK5 to measure its kinase activity in PC12 cell incubated either with MT2 or K562 culture medium and its densitometric quantification in (C). (B) Representative Western blots of PC12 cell lysates after 3 days of differentiation incubated either with MT2 or K562 culture medium and densitometric quantification of the bands detected in the Western blots for β-Catenin in (D), GSK3β-(pSer⁹) in (E), and PP2A in (F). Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used as loading control. Analysis of the PP2A activity in PC12 cell lysates after 3 days of differentiation incubated either with MT2 or K562 culture medium is shown in (G). Values are expressed as mean values ±SD. Statistical significance with *p < .05. Observed molecular weights of 23 kDa (H1 Histone), 95 kDa–45 kDa (higher to lower bands of β-Catenin), 57 kDa (GSK3β), PP2A (36 kDa), and 35 kDa (GAPDH).

MT2 cell culture medium does not affect the phosphorylation of motor proteins in differentiated PC12 cells

We followed the possible dysfunction in axonal transport by changes in the phosphorylation status of motor proteins in PC12 lysates cultured with media of MT2 or K562 cells.^{4,22–24,54,55} Expression levels of these proteins are shown in 3 A. We study charge modifications using comparative 2D electrophoresis followed by immunodetection by Western blot analysis. The migration distance in the second dimension of DIC and KHC is shown in Figure 3B (treated with MT2 medium) and in Figure 3D (control). KLC migration is shown in Figure 3C (MT2 medium) and in Figure 3E (control). The analysis of the relative migration distance did not show any statistical differences in the isoelectric point-dependent migration of these species, suggesting no changes in the phosphorylation status of these proteins (Fig. 3F–H).

FIG. 3.

Kinesin and dynein motor proteins characterized by 2D-electrophoresis and Western blot. (A) Representative Western blots of PC12 cell lysates after 3 days of differentiation incubated either with MT2 or K562 culture medium to detect kinesin heavy chain (KHC), kinesin light chain (KLC) and dynein intermediate chain (DIC). GAPDH was used as loading control. Representative Western blots of 2D-electrophoresis of PC12 cell lysates after 3 days of differentiation incubated either with MT2 culture medium (B and C) or K562 culture medium (D and E). In the vertical axis the molecular weight (kDa) standards and in the horizontal axis the pH gradient (from 3 to 11) are shown. In B and D the black arrow shows the DIC signal and the white arrow the KHC signal. In C and E the white arrow shows the KLC signal. Analysis of the relative migration distance obtained in (B), (C), (D), and (E) is in (F) for KHC, in (G) for KLC, and in (H) for DIC. Values are expressed as mean values ±SD. Statistical significance with *p < .05. Observed molecular weights of 120 kDa (KHC), 64 kDa (KLC), 74 kDa (DIC), and 35 kDa (GAPDH).

MT2 cell culture medium reduces tau phosphorylation at Thr¹⁸¹ but not MAP1B in differentiated PC12 cells

During neurite extension, MAP1B is essential, enhancing the rate of MT assembly. MAP1B function in MT dynamics is regulated by phosphorylation, involving the participation of GSK3β, CDK5, and PP2A.^{28,29,39,53,56,57} In Figure 4A,D, 4E we show no detectable changes in MAP1B phosphorylation in PC12 cells by Western blot using the antibody anti-pMAP1B mAb SMI-31.^53,56,57

FIG. 4.

Characterization of MAPs in PC12 model. Representative Western blots of PC12 cell lysates after 3 days of differentiation incubated either with MT2 or K562 culture medium (A). Densitometric analysis of the bands detected in (A) are shown for total Tau in (B), pT¹⁸¹-Tau in (C), total MAP1B in (D) and pMAP1B in (E). GAPDH was used as loading control. Values are expressed as mean values ±SD. Statistical significance with *p < .05. Observed molecular weights of 320 kDa (MAP1B), 65 kDa (Tau), and 35 kDa (GAPDH).

This cytoskeletal protein has several neurophysiological/pathological phosphorylation sites associated with CDK5 and GSK3β kinase activities and is dephosphorylated by PP2A. We found that HTLV-1 secreted proteins significantly decreased pThr¹⁸¹ levels in PC12 cells with no changes in total Tau levels (Fig. 4A–C). This result can be explained by the lower activity of CDK5 (Fig. 2C).

