The role of cyclin dependent kinase 5 (Cdk5) in neuropathic pain

Introduction

Neuropathic pain results from damage to the nervous system at the peripheral or central level, and it can have diverse origins including trauma, diabetes, viral infection, cancer, multiple sclerosis, among others [66; 200]. Neuropathic pain is an unpleasant chronic condition that lasts after the initial injury is resolved and is characterized by increased responsiveness that leads to allodynia and hyperalgesia. Allodynia is defined as pain in response to an innocuous stimulus and hyperalgesia, is defined as increased pain in response to a stimulus that normally produces pain. This type of pain is estimated to affect about 0.9% to 17.9% of the general population and reduces health-related quality of life in patients [164; 185].

Although the pathophysiology of neuropathic pain is far from being understood, an increasing body of literature suggests that protein kinases alter the phosphoregulatory “set-point” of proteins involved in pain signaling. Following inflammation or nerve damage, a wide variety of second intracellular messengers such as protein kinase A, B and C [2; 186; 221], mitogen-activated protein kinase (MAPK) [68; 106], calcium/calmodulin kinase II (CaMKII) [103], cyclin-dependent kinase 5 (Cdk5) [135; 136], among others, are activated. Cdk5 belongs to the family of “cyclin-dependent kinases” (Cdks) that phosphorylate serine and threonine residues and are involved in cell cycle regulation. Cdk5 shares a common 3-dimensional fold with other members of the Cdk family [199], with the ATP and substrate binding sites nested between the N-terminal β-sheet domain (N-lobe) containing a glycine-rich loop (G-loop) and the C-terminal helical domain (C-lobe) containing the activation loop (T-loop) (Figure 1A, B). Unlike other members of the Cdk family, Cdk5 kinase activity does not require phosphorylation in the T-loop [141; 143]. Instead, binding of either neuronal activators p35 or p39 (or their cleavage products p25 and p29, respectively) is both necessary and sufficient for activation [62; 90; 179]. Although these essential activators do not belong to the cyclin family, the binding interface is highly similar to Cdk-cyclins, with the p25 cyclin-box-like fold contacting the Cdk5 PSSALRE helix (Figure 1A, B) [23; 173]. In other Cdks both binding of cyclin and phosphorylation at Thr-160 are required for the T-loop to adopt the active conformation, forming the +1 site and the +3 site, where the negatively charged phosphate at pT160 coordinates the K/R/H side-chain of the peptide substrate [23]. The absence of T-loop phosphorylation raised fundamental questions regarding how Cdk5 is activated and how it retains the strong preference for the S/TPXK/R/H consensus sequence [168; 173]. The keys to these mysteries were unlocked by the first structure of Cdk5 with p25 bound [173] which revealed that p25 forms extensive interactions with the T-loop, stretching it into the active conformation (Figure 1B, C) [173]. Furthermore, p25 contributes a conserved glutamate to the +3 site, providing the negatively charged partner for K/R/H CDK5 Molecular Figure (Figure 1B, D) [109; 110; 173]. Although no structure is available of Cdk5 with peptide bound, the Cdk2 complex with cyclin A3 and peptide substrate permits identification of the location of the ATP phosphate moiety, the conserved DKS triad that stabilizes the target Ser/Thr hydroxyl and the +1 site formed by conserved Val162-Val163 (Figure 1B, D) [23]. Also consistent with other Cdk structures, binding of p25 results in a slight rotation of the N-terminal and C-terminal lobes away from each other, opening the target binding groove (Figure 1C) [173]. The Cdk5 structure with its inhibitor (R)-roscovitine with a clearly resolved G-loop revealed that the Y15 side chain is buried against the PSSALRE helix (Figure 1B), suggesting that phosphorylation would result in (or require) that the side-chain adopt an outward conformation [110] although it is not yet clear how this would activate Cdk5. In Cdk2, phosphorylation at this site inhibits activity by interfering with ATP and peptide substrate binding [7; 195]. Likewise, phosphorylation at T14 is thought to inhibit kinase activity by changing conformation of the G-loop, interfering with ATP binding [7].

Figure 1. Sequence and structure representations of Cdk5.

A. Diagram of p35 and Cdk5 sequences. Solid black bars designate regions of known structure. B. Structure of Cdk5/p25 complex with the inhibitor (R)-roscovitine bound in the ATP site (PDB ID 1unl, [110]). Circles mark locations of phosphate moiety (P) of ATP (predicted), DKS triad (0), hydrophobic +1 site, and negatively charged +3 site [23; 110; 165]. C. Comparison of Cdk5 structures with and without p25 bound. The T-loop is partially disordered and in an inactive conformation in the non-p25 bound structure (gray, PDB ID 4au8 [109]) and in an extended, active conformation in the p25 bound structure (pink PDB ID 1unl, [110], p25 omitted for clarity). Curved arrows indicate rotation of N- and C-terminal lobes with p25 binding. Both structures contain an inhibitor in the ATP site. D. Electrostatic surface representation of peptide binding groove from panel B (negative in red, neutral in white, positive in blue).

Under neurotoxic stress [88], inflammation [135] and neuronal injury conditions [57], p35 and p39 can be cleaved by calpain in response to increased calcium influx, generating p25 and p29, respectively. When these N-terminal truncated forms bind to Cdk5, the kinase is hyperactivated due to a decrease in protein turnover, and since they lack the myristoylation site, Cdk5/p25 or p29 complexes can move freely through the cytoplasm, thus phosphorylating different cell targets [138]. Different studies have shown that the formation of the Cdk5/p35 complex has multiple functions in immature neurons, including migration, differentiation, and synaptogenesis [67;84]. Likewise, Cdk5 participates in various vital neuronal functions such as neurotransmitter release, neuronal migration, cell differentiation, neuronal plasticity, apoptosis, memory, learning, addiction and pain, among others [43; 44; 92; 161]. Even though Cdk5 regulates these critical functions, its deregulation through calpain-dependent cleavage of its activator p35 to p25 has been implicated in neurodegenerative disorders such as Alzheimer’s [138], Parkinson’s [126; 166], Huntington’s disease [104], amyotrophic lateral sclerosis [126] and inflammatory [135] and neuropathic pain [57]. Cdk5 knockout and p35/p39 double-null mutant mice are associated with perinatal mortality stemming from neuronal migration defects [81; 130].

Cdk5 is emerging as an important kinase involved in nociceptive signaling in the peripheral nervous system. This enzyme and its activators p35/p25 are expressed in the spinal nerves [57; 174], dorsal root ganglia (DRG) [57; 135; 174; 207], in the dorsal horn of the spinal cord [135; 207], and in the trigeminal ganglia [135]. It is important to highlight that following chronic constriction injury (CCI), Cdk5 expression increases as there is a greater occupancy of phosphorylated cAMP response element-binding protein (CREB) in Cdk5’s promoter region in the spinal cord [93]. CREB is a cellular transcription factor that binds to DNA sequences called cAMP response elements (CRE) located in the promoter region of Cdk5. However, it was reported that pCREB favors histone H4 acetylation in the Cdk5 promoter region, thus decreasing the ability of histones to bind to DNA, allowing chromatin expression and gene transcription [93]. Hence, these findings suggest that there is potent epigenetic regulation of spinal Cdk5 during neuropathic pain, and that Cdk5 up-regulation contributes to the pathogenesis of chronic pain. Furthermore, immunoreactivity assays have shown that Cdk5 and p35 co-localize with transient receptor potential vanilloid 1 (TRPV1), a specific C-fiber nociceptive neuronal marker [135]. Interestingly, activity and expression of this complex is restricted to C fibers, whilst Aβ and Aδ fibers are spared with the kinase activity [135; 154]. Increased expression of Cdk5/p35 in primary sensory and dorsal horn neurons by peripheral inflammation contributes to heat hyperalgesia [135; 207]. Enhanced expression of Cdk5 and its activators in the DRG in the L5/L6 spinal nerve ligation (SNL) model of chronic neuropathic pain contributes to mechanical allodynia [57].

