Insight into the role of CRMP2 (collapsin response mediator protein 2) in T lymphocyte migration

Abstract

Lymphocyte migration into the central nervous system is a critical step in the physiopathology of a variety of neurological diseases, including multiple sclerosis and virus-induced neuroinflammation. To better understand the molecular mechanisms involved in cells migration, we focused our studies on collapsin response mediator proteins (CRMPs), a group of phosphoproteins that mediate neural cell motility. There is now evidence that collapsin response mediator protein 2 (CRMP2) plays critical roles in the polarization (uropod formation) of T lymphocytes and their subsequent migration. CRMP2 was known to respond to semaphorin, ephrin and neurotrophin signaling in neurons. The link between the chemokine CXCL12, CRMP2 activity and cell migration has been demonstrated in T lymphocytes. These observations and comparisons of the activity of CRMPs in immune and non-immmune cells are summarized here. The ability of a human retrovirus to enhance lymphocyte migration through the modulation of CRMP2 activity is also discussed. In conclusion, viruses have the ability to manipulate the lymphocyte motility machinery, intensifying neural tissue invasion in infected patients.

Lymphocytes are key players in the initiation and termination of most immune responses in lymphoid and non-lymphoid organs. They can stimulate the secretion of antibodies, increase phagocyte activity and directly destroy target cells. Naïve and effector T lymphocytes are able to enter and leave all organs, including the central nervous system (CNS), where they continuously patrol in search of foreign antigens. Their ability to cross the blood-brain barrier is determined by the activation stage of T lymphocytes rather than their antigen-specificity. When activated in situ in the presence of neuroantigen, T cells accumulate within the brain and control protective/deleterious immune responses, thus participating in chronic neuroinflammation. Understanding the mechanism that supports lymphocyte migration and neuroinvasion is essential for a variety of neurological diseases.

A Role for CRMP2 in Cell Fate and Motility Outside the Central Nervous System

Lymphocytes develop a polarized morphology when they undergo migration, activation and cell-to-cell contact during the immune response. This acquisition of polarity requires drastic cell morphological changes and the permanent remodeling of intracellular architecture. While searching for molecules involved in T lymphocyte polarization and migration, we identified collapsin response mediator protein-2 (CRMP2), a multifunctional adaptator protein first described in the CNS and thoroughly studied in neurons.^1-3 CRMP2 plays a key role in a number of cellular events in neurons, including axon specification and polarity, transport of specific vesicles as a cargo receptor and modulation of neurotransmitter release via regulation of both pre- and post-synaptic Ca²⁺ channels.^4-7 Thus, under semaphorin signaling, CRMP2 controls microtubule assembly in neurite outgrowth/retraction and dendrite/axon fate specification. Stimulation of tubulin GTPase activity and the direct binding and transport of tubulin in axons are involved in these processes.^8-10 Several observations provided evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte polarization and migration.¹¹ CRMP2 has the ability to bind to the cytoskeletal elements tubulin and vimentin redistributed at the uropod, the flexible structure of T cells. RNA-mediated crmp2/dpysl2 gene silencing and blocking antibody strongly reduces T cell polarization (uropod formation) and migratory rate. Conversely, increased CRMP2 expression promotes uropod formation and the migration of transfected lymphocytes. Thus, CRMP2 is a key player in cell behavior within a wider field than just the nervous system. Lymphocytes need CRMP2 to acquire a polarized morphology and to engage in subsequent migration. However, neural and immune cells differ in the signaling pathway regulation of CRMP2 activity.

CRMP2 Activity Is Modulated by Chemokines in T Lymphocytes

In neural cells, CRMP2 mediates signals from numerous extracellular cues such as semaphorins, ephrins, neurotrophins and reelin. In T lymphocytes, CRMP2 activity is modulated by chemokines. Chemokines play a major role in T lymphocyte traffic between organs and migration within tissues through cytoskeletal remodeling.^12,13 The chemokine stromal cell-derived factor-1α (SDF1/CXCL12), a potent chemoattractant for mature T-cells, binds to its cognate receptor, CXCR4. This activates a series of downstream signaling pathways, including heterotrimeric G-proteins, the Src family of tyrosines kinases and phosphoinositide 3-kinase (PI3K), leading finally to lymphocyte polarization.¹³ CRMP2 is implicated in this uropod-signaling pathway.¹⁴ CXCL12, in concert with vimentin, induces the re-localization of CRMP2 to the uropod, together with differential CRMP2 phosphorylation.