MT2 culture medium increases CRMP-2 phosphorylation at Ser⁵²² but not at Thr⁵⁵⁵ in differentiated PC12 cells

CRMP-2 is involved in neuronal development, regulates interaction between MTs and motor proteins, and is associated with Semaphorin signaling, being regulated by the degree and type of phosphorylation.^{19,30,37,38,41,43,44,58–61} We studied in differentiated PC12 cells the effect of viral proteins on CRMP-2 phosphorylation, finding significantly increased levels of CRMP-2-pSer⁵²² due to CDK5 (Fig. 5A,C), but no changes in CRMP-2-pThr⁵⁵⁵, discarding the effect of ROCK (Rho-associated kinase) (Fig. 5A,B). Total levels of CRMP-2 did not change under the conditions studied (Fig. 5A,D).

FIG. 5.

CRMP-2 phosphorylation in PC12 model. Representative Western blots of PC12 cell lysates after 3 days of differentiation incubated either with MT2 or K562 culture medium (A). Densitometric analysis of the bands detected in A for pT⁵⁵⁵-CRMP-2 in (B), pS⁵²²-CRMP-2 in (C) and total CRMP-2 in (D). GAPDH was used as loading control. Values are expressed as mean values ± SD. Statistical significance with *p < .05. Observed molecular weights of 68 kDa and 66 kDa (higher and lower band of CRMP-2) and 35 kDa (GAPDH).

Discussion

Neurotoxic effects of HTLV-1 secreted proteins, including Tax, which reduce neurite extension and branching, could imply (a) Tau malfunction in MT organization and dynamics in growth cones; (b) MAP1B/pMAP1B ratio alteration controlling MT-F-Actin cross-linkage; (c) abnormal phosphorylated CRMP-2 binding to Tubulin-Actin, leading to MT formation and stability of F-Actin; (d) Kinesin/Dynein malfunction of the axonal transport of synthesized compounds and removal of aging components.^{25,28,29,31,43,60–68} In PC12 cells, NGF binding to TrkA (tyrosine kinase receptor A) induces neurite outgrowth and branching and differentiation through the signaling pathways TrkA/Ras/PI3-K/(Cdc42 or Rac)/AKT/PAK(p21-activated kinase) that regulate the crosstalk between MT dynamics and F-Actin cytoskeleton.^62,69–74 Activation of the RhoA/ROCK/LIMK pathway increases phosphorylation of ADF/cofilin, reducing neurite extension and branching.^73–75 During an initial phase, when neurites are being extended, activated AKT phosphorylates GSK3β at Ser,⁹ reducing RhoA activity. Also, CDK5/p35 promotes neurite outgrowth through phosphorylation of PAK, downregulating its kinase activity and reducing ADF/cofilin phosphorylation.^45,72,74 Thus, the reduction of neurite extension and branching reported in this paper could be mediated by inhibition of PI3K pathway and/or activation of via RhoA-ROCK.

In neurodegenerative disorders such as AD, PK, HD, ALS, MS, HIVE, MS, and DLB, neuronal cytoskeleton and transport dysfunctions are associated with phosphorylation of Tau, MAP1B, CRMP-2, kinesin and dynein causing impairment of their neurophysiological functions. Considering the increase in GSK3β and PP2A activities, we explored for changes in charge of kinesin and dynein due to altered phosphorylation that could explain axonal transport dysfunction. Although we did not detect modification in the phosphorylation of these proteins (Fig. 3), we cannot rule out that the experimental strategy was not sensitive enough or that there could be multiple phosphorylation changes in different residues that do not significantly alter the net charge of the molecule, therefore not changing their migration in the 2D-electrophoresis. Considering the alterations in GSK3β CDK5 and PP2A activities, we also studied the phosphorylation of MAP1B, which could be related to the alterations in neurite outgrowth and extension that we report here.^{28,29,53,56,57,76} Even though we observed a tendency to decrease for MAP1B expression and its phosphorylation using an antibody that detects epitopes phosphorylated by GSK3β and CDK5 (53,56,57,74) in PC12 cells cultured with HTLV-1 secreted proteins with respect to the control (Fig. 4D,E), these differences were not statistically significant. It could be relevant to use other antibodies against different phospho-epitopes recently reported, such as Ser¹²⁶⁰ and Ser¹²⁶⁵.⁷⁷