Given the capacity of Cdk5 to drive the pathological events leading to inflammation and chronic pain, the pharmacological targeting of this kinase is attractive for chronic pain treatment. From the therapeutic point of view, three strategies toward inhibiting pathological Cdk5 activity have been studied [3]. These include: (i) small molecules like roscovitine and olomoucine [80; 159], that compete for the active ATP binding site within the kinase domain to inhibit enzyme activity; (ii) peptides that disrupt the Cdk5-p25 binding interface, like the Cdk5 inhibitory peptide (CIP) [217], (iii) and most recently, small molecule inhibitors like tamoxifen, that disrupt Cdk5-p25 interaction [40]. However, to our knowledge, none of these have entered clinical trials. On the other hand, to determine whether Cdk5’s activity is involved in nociceptive transmission, only kinase inhibitors such as roscovitine and olomoucine have been used. Nevertheless, their use has been limited to animal models due to their poor selectivity. In rodents, intrathecal treatment with roscovitine has been shown to reduce endogenous Cdk5 activity in the DRG and spinal dorsal horn [207] and alleviates complete Freund’s adjuvant (CFA)-induced heat hyperalgesia [202;207] and formalin-induced nociceptive response [188]. Likewise, intrathecal delivery of roscovitine substantially attenuates mechanical allodynia in rats with CCI [93]. Furthermore, chronic intrathecal injection of olomoucine partially reverses mechanical allodynia in rats with L5/L6 SNL [57], suggesting the important role of Cdk5 activity in the maintenance of inflammatory and neuropathic pain. Therefore, this narrative review will present an overview of the regulation of Cdk5 activity and a snapshot of key substrates that have been implicated in neuropathic pain conditions.

Regulation of Cdk5 activity in pain signaling

Many of the secondary messengers that are activated following nerve damage related inflammation play a role in the activation of Cdk5. These messengers regulate Cdk5 activity through three mechanisms: (i) phosphorylation of Cdk5, (ii) increasing p35 interaction with Cdk5, and (iii) cleavage of p35 into p25 (Figure 2). The main group of these activators enhances p35 expression via the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). ERK activation by phosphorylation can be detected in primarily small-to-medium diameter DRG sensory neurons following noxious stimuli but not following innocuous stimuli [69; 129]. ERK1/2 activation will in turn increase the expression of the transcription factor early growth response 1 (Egr-1), which will induce p35 expression and ultimately increase Cdk5 activity [60; 183]. After a nerve injury, ERK1/2 is activated secondary to the release of neurotrophins and cytokines. Nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) can induce a robust activation of ERK within 15 minutes of application to DRG neurons [21]. Cytokines including transforming growth factor-beta (TGF-β), tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ), interleukin 6 (IL-6) and bradykinin similarly provoke ERK 1/2 activation [145; 167; 183]. Inhibition of MEK, a kinase upstream of ERK1/2, using U0126, revealed a decrease in protein levels of p35 while ERK1/2 and Cdk5 protein levels remained the same [218]. Further, Cdk5 activity was found to decrease significantly following U0126 treatment. Thus, neurotrophin and cytokine signaling after injury, promote Cdk5 activity through ERK1/2 activation.

Figure 2. Molecular Regulation of Cdk5 activity.

Following nerve injury, cytokines including Transforming growth factor beta (TGF- β), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), Interleukin 6 (IL-6) and bradykinin are released. All of these activate the mitogen-activated protein kinase (MAPK) signaling pathway by activating extracellular-signal-regulated kinase 1/2 (ERK 1/2). Neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) are also released. Of these, NGF and GDNF have been found to also activate the MAPK signaling pathway via activation of ERK. ERK subsequently activates early growth response 1 (Egr-1) which promotes the expression of p35. This allows for increased association with Cdk5 and activation. GDNF also, by interaction with GDNF family receptor alpha-1 (GFRα1), activates phosphoinositide 3-kinase (PI3K) pathway which also activates p35 into increased association with Cdk5. BDNF, another neurotrophin released, can activate Cdk5 via tyrosine receptor kinase B’s (TrkB) phosphorylation of Cdk5. This phosphorylation increases Cdk5 activity and can be catalyzed by Fyn, a tyrosine kinase. Alternately, BDNF activates Cdk5 by activating protein kinase C δ’s (PKCδ) promotion of p35 expression. Following excitatory glutamate signaling, N-methyl-D-aspartate receptor (NMDAR) and kainite receptors are stimulated to permit a calcium influx. Long-term signaling and high calcium influx will promote the association of calcium/calmodulin-dependent protein kinase II α (CAMKIIα), p35, calpain and GluN2B subunit. This will induce calpain cleavage of p35 to p25 resulting in hyperactive Cdk5. Short-term signaling will transiently activate Cdk5 via calmodulin activation, thus phosphorylating p35 and destining it for degradation by the ubiquitin protease rendering Cdk5 inactive. G-tubulin binding to Cdk5 prevents Cdk5 association with p39 and p35 and promotes its inactive state. Finally, α- actinin-1 and CAMKIIα are known to interact with both p35 and p39. These complexes can then interact with Cdk5/p35 and Cdk5/p39 complexes and are suspected to affect Cdk5 activity.

During neuronal differentiation and brain development, levels of Cdk5 and p35 gradually increase. At this time, ERK 1/2 is known to translocate to the nucleus upon activation to stimulate the transcription of c-fos. CREB is activated by both PKA and ribosomal S6 protein kinase (RSK), an intermediary kinase between ERK1/2 and CREB [87; 175]. CREB and c-fos are known to bind the CRE and AP-1 binding sites, respectively, both of which are present in the Cdk5 gene’s promoter region [87; 175]. Transfecting cells with c-fos and CREB cDNAs increased Cdk5 expression [87; 175]. PKA and ERK1/2 pathways have been implicated with inducing Cdk5 and p35 expression thus creating more opportunities for Cdk5’s activity which has been linked by various targets to nociception.

GDNF also modulates p35 and Cdk5 activity via its interactions with GDNF family receptor α1 (GFRα1) on non-peptidergic nociceptors [13]. GDNF signaling through its receptor GFRα1 increase the transcription and translation of Cdk5 and p35 through the PI3K and the MEK/ERK1/2 pathway [86]. The resulting increase in Cdk5 and p35 availability implicates a link between Cdk5 activation and pain.