Regulation of CRMP2 activity by sequential phosphorylation has been well described in neural cells under semaphorin-3A (Sema-3A) treatment.^15,16 Sema-3A-induced retraction of neurites is coupled with activation of cyclin-dependent kinase-5 (Cdk5), which phosphorylates CRMP2 at Ser-522. This primes the protein for subsequent phosphorylation by glycogen synthase kinase 3β (GSK3β) at Thr-509 and Thr-514.¹⁷ In resting T lymphocytes, CRMP2 is phosphorylated at Ser-522 and Thr-509/514. The newly reported CRMP2-(pTyr479) form, phosphorylated by Yes kinase, is weakly expressed.¹⁴ Treatment with CXCL12 induces a rapid drop in CRMP2-(pThr509/514) balanced by a rapid increase in CRMP2-(pTyr479) (Fig. 1, left). Stabilization of CRMP2-(pTyr479) is closely associated with enhanced CRMP2 affinity for cytoskeleton as well as increased lymphocyte migration rates. Additional studies recently showed that the chemokines CCL5, CXCL10 and CCL22, involved in pathological immune cell neuroinvasion, induce similar posttranslational modulations in T lymphocytes (unpublished). Thus, differential CRMP2 phosphorylation by GSK3β and Yes modulates the contribution of CRMP2 to cytoskeletal re-organization during chemokine-directed T lymphocyte migration. Interestingly, the CRMP2-(pThr509/514), CRMP2-(pSer522) and CRMP2-(p479) forms are expressed in both lymphocytes and neural cells. This suggests the potential involvement of chemokine and CRMP2-associated molecular pathways in neural cell migration.

Figure 1. CRMP2 activity in T lymphocytes: alteration by viral infection. CRMP2 belongs to a molecular pathway downstream of chemokine receptors, notably PI3K/GSK3/CRMP2 signaling. Left: Treatment by the chemokine CXCL12/SDF1 activates lymphocyte migration by switching the CRMP2 phosphorylation status: inhibition of CRMP2(p509/514), phosphorylated by GSK3β vs. induction of Tyr479 phosphorylation by Yes kinase. HTLV-1 infection results in CRMP2 hyperphosphorylation. Right: Proteolytic processing controls CRMP2 activity: the short cleaved form (58 kDa) antagonizes the full-length form (62 kDa), acting as a physiologically dominant-negative signal. HTLV-1 infection causes enrichment of the active (full-length) CRMP2 form in T lymphocytes and inhibits activity of the cleaved form. Infection also activates the PI3K/Akt/CRMP2 signaling pathway, intensifying the migratory potential of infected T lymphocytes.

CRMPs and Non-Immune Cell Migration

The CRMP family comprises five cytosolic phosphoproteins (CRMP1–5). Their expression in the developing and adult central nervous system correlates with stages of neuronal differentiation and migration.^18,19 Although CRMPs were identified as signaling molecules of axonal outgrowth and neurite maturation, in vivo studies recently showed their specific involvement in the control of neuronal migration.^20-23 CRMP1 regulates radial neuronal migration in the developing cortex by mediating reelin signaling. Cortical neuronal migration is retarded in crmp1^–/– mice and the presence of reelin correlates with the level of CRMP1 expression and phosphorylation.²¹ CRMP1 has also been associated with the migration of non-neuronal cells and defined as a cancer invasion suppressor.²⁴ In fact, CRMP1 can suppress cancer cell invasion through F-actin depolymerization, while its isoform, LCRMP1, can promote cancer cell migration through actin polymerization and filopodia formation.²⁵ This is an interesting example of a negative feedback mechanism by protein isoforms in a living cell.

CRMP2 is another key regulator of cortical development.²⁶ CRMP2 knockdown in utero inhibits multipolar to bipolar transition required for the directional migration of neurons from the ventricular zone to the different layers of the cortex. Moreover, the canonical BMP-SMAD signaling pathway, known as a regulator of neural progenitor differentiation, controls neuronal migration by suppressing CRMP2 transcription. CRMP2 activity can also be modulated by the brain-specific cytoskeletal regulator, α2-chimearin, in the control of neuronal migration in the neocortex.²² In utero knockdown of CRMP2 and α2-chimearin resulted in accumulation of migrating neurons in the intermediate cortical zone. In conclusion of these observations, CRMP1 and CRMP2 modulate multiple phases of cell behavior and are essential for migration, at least of neural cells. CRMP1 is suspected of having a potential role in T lymphocyte migration, as mRNA and protein are expressed in T cells (unpublished).