In neurodegenerative disorders CDK5 shows an increase in its hyperactive form, CDK5/p25 complex, which has a longer protein half-life.⁷⁸ However, we observed that viral proteins produced a reduction of CDK5 activity in PC12 cells (Fig. 2C). Neurophysiological CDK5 activity depends on two regulatory subunits, p35/p39, which during pathological conditions (abnormal rise in Ca²⁺) induce calpain cleavage, producing p25/p29 proteins, respectively.^{25,45,46,78–80} Further studies could elucidate the role of these regulatory subunits. The deficiency in CDK5 activity could be considered a neurotoxic effect of viral proteins leading to Tau-pThr¹⁸¹ level reduction (Fig. 2C; Fig. 4C). Normal phosphorylation of Tau at specific residues regulates MT assembly since its positively charged binding regions improve interactions with negatively charged MTs.^{26,27,40,49–51,81} Increase or reduction of Tau phosphorylation changes its tridimensional structure, modifying neuronal cytoskeleton dynamics, reducing neurite length, and altering branching.^26,40,50,51 We previously evaluated nine reported hyperphosphorylated Tau residues in CSF of HAM/TSP patients, finding only pThr¹⁸¹.⁸² Previously, using a neurite retraction model (with SH-SY5Y cells treated with MT2 cell medium), we found an increase in both Tau-pThr¹⁸¹ and CDK5 activity.⁴⁷

GSK3β mediates extracellular signals that control axon extension and branching, being modulated by phosphorylation (active form with pTyr²¹⁶ and inactive with pSer⁹).^{39,47,83–85} GSK3β phosphorylates substrates (Tau, MAP1B and CRMP-2) that control the stability and dynamics of axonal MTs.^83,84 PP2A, the main brain phosphatase, is composed of three subunits: PP2Ac (catalytic), PP2Ab (regulatory), and PP2A (structural/scaffold), and it is regulated by phosphorylation and methylation.^{34,35,38,86–90} As we mentioned above, upon activation of PI3K/AKT through NGF signaling, GSK3β is inactivated by AKT phosphorylation, and PP2A reverses the process, and in turn, active GSK3β facilitates the activating methylation of the catalytic subunit of PP2A.^86–89 PC12 cells treated with viral proteins showed significantly reduced PP2A activity (Fig. 2F) like in some neurodegenerative diseases including AD, PD and HD. Thereby, PP2A might contribute to the neurotoxic effects of HTLV-1 viral proteins, enhancing the hyperphosphorylation in CRMP-2.

CRMP-2 kinases include GSK3β (at Ser⁵¹⁸/Thr⁵¹⁴/Thr⁵⁰⁹), CDK5 (at Ser⁵²²), and ROCK (at Thr⁵⁵⁵).^{17,19,30,37,38,41,60,61} Hyperphosphorylation of these residues causes inhibition of the neurophysiological functions because the charge increase produces tridimensional structure changes, reducing the affinity toward tubulin dimers and actin (altering cytoskeleton depolymerization), motor proteins, causing growth cone collapse and neurite retraction.^{17,19,30,37,38,41,43,44,61,91,92} In PC12 cells we found hyperactivation associated with CDK5 (Fig. 5C) but not with ROCK (Fig. 5B) because we found changes in CRMP-2-pSer⁵²² but not in CRMP-2-pThr⁵⁵⁵ levels in the presence of viral products, which is different from disorders such as AD, PD, ALS, and HD, with high levels of this phosphorylation found in postmortem brain, biopsy, or blood samples.^37,38,61,93 Therefore, we might discard, among the neurotoxic effects of HTLV-1 proteins, the participation of RhoA/ROCK pathway that regulates MTs and F-Actin cytoskeletons by phosphorylation of associated proteins, including Tau, MAP2 and CRMP-2, leading to axon growth in other cases.⁹⁴ The remaining activity of CDK5 and the lower PP2A activity are enough to produce the required phosphorylation priming of CRMP-2 at Ser⁵²², which facilitates further deleterious phosphorylation by GSK3β.^19,41,44 Therefore, the neurotoxic effect of CRMP-2-(pSer⁵²²) and probable increase in other phosphorylation sites such as Thr^514/509 may lead to the reduction in neurite extension and decrease of secondary neurite number. HTLV-1 infected T cell lines and PBMCs from HAM/TSP have shown accumulation of CRMP-2-(pThr⁵¹⁴/⁵⁰⁹).^15,16 We determined that CRMP-2-pSer⁵²² levels in PBMCs from HAM/TSP patients are significantly higher compared with those in ACs, with also infected PBMCs showing a chemotactic effect toward sSEMA-4D.¹⁰ These results suggest that neuropathological effects of extracellular Tax-sSEMA-4D complex on neurite growth and branching could occur through SEMA-4D-Plexin-1B/PI3K/AKT/GSK3β/CRMP-2 pathway, although participation of the SEMA-3A-Plexin-A1 signaling, which stimulates the same pathway, cannot be discarded.^{17,18,58,59,61,95,96} The reduction of CDK5 activity required in the priming phosphorylation of GSK3β may also play a role in HAM/TSP pathology.^41,43,44,97