Brain-derived neurotrophic factor (BDNF) is another neurotrophin that can regulate Cdk5 activity by 1- enhancing p35 expression and 2- increasing Cdk5 phosphorylation [34;176;216]. By activation of PKCδ phosphorylation on Y311 [153;216], BDNF signaling leads to increased protein level of p35 andCdk5 activity [216]. BDNF also enhances TrkB’s association with and phosphorylation of Cdk5 at Y15 thus increasing its kinase activity [34]. This phosphorylation of Cdk5 at Y15 can be catalyzed by Fyn, a tyrosine kinase also involved in the phosphorylation of collapsin response mediator protein 2 (CRMP2), a target of Cdk5 [181;203]. After a nerve injury, increased BDNF expression is required for the transition to chronic neuropathic pain [219]. Thus, in neuropathic pain, increased BDNF expression activates PKCδ, resulting in an increase of p35 expression and Cdk5 activation by phosphorylation.

Aside from participating in activation of calpain cleavage of p35, CaMKIIα – a serine/threonine-specific protein kinase linked to long-term potentiation (LTP), addiction and memory – is known to bind p39 and form a complex with Cdk5/p39 as well as with Cdk5/p35 when bound to p35. Similarly, α-actinin-1, an actin-binding protein with diverse functions, associates with p35 and p39 and can form complexes with Cdk5/p35 and Cdk5/p39 respectively. The potential for these interactions to regulate Cdk5 activity is unclear though it is speculated to play a role in preventing p35 and p39 cleavage and the resulting hyperactivation of Cdk5 [44]. Cdk5 is also regulated by the direct binding of G-actin. G-actin is the monomeric and globular form of actin which is assembled into long filament polymers, F-actin. Affinity precipitation and immunoblotting revealed G-actin’s interaction with p35, p25 and Cdk5. GST pull-down and further immunoblotting clarified that G-actin does not compete with p35 or p25 binding to Cdk5. Instead, it has been found that G-actin directly binds to Cdk5 and inhibits Cdk5/p35 and Cdk5/p25 activity independent of Cdk5’s activators [205]. The consequences of this relationship between G-actin and Cdk5 activity is not yet clear in relation to pain, though the actin cytoskeleton, here shown to regulate Cdk5 activity, is implicated in several downstream targets of Cdk5 that affect nociception.

Cdk5’s targets and downstream events implicated in pain signaling

Several known proteins that interact with or are substrates of Cdk5 have been linked to nociceptive pathways, including TRPV1 channels, synaptophysin, voltage-gated calcium channels (VGCCs), collapsin response mediator protein 2 (CRMP2), glutamate receptors, purinergic receptors, opioid receptors, among others, suggesting that Cdk5 may be involved directly in nociception (Figure 3, Table 1).

Figure 3. Cdk5 targets and relevant downstream events implicated in neuropathic pain.

p35 or p25 bound Cdk5 phosphorylates the voltage-gated calcium channels: CaV2.1, CaV2.2 and CaV3.2 thus affecting their downstream interactions and consequent levels of neurotransmitter release as well as levels of surface localization. Cdk5 also phosphorylates collapsin response mediator protein 2 (CRMP2) which affects its association with CaV2.2 and in turn CaV2.2 membrane expression. The phosphorylation of CRMP2 also affects its ability to be SUMOylated and subsequently interact with NaV1.7 to promote surface expression. Cdk5 has also been found to increase expression of vesicular glutamate transporter 2 (VGLUT2) thus increasing glutamate transport to synaptic vesicles. It also affects N-methyl-D-aspartate receptors (NMDRs) by increasing the expression of its GluN2A subunit, affecting incidence of long-term potentiation (LTP), as well as phosphorylating the GluN2B to favor its expression. Cdk5 phosphorylates metabotropic glutamate receptor 1 and 5 (mGluR1 and mGluR5) which affects their membrane localization and association with downstream effectors. It also phosphorylates purinergic receptors (P2X2R and P2X3R) affecting receptor sensitization and downstream activity. Cdk5 also phosphorylates delta opioid receptor affecting its membrane expression and opioid tolerance. Further, Cdk5 phosphorylates the μ2 subunit of the AP2 complex affecting clathrin-mediated endocytosis of transient receptor potential vanilloid 1 (TRPV1). It also promotes TRPV1 membrane localization by phosphorylating the forkhead-associated (FHA) domain of the kinesin-3 family member 13B (KIF13B), as well as by direct phosphorylation of TRPV1. Finally, Cdk5 interacts with synaptophysin (Syp) to affect neurotransmitter release.

Table 1.

Biological effects of Cdk5-mediated phosphorylation of key target proteins involved in neuropathic pain.

Targets	Regulatory mechanisms	Molecular and cellular effects	References
TRPV1	Phosphorylates Thr-407 (mouse/humans) or Thr-406 (rat)	Promotes surface localization of TRPV1
KIF13B-FHA domain	Phosphorylates FHA domain of KIF13B at Thr-506	Promotes TRPV1 trafficking to the plasma membrane by mediating KIF13B-TRPV1 association
AP2-μsubunit	Phosphorylates μ2 subunit of AP2 complex at Ser-45	Reduces TRPV1-μ2 interaction and inhibits TRPV1 internalization
Synaptophysin	Interacts with synaptophysin	Increases synaptophysin expression and enhances neurotransmitter release by favoring the interaction of synaptophysin with SNARE proteins
CaV2.1	Phosphorylates the intracellular loop connecting domains I-II of the α1 subunit of CaV2.1 channels	Inhibits the interaction between CaV2.1’s synprint site and SNARE proteins and reduces neurotransmitter release
CaV2.2	Phosphorylates de C-terminus domain of the α1 subunit of CaV2.2 channels	Increases channel open probability and neurotransmitter release by enhancing CaV2.2-RIM1 interaction
CaV3.2	Phosphorylates Ser-561 and Ser-1987 of the α1 subunit of CaV3.2 channels	Enhances the trafficking of CaV3.2 channels to the plasma membrane
CRMP2	Phosphorylates Ser-522	Enhances CRMP2 binding to CaV2.2 which increases CaV2.2 trafficking to the plasma membrane Permits CRMP2 SUMOylation and promotes CRMP2-NaV1.7 binding which augments channel trafficking to the plasma membrane
VGLUT2	Interacts with VGLUT2	Increases VGLUT2 expression and favors glutamate transport to synaptic vesicles
GluN2A subunit	Phosphorylates Ser-1232	Increases expression /activity of NR2A and enhances LTP
GluN2B subunit	Phosphorylates NR2B subunit (unknown residue)	Favors NR2B expression in the spinal cord but decreases NR2B expression in the prefrontal cortex
mGluR1 and mGluR5	Phosphorylates Ser-1167	Enhances mGluR binding to homer protein and promotes receptor clustering in the plasma membrane
P2X2aR	Phosphorylates Thr-372	Prevents receptor desensitization
P2X3R	Phosphorylates P2X3R (unknown residue)	Decreases P2X3 activity
DOR	Phosphorylates Thr-161	Enhances DOR bioavailability at the cell surface and favors DOR interaction with MOR

TRPV1 channels

TRPV1 is a Ca²⁺ permeable nonselective cation channel that is predominately expressed in small sensory C-fibers and to a lesser extent in medium Aδ-fibers [58]. In the spinal cord, TRPV1 is localized in both pre- and post-synaptic neurons in lamina I and II, as well as in astrocytes [47]. In addition to its location at the spinal level, TRPV1 receptor is found at supraspinal levels and contributes to descending modulation of nociception [108]. TRPV1 is activated by heat (>43°C), protons (pH <5.9), eicosanoids and endogenous ligands termed “endovanilloids” like capsaicin [4;29] and is targeted by many kinases that influence its trafficking and activity [142], among which is Cdk5. Cdk5 regulation of TRPV1 membrane trafficking is a fundamental mechanism controlling heat sensitivity of nociceptors. In vivo studies indicate that peripheral inflammation produced by CFA injection induces activation of Cdk5 in DRG [207], favoring the anterograde transport of TRPV1 and contributing to the development of heat hyperalgesia [202]. Mice with reduced Cdk5 activity show higher tolerance to TRPV1-mediated painful stimuli, whereas mice with increased Cdk5 activity are less tolerant to the same stimulus [65].