Impact of Retroviral Infection on CRMP2 Expression and the Migratory Rate of T Lymphocytes

Lymphocyte activation by human pathogens and their infiltration into the CNS is a key step in the development of immune-mediated neurological diseases.^27,28 One prototype of virus-induced neuroinflammatory disease is tropical spastic paraparesis (TSP), also called human T-cell lymphotropic virus type-1 (HTLV-1)-associated myelopathy (HAM).²⁹ T lymphocytes chronically infected by HTLV-1 display a chronic activation status. They migrate from the periphery into the CNS and constitute both the main viral reservoir in the brain and the main effectors of pathological changes.^30-32 Previous studies have underlined the contribution of virus-infected T lymphocyte secretion in demyelination and axonal loss, whatever their antigenic specificity. For example, lymphocyte cytokines and semaphorins can modify astrocyte glutamate metabolism and damage oligodendrocytes, the myelinating cells of the CNS.^33-35 In addition, the secreted viral protein, Tax, can promote or alter a number of cellular factors involved in lymphocyte and neural cell behavior.³⁶ This paradigm has been used to approach the potential contribution of a virus in the complex molecular mechanism that controls the migration and infiltration of immune cells in the context of infection.

CRMP2 is expressed in virtually all peripheral blood mononuclear cells (PBMC) of HTLV-1-infected patients and healthy blood donors (HD). However, the expression level is higher in cell subsets bearing the activation markers CD69⁺ and HLA-DR⁺,¹¹ a T cell feature clearly promoted by viral infection. In addition, CRMP2 expression is higher in virus-infected CD4⁺ T cell (vs. non-infected) clones isolated from HTLV-1 infected patients. High CRMP2 levels confer high lymphocyte motility. CRMP2 expression levels correlate with the migratory rate of activated T lymphocytes. Moreover, CRMP2 knockdown in infected T lymphocytes strongly reduce their migration.^11,37

Retroviral Infection Modulates CRMP2 Phosphorylation

It is now generally recognized that CRMP expression levels are not sufficient to affect cell activity, post-translational modifications also appearing to be crucial. CRMPs are some of the major phosphoproteins in the nervous system and dynamic regulation of their phosphorylation status is involved in diverse stages of neuronal development (reviewed in ref. ³⁸). CRMP2 is the target of a number of kinases, including RhoK, GSK3, CDK5, Fyn, Fes, Yes and CaMKII.^{2,14-16,39-41} Switching the CRMP2 phosphorylation status is thought to change its binding partners and functions. Depending on the residues phosphorylated, CRMP2 can modulate neuronal polarity, axon guidance, dendritic projection and the migration of neurons and T lymphocytes. CRMP2 hyperphosphorylation has regularly been associated with neuronal injury and degeneration.^42-44

CRMP2 hyperphosphorylation could be involved in the enhanced migratory rate displayed by HTLV-1-infected T lymphocytes, as suggested by accumulation of the CRMP2-(pSer522), CRMP2-(pThr509/514) and CRMP2-(pTyr479) forms.³⁷ The reason for such an alteration remains unknown. Transfection of T lymphocytes to induce expression of the multifunctional viral protein, Tax, did not significantly modify the CRMP2 phosphorylation status. Nevertheless, CRMP2 hyperphosphorylation could reflect the integration of multiple extracellular signals stimulated in T cells by viral infection, resulting in the activation of molecular pathways upstream of CRMP2. Interestingly, CRMP2 hyperphosphorylation was also observed during infection with another human retrovirus, HIV (human immunodeficiency virus).⁴⁵ In the hippocampus of HIV patients with encephalitis, enhanced CRMP2-(pSer522) and gp120 viral protein expression were detected in endogenous neuronal progenitor cells. A role for CRMP2 in altering the neurogenic niche in infected patients with encephalitis has been proposed.

Retroviral Infection Modulates CRMP2 Cleavage to Enhance Lymphocyte Motility

The importance of proteolytic processing of CRMP2 in controlling protein activity was previously observed both in neural cells⁴⁶ and T lymphocytes.³⁷ CRMP2 is expressed in full-length and short processed forms, resulting from cleavage of the C-terminus by the calcium-dependent protease, calpain.⁴⁷ Multiple putative phosphorylation sites are located on the C-terminus. Elimination by cleavage is supposed to limit the association of CRMP2 with molecular partners, preventing axon elongation in neurons, and possibly cytoskeletal reorganization in migrating lymphocytes. In fact, the short-processed CRMP2 (58 kDa) antagonizes the full-length protein (62 kDa). Cell transfection with CRMP2wt and CRMP2Δ503 (coding for the short form) had opposite effects (increase vs. decrease, respectively) on neurite maturation and T cell migratory rates. Thus, short CRMP2 acts as a physiologically dominant-negative signal to reduce process extension in neural cells, and uropod formation in T lymphocytes. This is a negative feedback mechanism by CRMP2 protein isoforms, additional to that described for CRMP1.²⁵