Patients with HIV-Encephalopathy, infected with another retrovirus of the family Retroviridae, which secretes Tat (Tax analogous), present gait abnormalities.^98,99 In brain samples of these patients the high CRMP-2-pSer⁵²² levels were associated with CDK5/p25 hyperactivation.^30,45 Upregulation of CDK5 activity agrees with previously reported results in our neurite retraction model (SY-SY5Y cells).⁴⁷ Some studies on other neuronal cells show that CDK5 deficiency deregulates proliferation and neurite outgrowth, causing an increase in primary and secondary neurite extension.¹⁰⁰ Thereby, this observed deficiency could be considered a neurotoxic effect of HTLV-1 proteins.

Conclusions

Possible neurotoxic effects of HTLV-1 secreted proteins include (a) reduction of CDK5 activity producing alteration in neurophysiological phosphorylation of Tau-(pThr¹⁸¹), (b) hyperphosphorylation of CRMP-2 in Ser⁵²², and (c) reduction of PP2A activity contributing to hyperphosphorylations in CRMP-2. Extracellular effects of Tax-sSEMA-4D are probably transduced through PI3K/AKT/GSK-3β/CRMP-2 signaling. Therapeutic strategies could be addressed similarly to other neurodegenerative disorders, increasing PP2A activity,^38,101,102 reducing GSK-3β^22,39,42 and increasing CRMP-2 activity^41,97,103 using compounds with moderate-to-weak inhibition or activation properties, ideally without interfering with its normal functions’ activity.

Authors’ Contributions

S.Q., M.A.V., E.R., and J.P. contributed to the conception of ideas and formulation of research goals. S.Q., M.A.V., M.E.P., M.R., J.R., C.V., and E.U. performed experiments, data collection, and data analysis. M.A.V. had management and coordination responsibility for the research activity. S.Q., M.A.V., J.P., E.U., and M.E.P. prepared the original draft. S.Q., M.A.V., and J.P. reviewed and edited the article to its final form.

Footnotes

Acknowledgments

Our research group wishes to thank all the scientific contributions made by Dr. Luis Cartier, MD, from Departamento de Ciencias Neurológicas, Facultad Medicina, Universidad de Chile, Chile, who was responsible for the initiation and development of this research based on providing molecular knowledge of possible causes of axonal degeneration in patients with HAM/TSP. The authors are also grateful to Dr. Scott Brady for the generous donation of antibodies against DIC and KLC. The authors also wish to thank Dr. Pascale Giraudon (INSERM, France), Dr. Charles R. Bangham (Imperial College London, UK), and Dr. Gerardo A. Morfini (University of Illinois at Chicago, USA), who kindly visited our laboratories to exchange ideas and knowledge about our research.

Author Disclosure Statement

All authors declare that they have no conflicts of interest.

Funding Information

We are grateful to Fondecyt, which supported this study (Grant #108-0396), and to Conicyt grant #22110639.

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HTLV-1 Secreted Proteins Induce Axonal Degeneration Through Kinase and Phosphatase Activity Dysregulation

Abstract

Keywords

Introduction

Materials and Methods

MT2 and K562 cell cultures

Differentiation of PC12 cells and treatment with MT2 or K562 culture medium

Neurite outgrowth evaluation

CDK5 activity assay

Indirect measurements of GSK3β activity

PP2A activity assay

Cell lysis, protein determination, SDS-PAGE and Western-blot analysis

Cell lysis

Protein determination of cell lysate

Western-blot analysis

Two-dimensional (2D) electrophoresis

Statistical analysis

Results

MT2 cell culture medium decreases branching in differentiated PC12 cells

MT2 cell culture medium affects kinase and phosphatase activities in differentiated PC12 cells

MT2 cell culture medium does not affect the phosphorylation of motor proteins in differentiated PC12 cells

MT2 cell culture medium reduces tau phosphorylation at Thr181 but not MAP1B in differentiated PC12 cells

MT2 culture medium increases CRMP-2 phosphorylation at Ser522 but not at Thr555 in differentiated PC12 cells

Discussion

Conclusions

Authors’ Contributions

Footnotes

Acknowledgments

Author Disclosure Statement

Funding Information

References

MT2 cell culture medium reduces tau phosphorylation at Thr¹⁸¹ but not MAP1B in differentiated PC12 cells

MT2 culture medium increases CRMP-2 phosphorylation at Ser⁵²² but not at Thr⁵⁵⁵ in differentiated PC12 cells