It has been reported that Cdk5-mediated phosphorylation of TRPV1 at T407 (in mouse and human) [134] and T406 (in rat) [99] promotes receptor trafficking to the plasma membrane [99;134] (Figure 3, Table 1) and modulates agonist-induced Ca²⁺ influx [134]. Furthermore, disrupting the phosphorylation of TRPV1 at T406 significantly decreased activation kinetics and the receptor desensitization was reduced or even eliminated [65]. Inhibition of Cdk5 activity with roscovitine in cultured rat DRG neurons results in a significant reduction of capsaicin-mediated Ca²⁺ influx, and this effect was reversed by restoring Cdk5 activity [134]. In the same way, intrathecal delivery of an interfering peptide (TAT-T406) against the phosphorylation of T406 alleviates heat hyperalgesia and reduces the surface levels of TRPV1 in a rat model of CFA-induced inflammatory pain [99]. Additionally, C-fiber neuron-specific Cdk5 conditional-knockout mice showed decreased TRPV1 phosphorylation, resulting in decreased paw an tail withdrawal latency in response to noxious thermal stimulation [134]. Therefore, Cdk5-mediated phosphorylation of TRPV1 at T406 plays an important role in the transduction of nociceptive stimuli and pain signaling.

Trafficking of channel proteins to the membrane and their internalization co-exist to maintain equilibrium conditions. The constitutive internalization of ion channels contributes to the stability of membrane protein distribution, which is important for ion channel function. Clathrin-mediated endocytosis is the major route of integral membrane protein internalization and TRPV1 internalization occurs through a clathrin-dependent mechanism [98]. In the clathrin-dependent internalization pathway, the AP2 complex plays a crucial role by interacting with clathrin and the target protein. In this process, the μ2 subunit of the AP2 complex is responsible for binding to the target protein [133]. Clathrin and TRPV1 were recently reported to colocalize in HEK-293 cells, and the TRPV1 N-terminal is the domain that interacts directly with μ2 [98]. Notably, in the same study it was reported that Cdk5 phosphorylates μ2 at S45 reducing TRPV1-μ2 interaction, which negatively regulates receptor internalization (Figure 3, Table 1). Furthermore, roscovitine enhanced the interaction between TRPV1 and μ2 and consequently TRPV1 internalization which suggests a clathrin-mediated endocytosis [98;99]. Specific interference of Cdk5 phosphorylation of μ2 at S45 with a TAT-peptide (TAT-S45) reduced the phosphorylation of μ2, enhanced TRPV1 internalization and attenuated spontaneous pain and inflammatory thermal hyperalgesia in capsaicin and CFA-induced inflammation in rats [98]. These data indicate that the activity of Cdk5 has a negative regulatory effect on TRPV1 internalization.

Interestingly, Cdk5 has been found to regulate receptor trafficking to the plasma membrane through kinesin-3 family member 13B (KIF13B). A phosphorylation site for Cdk5 at T506 located in the forkhead-associated (FHA) domain of the kinesin-3 family motor protein GAKIN (guanylate kinase-associated kinesin)/KIF13Bwas found [202]. TRPV1-containing vesicles bind to the FHA domain of KIF13B and are consequently delivered to the cell surface [202]. In connection with the above, KIF13B transports TRPV1 channels to the membrane of DRG neuron and Cdk5/p35 promotes the trafficking process by mediating KIF13B–TRPV1 association (Figure 3, Table 1), that is at least partially dependent on Cdk5-mediated phosphorylation of KIF13B at T506 [202]. It is worth mentioning that inhibition of T506 phosphorylation alleviates heat hyperalgesia in rats [202], and that mutating T406 of the TRPV1 channel to alanine, reduces the interaction of TRPV1 with cytoskeletal elements and decreases KIF13B-TRPV1 interaction, which leads to reduced surface expression of TRPV1, suggesting that Cdk5 phosphorylation of T406 is obligatory for recognition of the KIF13B-FHA domain and, at least partially, regulates TRPV1 transport [99], thus, Cdk5 regulation of TRPV1 trafficking to the plasma membrane is an underlying mechanism that controls heat hyperalgesia. Taken together, these previous reports suggest that Cdk5, whether directly or indirectly regulating the receptor, is required for normal functional expression of TRPV1 channels in heat sensitivity of nociceptors.

Synaptophysin

Neurotransmitter release is the chemical signal necessary for information transfer among neurons. Neurotransmitters are stored in synaptic vesicles (SVs) at the presynaptic terminal, from which they are released at the arrival of the nerve impulse by an increased Ca²⁺ influx into the synaptic plasma membrane through voltage-gated calcium channels. Following Ca²⁺ influx, SVs are fused with the synaptic plasma membrane to release neurotransmitters. Synaptophysin has been shown to play a primary role in mediating the exocytosis-endocytosis of synaptic vesicles and in regulating the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex formation [27;191]. SNARE protein complex is essential for the fusion of vesicles to the plasma membrane [184], thus, it is involved in neurotransmission [64;96]. Synaptophysin co-localizes with the key SNARE components VAMP-2, SNAP-25, and syntaxin-1A in rat spinal dorsal horn neurons [213]. Previous studies have revealed that overexpressing synaptophysin results in increased neurotransmitter release by affecting the probability of vesicular exocytosis and/or the number of synaptic vesicles initially docked at the active zone [1]. In addition, synaptophysin’s expression is increased in the ipsilateral dorsal horn of the spinal cord of rats with sciatic nerve transection [95] and CCI [36]. Moreover, Cdk5 co-localizes with its activator p35 and synaptophysin in L4-L5 dorsal horn neurons, and with VAMP-2 in the cytoplasm of neurons [213], suggesting that Cdk5 may interact with synaptophysin/VAMP-2. During SNARE formation, VAMP-2 docks with SNAP-25/syntaxin-1A to make synaptic vesicles fuse with the plasma membrane [33]. Blocking Cdk5 function by intrathecal administration of roscovitine, resulted in a significant decrease in the spinal amount of synaptophysin. This correlated with a relief of heat hyperalgesia induced by intraplantar CFA injection [213]. Previous studies revealed that overexpression of synaptophysin resulted in increased glutamate release [1]. Since the glutamatergic neuron system plays a key role in mediating inflammatory hyperlagesia [59], it is reasonable to propose that synaptophysin could mediate the release of glutamate. Therefore, Cdk5 activity may mediate heat hyperalgesia by promoting SNARE complex formation and excitatory neurotransmitter release (Figure 3, Table 1).