In the context of HTLV-1 infection, the levels of full-length CRMP2 were always higher (compared with the short version) in infected compared with uninfected T cell clones.³⁷ Importantly, this correlated with the higher migratory rate of infected T cell clones. In addition, these cells displayed a more pronounced adhesion of full-length CRMP2 to the cytoskeletal components, tubulin and actin. This could be directly linked to viral infection, as transfection to induce Tax protein expression in T lymphocytes enhanced full-length CRMP2 levels and resulted in a high migratory rate. Conversely, knockdown of the expression of Tax protein was associated with a reduction in full-length CRMP2. In addition, cells co-transfected with CRMP2Δ503 and Tax showed no reduction in their migration rate compared with the control, contrary to what was observed with CRMP2Δ503 alone. Moreover, Tax expression restored the level of endogenous full-length CRMP2 previously decreased by CRMP2Δ503. Thus, by promoting full-length CRMP2 in infected T lymphocytes, Tax expression conferred a migratory advantage, possibly by preempting the negative effect of short CRMP2 (Fig. 1, right). In conclusion, we propose that viruses, in particular HTLV-1, have the ability to affect CRMP2 processing, favoring the presence of active CRMP2 in T lymphocytes.

Retroviral Infection Activates the PI3K/Akt/GSK3/CRMP2 Axis in T Lymphocytes

CRMP2 belongs to the PI3K/Akt/GSK3β molecular pathway.⁴⁸ As this pathway is involved in regulating cell migration, HTLV-1 was suspected of affecting this cell machinery in T lymphocytes. The ability of CRMP2 to bind to several proteins that are active downstream of chemokine receptors in T lymphocytes (Vav-1, Yes kinase, PLCγ and PI3KR1/p85)¹⁴ backed up this hypothesis. Comparative genomic analyses performed on CD69⁺ cells selected from HD and HTLV-1 infected patients suffering from TSP/HAM indicated that a number of genes involved in signaling pathways, downstream of chemokine receptors and cell migration, were specifically present in patients.³⁷ Patient cells exhibited a distinct profile, with 22 genes uniquely expressed and 10 others with a ≥ 3-fold increased expression. Several are involved in G protein and PI3K signaling. Others act in intracellular trafficking and secretion. Global activation of the chemokine receptor pathway was associated with high CRMP2 expression and elevated migratory rates of selected cells. These data clearly showed that HTLV-1 infection influences the PI3K/Akt/GSK3β/CRMP2 axis in T lymphocytes, thus positively controlling cytoskeletal organization and lymphocyte migration. To conclude, this study on infected T cells freshly isolated from patients complements other studies showing that HTLV-1 activates the CXCL12/CXCR4/G protein axis in virus-infected T cell lines.⁴⁹

Concluding Remarks

Modulation of CRMP2 activity by a virus is not restricted to HTLV-1, as protein alterations have been reported in two models of virus-induced neuroinflammation. Prominent infiltration of activated lymphocytes and dendritic cells with high CRMP2 levels was observed in the CNS of mice infected either with paramyxovirus CDV or rabies virus.⁵⁰ This correlated the degrees of clinical manifestation. By extrapolation, one can suppose that chronic activation of the CRMP2-associated signaling pathway by a virus could amplify immune cell motility and subsequent neuroinvasion. For example, although no virus has been identified so far as the etiological agent of multiple sclerosis (MS), it is now accepted that a variety of common viruses could be responsible for triggering autoimmune T and B cells at the periphery and sustain subsequent inflammation in the CNS of patients.^34,51 The study of CRMP2 expression and posttranslational modifications in MS patients could shed light on these mechanisms. In conclusion, the evidence of viral impact on CRMP2 activity provides clues to understanding the mechanisms of CNS infiltration by T lymphocytes during a silent or detectable infectious episode.

Glossary

Abbreviations:

CRMP2

collapsin response mediator protein 2

CNS

central nervous system

HTLV-1

human T leukemia virus type 1

HAM-TSP

HTLV-1-associated myelopathy/ tropical spastic paraparesis

Varrin-Doyer M, Nicolle A, Marignier R, Cavagna S, Benetollo C, Wattel E, et al. Human T lymphotropic virus type 1 increases T lymphocyte migration by recruiting the cytoskeleton organizer CRMP2. J Immunol. 2012;188:1222–33. doi: 10.4049/jimmunol.1101562.