Voltage-gated calcium channels (VGCCs)

VGCCs are expressed in DRG neurons and in the spinal dorsal horn where they play an important role in nociception [123;212]. In the nervous system, neurotransmitter release from active zones at the presynaptic terminals is triggered by Ca²⁺ influx through VGCCs [30], mainly via high voltage-activated (HVA) Ca²⁺ channels CaV2.1 (P/Q-type) and CaV2.2 (N-type) [146;197]. HVA Ca²⁺ channels contain a synaptic protein interaction “Synprint” site located in the large intracellular loop connecting domains II and III of the α1 subunits of these channels that interacts with proteins involved in neurotransmitter release such as the SNARE proteins, syntaxin, synaptotagmin, and SNAP-25 [30]. Cdk5/p35 regulates neurotransmitter release through phosphorylation of CaV2.1 channels [178]. Disrupting the interactions between SNARE proteins and the synprint site of VGCCs inhibits neurotransmission [30]. Cdk5/p25 dependent phosphorylation of the intracellular loop connecting domains II and III (between residues 724 and 981) of the α1 subunit of CaV2.1 inhibits the interaction between the synprint site and SNARE proteins (Figure 3, Table 1), SNAP-25 and synaptotagmin I, which are required for neurotransmitter release. Therefore, Cdk5 inhibits neurotransmitter release through phosphorylation of CaV2.1 channel and down-regulation of the channel’s activity [178].

Inhibition of CaV2.2 channel activity has been shown to have a significant analgesic effect in humans [6], making these channels an important target for the treatment of acute and chronic pain. Under chronic pain conditions, there is an increase of CaV2.2 channel expression in primary afferent fibers and in the dorsal horn of the spinal cord [37]. As with CaV2.1, CaV2.2 are also substrate for Cdk5. The pore-forming α1 subunit of the CaV2.2 channel is phosphorylated within its C-terminal domain by Cdk5/p35. Interestingly, this phosphorylation results in enhanced Ca²⁺ influx due to increased channel open probability without affecting the surface expression of these channels [170]. Furthermore, inhibiting Cdk5 activity using a dominant-negative Cdk5, reduced CaV2.2 current density, which suggests that Cdk5 is responsible for the channel phosphorylation and increased Ca²⁺ current density [170]. To determine whether CaV2.2 channel phosphorylation by Cdk5 favors neurotransmitter release, primary neurons were transduced with a bicistronic herpes simplex virus (HSV) expressing wildtype CaV2.2 and the miniature excitatory postsynaptic currents (mEPSC) were recorded. An increase in the frequency of mEPSCs compared to those expressing a channel-less control (GFP HSV) was found [170]. This suggests that Cdk5-mediated phosphorylation of wildtype CaV2.2 modulates presynaptic function by enhancing vesicle release. Hence, unlike P/Q-type (CaV2.1) Ca²⁺ channels, phosphorylation of CaV2.2 by Cdk5 favors neurotransmitters release. The molecular mechanisms underlying the effects of Cdk5-mediated CaV2.2 channel phosphorylation on neurotransmitter release are mediated by an enhanced interaction between CaV2.2 channels and Rab interacting molecule 1 (RIM1) [170]. RIM1 directly binds and tethers CaV2.2 channels to the synaptic cleft to facilitate neurotransmitter release [70;77], this finding indicates that Cdk5-mediated phosphorylation of CaV2.2 may play a role in modulating CaV2.2 and RIM1 binding, thereby affecting neurotransmitter release (Figure 3, Table 1).

Another family of Ca²⁺ channels that are regulated by Cdk5 are the low-voltage gated CaV3 or T-type Ca²⁺ channels. T-type Ca²⁺ channels regulate neuronal excitability in the peripheral and central nervous system [57;192;198], hormone secretion [55] and are capable of associating with the synaptic vesicle release machinery [194], while their malfunction is related to certain pathological conditions among which is inflammatory and neuropathic pain [15;35]. All three members of T-type Ca²⁺ channels (CaV3.1–3.3) are present in the spinal dorsal horn [171] and in the DRG [15;101;171], albeit the most abundant isoform of T-type channels in small and medium diameter nociceptive neurons is CaV3.2 [171]. Despite the fact that Cdk5 regulates the activity of CaV3.1 channels [28], only CaV3.2 and CaV3.3 channels play an important role in the pathogenesis of neuropathic pain [15;196]. CaV3.2 channels are found at the central terminals of peptidergic and non-peptidergic fibers that synapse in the superficial laminae of the dorsal horn of the spinal cord. [15]. Their expression and activity is increased in DRG neurons and in the spinal dorsal horn in the SNL [57;210], CCI [63], spared nerve injury (SNI) [71], and partial sciatic nerve ligation [51] models of neuropathic pain. Over-expressing Cdk5/p35 in HEK-293 cells that stably express CaV3.2 channels increased Ca²⁺ current density, which could be prevented by inhibiting Cdk5 activity with olomoucine [57]. Site-directed mutagenesis showed that the relevant sites for this regulation are S561 and S1987, located in the α1 subunit of CaV3.2, resulting in increased membrane expression of these channels (Figure 3, Table 1). S561 is located in the central portion of the intracellular loop that connects repeats I and II, which play important functional roles in CaV3.2, including trafficking of the channels to the plasma membrane [187]. Importantly, just as CaV3.2 and Cdk5 interact in HEK-293 cells, these two proteins also interact in rat DRG [57]. CaV3.2, Cdk5 and p35/p25 expression are up-regulated in the DRG after SNL. In vivo inhibition of Cdk5 with olomoucine partially reversed mechanical allodynia and decreased the area of the compound action potential recorded from sensory C fibers from an injured spinal nerve-DRG-dorsal root of SNL rats [57], suggesting that the inhibition of Cdk5 activity decreases the excitability of nociceptive C fibers. Therefore, these data indicate that Cdk5-mediated phosphorylation of S561 and S1987, secondary to an exacerbated activity of Cdk5 after SNL, enhances the membrane localization and clustered distribution of the channels in the soma of sensory neurons consequently contributing to neuronal excitability in pain.

Collapsin response mediator protein 2 (CRMP2)

CRMP2 is a member of the collapsin response mediator protein family. CRMP2 was first identified as a mediator of semaphorin-induced growth-cone collapse and thus, became known to specify axon/dendrite fate, neurite growth and retraction, as well as axonal growth [54]. In the sensory system, CRMP2 RNA is expressed in DRG as well as in both dorsal and ventral horns of the spinal cord during the E13 stage of development, through the E15 stage of development, until birth where CRMP2 is observed at only low levels in the gray matter while maintaining expression in nearly all DRG neurons throughout development and into adulthood [189]. Accumulating evidence has charted the interactome of CRMP2 and shown that it acts mostly through interactions with tubulin, Sra-1, Numb, α2-chimaerin, phospholipase D, and most relevantly with CaV2.2 and the voltage-gated sodium NaV1.7 channel to affect microtubule dynamics, vesicle recycling, protein endocytosis, synaptic assembly and pain responses [22;54;73; 89; 127]. CRMP2 commonly undergoes post translational modifications to its unfolded C-terminal tail [5; 72; 139; 180]. These include phosphorylation by Cdk5 at S522 [20].

CaV2.2 channel expression can be modulated by CRMP2 binding via its Ca²⁺ channel binding domain 3 (CBD3) to two intracellular regions within CaV2.2 [20]. CRMP2’s interactions with CaV2.2 neurons have been found to enhance CaV2.2 channel surface expression (Figure 3, Table 1) and Ca²⁺ current in DRG [19; 53]. Consequently, when CRMP2 and CaV2.2 interaction is disrupted, neuropeptide release from sensory neurons, dependent on Ca²⁺ currents, is inhibited, and in turn, excitatory synaptic transmission in the spinal dorsal horn is inhibited [19]. Furthermore, a reduction in neuropathic pain responses has been observed [19; 53; 118; 140; 149; 208]. Evidence suggests that this interaction can be increased by Cdk5 phosphorylation of CRMP2 at S522 [180]. Confocal imaging and electrophysiological recordings of phosphomimetic (S522D) and phospho-null (S522A) mutants of CRMP2, demonstrated that Cdk5 phosphorylation of CRMP2 at S522, controls CaV2.2 localization [20; 209]. Cdk5/p25 phosphorylation of CRMP2 increases CRMP2’s binding to CaV2.2, ultimately resulting in a persistence of CaV2.2 membrane localization which results in enhancement of currents via CaV2.2 [20]. It also reduces CRMP2’s affinity to tubulin and alters neuronal functionality by halting neurite growth [39; 180]. Further, expressing CRMP2-S522D the spinal cord and DRG of naïve rats induced mechanical allodynia [118]. This suggests that phosphorylation of CRMP2 by Cdk5 is sufficient to elicit neuropathic pain by regulating CaV2.2 membrane localization and function [117; 119; 209].

The same relationship is found between CRMP2 phosphorylation by Cdk5 and NaV1.7 localization [117; 119; 209] (Figure 3, Table 1). Cdk5 is an obligatory priming kinase required for subsequent small ubiquitin-like modifier (SUMO)ylation of CRMP2 and other phosphorylation events [49; 117]. The binding between CRMP2 and NaV1.7 is enhanced by SUMOylation, and SUMOylation of CRMP2 is increased during neuropathic pain [117]. NaV1.7 is a voltage-gated Na⁺ channel highly expressed in nociceptive neurons. It is found in the DRG and trigeminal ganglion, as well as in sympathetic ganglion neurons [11; 17; 177; 193]. It is a fast-activating and inactivating channel with a slow repriming current (TTX-S) enabling it to act as a threshold channel to propagate action potentials in response to depolarizations of sensory neurons by noxious stimuli [79; 152]. Previous research has established NaV1.7 as necessary and sufficient for pain sensitivity [41; 45; 113; 125]. During neuropathic pain, NaV1.7 surface localization is increased, causing an increase in DRG excitability by amplifying subthreshold depolarizations [45]. Non-Cdk5 phosphorylated can trigger NaV1.7 internalization via recruiting the endocytic machinery complex made up of the protein Numb, the E3 ubiquitin ligase Nedd4–2, and epidermal growth factor receptor pathway substrate 15 (Eps15) [49]. Cdk5 phosphorylation of CRMP2 inhibits these interactions thereby protecting NaV1.7 from endocytosis. This unravels a novel mechanism that acts on clathrin-mediated endocytosis to regulate NaV1.7 expression and function. This has significant implications for most common cancer therapies like paclitaxel, which up-regulates NaV1.7 expression and induce peripheral neuropathy [94]. These reports suggest that on top of promoting CaV2.2 membrane localization, Cdk5 dependent phosphorylation of CRMP2 promotes NaV1.7 membrane localization, thus increasing neuronal excitability.

Neurofibromatosis type 1 is a condition characterized by benign tumors in the peripheral nervous system that also produces chronic pain. Rodent models of NF1 show an up-regulation in CaV2.2 and NaV1.7 channels. Further, they show increased levels of calcitonin gene-related peptide (CGRP) transmission to spinal dorsal horn neurons, demonstrating a facilitation of nociceptive signaling consistent with the pain reported by patients [9; 116]. Neurofibromin, the protein product of the NF1 gene which is mutated in patients with Neurofibromatosis type 1, is a Ras-GAP tumor suppressor [204]. It binds CRMP2 at its C-terminal domain, suppressing CRMP2 phosphorylation and activity. Suppression of NF1 by siRNA or CRISPR in turn up-regulated CRMP2 phosphorylation by Cdk5 [122; 137]. Inhibition of CRMP2 phosphorylation by Cdk5 (using (S)-Lacosamide) reversed the increased CaV2.2 and NaV1.7 functions after loss of Neurofibromin [122]. Consequently, NF1-related hyperalgesia was reduced in both male and female rats [122]. Similar effects could be achieved by targeting CRMP2 interaction with Neurofibromin with a blocking peptide [120; 121]. Increased Cdk5 phosphorylated CRMP2 was also found in a porcine model of NF1 [76]. Altogether, the relief of inhibition of CRMP2 phosphorylation by Cdk5 in rats with CRISPR-mediated loss of NF1 demonstrated that the subsequent increase of phosphorylated CRMP2 led to a concomitant gain of CaV2.2 and NaV1.7 functions. Consequently, DRG neuron excitability and spinal release of the nociceptive neurotransmitter CGRP were sensitized to elicit hyperalgesia in rats. Thus, underlying heightened pain sensations in patients with NF1.

Glutamate transporter and receptors

Glutamate is the major excitatory neurotransmitter in the central nervous system and plays a key role in pain processing. This neurotransmitter is transported into SVs by vesicular glutamate transporters (VGLUTs) prior to its release from excitatory synapses [10]. VGLUTs include proteins VGLUT1–3. Among these, VGLUT2 is expressed in primary afferent neurons [158] and participates in mediating pain hypersensitivity induced by inflammation and peripheral nerve injury [150; 158]. This glutamate transporter co-localizes with CGRP and TRPV1 in small and medium size DRG neurons. Additionally, conditional knockout mice in which expression of VGLUT2 is disrupted specifically in nociceptors, have reduced firing of lamina I spinal cord neurons in response to noxious heat and in a model of nerve injury-induced neuropathic pain, the magnitude of heat hypersensitivity was diminished in these mice [158]. Notably, co-expression of Cdk5 and VGLUT2 is elevated in small- and medium-diameter L4-L6 DRG neurons and in L4-L6 spinal cord segments after intraplantar injection of CFA. Similarly, the expression of p25, but not p35, is increased in the spinal cord. Moreover, intrathecal injection of roscovitine decreased VGLUT2 protein expression in the DRG and in the spinal cord as well as p25 [172]. Hence, Cdk5 can modulate neurotransmitter release in nociceptors by closely interacting with VGLUT2 (Figure 3, Table 1).

NMDARs are glutamate ligand-gated ion channels widely expressed in the central nervous system [115]. These receptors are heterotetramers composed of the ubiquitous GluN1 subunit and either GluN2 (A–D) and/or GluN3 (A and B) subunits. At most synapses, functional NMDA receptors are formed by two GluN1 and two GluN2 or GluN3 subunits [83]. In the sensory system, GluN1 and GluN2B are expressed in nociceptive neurons [105; 111; 157], whereas GluN1 has been found in the central terminals of primary afferent fibers [97]. Nevertheless, GluN1, GluN2A, GluN2B and GluN2D are located in lamina I and II of the dorsal horn [61; 162; 169; 211] and are involved in the development of chronic pain such as inflammatory and neuropathic pain [78; 182]. Ca²⁺ and Na⁺ influx through NMDARs modulate synaptic plasticity by increasing intracellular Ca²⁺ concentration and depolarizing the synaptic membrane to generate excitatory postsynaptic potentials (EPSP) [16]. The Ca²⁺ influx rise activates different kinases including PKA, PKC, ERK, CaMKII, and Cdk5, which leads to a phosphorylation-mediated increase in channel open probability of excitatory NMDARs and modulation of its trafficking [131; 160; 190; 214], this subsequently leads to central sensitization [74] which is an important phenomenon involved in chronic pain states responsible of mediating allodynia, and primary and secondary hyperalgesia [85].

Cdk5 can associate with and phosphorylate NMDARs on the S1232 of the GluN2A subunit and this phosphorylation can be inhibited by roscovitine [92]. Notably, the Cdk5-GluN2A pathway plays a relevant role in neuropathic pain. In a rat model of chronic compression of dorsal root ganglion (CCD) it was found that the expression levels of GluN2A subunit were significantly increased in the DRG [206]. Moreover, after intrathecal administration of roscovitine, the expression levels of GluN2A in the DRG significantly decreased, and this was accompanied by a reduction in nociceptive behavior [206]. Similarly, phosphorylation of GluN2A at S1232 was significantly up-regulated in the spinal dorsal horn after intraoperative infusion of remifentanil in a rat model of postoperative pain [102]. Furthermore, these increases were attenuated by pretreatment with roscovitine [102]. LTP in the hippocampus is the strengthening of synaptic efficacy that is considered to be a basic mode of learning and memory [12], in nociceptive pathways, constitutes a form of synaptic facilitation that leads to pain amplification following trauma, inflammation, and nerve injury [156]. The best understood form of LTP is induced by the activation of the NMDARs. Mice lacking GluN2A subunit show defective LTP and impaired spatial memory [155], indicating that GluN2A is an important modulator during LTP. Inhibition of Cdk5 activity with olomoucine, prevents induction of LTP in CA1 pyramidal neurons of rats [92]. Therefore, this evidence indicates that the observed changes of NMDAR activity in neurons are caused in part by GluN2A subunit phosphorylation by Cdk5 and could lead to the amplification of pain (Figure 3, Table 1).

GluN2B subunit has an important function in spinal dorsal horn neurons. It has been reported that this subunit is up-regulated in the superficial dorsal horn in a CCD model [215]. Selective GluN2B receptor antagonists produced analgesia in rat models of inflammatory and neuropathic pain [144; 215] by decreasing the expression of GluN2B in the spinal cord [215] and by inhibiting LTP in the C-fiber responses of wide dynamic range neurons [144], which indicate that activation of the dorsal horn GluN2B receptors are crucial for the spinal nociceptive synaptic transmission and for the development of long-lasting spinal hyperexcitability. Interestingly, phosphorylation of GluN2B was significantly up-regulated in spinal dorsal horn after intraoperative infusion of remifentanil in a rat model of postoperative pain [102]. Pretreatment with roscovitine in these rats reduced the up-regulation of GluN2B phosphorylation in the spinal cord and alleviated mechanical allodynia and thermal hyperalgesia [102], suggesting that Cdk5-mediated phosphorylation of GluN2B subunit is an important mechanisms underlying nociception.

In addition to NMDAR, metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors, modulate synaptic transmission through a variety of intracellular second messenger and play an important role in nociceptive processing and central sensitization [46; 147]. mGluRs can be divided into group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) receptors. The activity of serine-threonine kinases has been shown to greatly impact the activity of group I mGluRs [132]. This group of receptors is often located at the periphery of synaptic zones, where they are clustered and tethered to the postsynaptic terminal by their interaction with homer proteins which help create signaling complexes that regulate mGluR signal transduction [48; 50]. Furthermore, in vitro kinase assays have revealed that the homer-binding domain of mGluR5 contains active Cdk5 phosphorylation sites [132]. While no Cdk5 phosphorylation consensus sequence exists in the intracellular loops of mGluR1 or mGluR5, phosphorylation occurs in the most distal half of the C-terminal of mGluR5, specifically at T1164 and S1167, and this phosphorylation enhances mGluR5 binding with homer [132]. Using a phosphospecific mGluR antibody, it was shown that the homer-binding domain of both mGluR1 and mGluR5 is phosphorylated in vivo at S1167, and that inhibition of Cdk5 with siRNAs decreases the amount of phosphorylated receptor [132]. Thus, Cdk5- mediated phosphorylation of mGluRs might alter synaptic plasticity by enhancing mGluR1 and mGluR5-Homer interaction and cell-surface localization (Figure 3, Table 1). Intraoperative infusion of remifentanil in a rat model of postoperative pain up-regulated the expression of Cdk5, p35 and p25 in the spinal dorsal horn and favored the phosphorylation of mGluR5 at S1167. In the same way remifentanil significantly enhanced mechanical allodynia and thermal hyperalgesia induced by plantar incision. This up-regulation was decreased by intrathecal pretreatment with roscovitine at the same time that attenuated nociceptive responses [104]. Overall, these findings have shown that Cdk5 may contribute to nociceptive processing via regulating glutamate release, and phosphorylation of NMDAR and mGluRs in the spinal dorsal horn.

Purinergic Receptors

Adenosine 5’-triphosphate (ATP) is a key sensory signaling molecule that activates purinergic P2X ligand-gated ion channel receptors and P2Y G-protein coupled receptors. Unlike P2Y receptors, P2X receptors are nonselective cation channels permeable to Ca²⁺, Na⁺, and K⁺ [128]. There are seven P2X receptor subunits (P2X1–7) that can be assembled as homomeric or heteromeric complexes [75]. The activation of these receptors by ATP is implicated in a wide range of physiological processes including pain [24]. During inflammation or tissue damage, there is a significant increase in extracellular ATP levels [82] which is consistent with the implication of P2X receptors in chronic inflammation and neuropathic pain [24]. P2X2 receptor (P2X2R) is one of the most abundant subtypes in the nervous system and its subunits can form homo or heteromeric channels with P2X3 subunits, which are expressed in nociceptive neurons and whose activation is involved in pain signaling [31; 91]. Previous studies have documented that phosphorylation of P2X2R by PKA, PKC [14] and Cdk5 [38] regulate its gating properties. Cdk5/p35 complex interacts with P2X2R in heterologous overexpression systems, in primary cultures of trigeminal ganglion and DRG neurons, and in adrenal pheochromocytoma (PC12) cells that endogenously express Cdk5/p35 and P2X2R [38]. Several functional splice variants of P2X2R have been described [128]. P2X2aR is the full-length variant that desensitizes slower than the shorter variant P2X2bR which lacks a 69 amino acids in the intracellular C-terminal domain [18; 163]. Cdk5 regulates P2X2R activity by phosphorylating T372, which is exclusively present in the full-length P2X2aR, but absent in P2X2bR [38]. Moreover, Cdk5 mediated phosphorylation of T372 does not affect the number of functional channels but it can prevent receptor desensitization (Figure 3, Table 1) after repetitive ATP applications in both heterologous and endogenous systems [38], suggesting that Cdk5 could increase the activity of P2X2aR during pain conditions.

Contrary to P2X2aR, Cdk5 activation seems to down-regulate P2X3R function (Figure 3, Table 1) [124]. P2X3 subunits, in addition to forming heteromeric channels with P2X2 subunits, form homomeric complexes [38]. P2X3R are primarily expressed in the trigeminal ganglion and DRG sensory neurons [42] where they are found mainly on small- and medium-sized neurons [52]. Cdk5/p35 complex induces an increase in serine phosphorylation of P2X3R without affecting total P2X3R expression [124]. On the other hand, HEK-293 cells co-transfected with Cdk5, p35, and P2X3 showed that P2X3R currents evoked by a selective P2X3 agonist were smaller than those recorded from HEK-293 cells expressing P2X3R alone [124]. This evidence shows that Cdk5 down-regulates P2X3R function and that the opposing regulation of P2X2R and P2X3R by Cdk5 might regulate heteromeric P2X2/3R, an important complex expressed in nociceptive neurons, differently. Although the in vivo regulation has not been investigated, Cdk5 activity by down-regulating P2X3 function is predicted to bias pain perception.

Delta opioid receptors

It is known that opioid analgesics like morphine, are the most effective analgesic in the clinic. However, morphine use is limited by the development of tolerance and dependence, processes that are related to opioid addiction. Although morphine acts primarily via μ-opioid receptor (MOR) [112], evidence indicates that δ-opioid receptor (DOR) is also critical for the development of morphine antinociceptive tolerance [220]. Interestingly, the up-regulation of DOR, either by inflammation or by morphine application, has been found in vivo to involve activation of Cdk5. The regulation of DOR by the kinase seems to negatively regulate MOR receptor functions [8]. On the other hand, intraplantar injection of CFA or capsaicin promotes the trafficking of DOR to the plasma membrane of DRG neurons [25]. Under conditions of inflammatory injury, DOR and MOR mRNA and protein levels are increased in the ipsilateral dorsal horn [26; 107]. Chronic morphine treatment up-regulates DOR [114], and DOR knockout mice do not exhibit morphine tolerance [220]. Likewise, MOR–DOR heterodimerization has been shown to play an important role in the development of tolerance [56]. Phosphorylation of opioid receptors is the first step in opioid receptor activation and the protein kinases responsible for agonist-induced phosphorylation of DOR include PKA, PKC, CaMK-II, G-protein coupled receptor kinase (GRK) and MAPK [100]. In addition, a growing body of evidence suggests that Cdk5/p35 plays an important role in morphine tolerance [136; 201]. Transgenic p35 mice (Tgp35), with elevated levels of Cdk5 activity are more susceptible to opioid tolerance as compared to p35 knockout mice which exhibit significantly decreased Cdk5 activity [136]. Intrathecal injection of roscovitine prevented the development of morphine tolerance and enhanced morphine’s antinociceptive effect in tolerant rats [201]. Moreover, inhibiting Cdk5 activity in NG108–15 cells, which endogenously expressing only DOR but not MOR, attenuates the DOR-mediated Ca²⁺ influx [201]. Interestingly, inhibiting and/or silencing Cdk5 activity decreases the bioavailability of DOR on the cell surface without affecting the total expression level of DOR protein [201]. It is worth noting that the single putative Cdk5 phosphorylation site is at T161 which is located in the second intracellular loop, and Cdk5 phosphorylates this residue in DRG neurons [201]. Importantly, phosphorylated DOR at T161 in DRG neurons increases in both the surface-localized and the total amount in CFA-induced inflammatory pain [32], suggesting that this might be involved in CFA-induced hypersensitivity. Conversely, Cdk5 does not phosphorylate the corresponding residue in MOR, yet it is required for the formation of DOR–MOR heterodimers [201]. Therefore, in addition to the C-terminal, T161 of the second intracellular loop domain of DOR is required for trafficking and morphine antinociceptive tolerance [148; 220] and several studies have found that blocking DOR attenuates this tolerance [151; 220]. Cdk5 activity and expression increase after morphine treatment, which then increases DOR phosphorylation level at T161. Intrathecal delivery of a construct expressing T161A mutant of DOR, attenuated morphine antinociceptive tolerance in rats, suggesting that T161 phosphorylation of DOR contributes to Cdk5-mediated morphine antinociceptive tolerance [201]. Chronic morphine treatment induces the translocation of DOR from intracellular compartments to plasma membranes, enhancing the number of functional receptors [25]. Furthermore, intrathecal injection of an engineered Tat fusion-interfering peptide corresponding to the second intracellular loop of DOR reduces the cell surface expression of DOR, disrupts the formation of DOR–MOR heterodimers, and attenuates the development of morphine antinociceptive tolerance [201]. Similarly, intrathecal delivery of the Tat fusion-interfering peptide increased inflammatory hypersensitivity, and inhibited DOR- but not MOR-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of this peptide postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain [32]. Overall, these data indicate that Cdk5-mediated phosphorylation of DOR at T161 enhances its membrane localization and function (Figure 3, Table 1), thus mediating the development of morphine antinociceptive tolerance.

Summary

The present review provided information regarding Cdk5-mediated regulation of several proteins that are known to play an important role in neuropathic pain. These molecular targets range from cytoplasmic proteins and integral membrane proteins to protein complexes like ion channels and membrane receptors. In this review, we also sought to highlight Cdk5’s regulation which is driven primarily by three mechanisms: phosphorylation of Cdk5, increasing p35 interaction with Cdk5, and proteolytic cleavage of p35 into p25 by calpain. Although its regulation has been extensively studied, it is important to point out that in neuropathic pain conditions, the main cause of exacerbated Cdk5 activity is its association with its activator p25. The co-complex stabilizes the kinase consequently increasing its function, changing its cellular location, and altering its substrate specificity.

The evidence presented in this review also shows that Cdk5 interacts with proteins like synaptophysin and VGLUT2 and increases their expression, but the possibility that this could be the result of a direct effect of Cdk5 on these proteins is unlikely since Cdk5 seems not to affect the activity of its targets by a mechanism independent of its kinase activity. Finally, evidence has been presented to demonstrate that although Cdk5 enhances the function of most of its targets, in a few cases such as for CaV2.1 and P2X3R, the opposite occurs. However, the overall result of Cdk5-mediated regulation of its targets is to enhance pain hypersensitivity, either by (i) enhancing the trafficking of proteins to the plasma membrane, (ii) by increasing their expression and activity, (iii) by promoting the interaction of its target proteins with others and thus enhancing their function or (iv) by aiding in the development of morphine nociceptive tolerance. Finally, the trafficking of Cdk5 substrates to the plasma membrane seems to be the main effect of Cdk5-mediated phosphorylation of these proteins. It is envisioned that via this action Cdk5 contributes to the development and maintenance of neuropathic pain.