α2δ in calcium channel localization and function

α2δ subunits are important for trafficking VGCC α1 subunits to the plasma membrane (Figure 1), a key step in regulating calcium conduction at the neuronal surface [4]. There are four α2δ isoforms encoded by four distinct genes (Cacna2d1, Cacna2d2, Cacna2d3, andCacna2d4), each encoding a single polypeptide that is later cleaved into α2 and δ fragments post-translationally [6]. In a well-known sensory circuit in spinal cord, α2δ-1 directs Ca_v2.2 (an N-type VGCC) to the surface of dorsal root ganglions (DRGs) as well as to presynaptic terminals in the dorsal horn [7]. Conversely, genetic knockout (KO) of α2δ-1 significantly inhibits Ca_v2.2 expression in this pathway. In rat superior cervical ganglion neurons, Ca_v2 membrane expression is differentially regulated by α2δ subunits, with α2δ-1 and α2δ-2 promoting the displacement of Ca_v2.2 with Ca_v2.1 (α2δ-1 also allowed Ca_v2.2 to 2.3 displacement), while α2δ-3 protects Ca_v2.2 from removal [8]. Ca_v2.2 may therefore act at the presynapse of newly formed or developing connections and be replaced during maturation to regulate synaptic strength in an α2δ-dependent manner [9]. In addition to binding α2δ, Ca_v2.2 coimmunoprecipitates with the α2δ-like protein, Cachd1, which increases Ca_v2.2 surface expression and current by reducing its endocytosisin vitro [10]. The interaction with Cachdl inhibits Ca_v2.2 binding to α2δ-1, suggesting a regulatory mechanism to keep Ca_v2.2 in the membrane. α2δ-1 is prevented from interacting with Ca_v2.2 through its association with BK channels (i.e. large conductance, calcium-activated potassium channels), though this study showed the effect of reduced Ca_v2.2 surface levels when α2δ was sequestered by BK [11]. The distinction between the two outcomes may potentially be explained by functional differences provided by the proteolytic cleavage of α2δ subunits, as uncleaved α2δ (or ‘pro-α2δ’) actually inhibits native Ca²⁺ currents [12]. Cleavage into the separate α2 and δ peptides is required to permit the trafficking of mature Ca_v2.2 to the synapse and subsequently allow Ca²⁺ entry, creating a posttranslational gating mechanism for the trafficking and stabilizing properties of α2δ. Interestingly, calcium permeability is not a prerequisite for α2δ interactivity, as selectivity filter Ca_v2.2 mutants impermeable to calcium were still shown to bind to and traffic with α2δ subunits [2].

In addition to regulating the localization of calcium channels, α2δ are highly involved in tuning specific channel properties. In Ca_v1.2 (L-type) VGCCs, α2δ causes a structural rearrangement that increases the coupling of voltage-sensing domains I–III, but not IV, to the channel pore [13]. This shift then permits channel operation in the physiological membrane potential range of excitable cells, thereby favoring the active state of the channel. To investigate whether increased α2δ-1, as can result from nerve injury, contributes to increased calcium influx at physiological membrane potentials, Alleset al. [14] performed chronic sciatic constriction injury in rats and observed decreased spontaneous excitatory postsynaptic currents (EPSCs) by blocking L-type VGCCs. α2δ-transfected HEK293F cells treated with the antiepileptic, anti-pain α2δ-1 ligand, gabapentin (GBP), showed reduced Ca_v1.2/α2δ-1 peak Ca²⁺ currents, consistent with an α2δ-1-mediated increased influx through L-type channels [14]. α2δ subunits may also influence calcium dynamics by regulating a developmental VGCC switch, which has been demonstrated at several central synapses including the calyx of Held [15]. At postnatal (P5), before the onset of hearing at P12, genetic silencing of α2δ-3 in spinal ganglion neurons results in decreased Ca²⁺ currents through L-type and N-type channels. However, the channel selectivity switches with maturity by P20, with compromised P/Q-type and R-type channel activity.

Control of functional α2δ expression appears to be governed, at least in part, by the neurexin (Nrxn) family of synaptic cell adhesion molecules. In hippocampal neurons from αNrxn-1,2,3 triple KO mice, increased α2δ-1 surface mobility on axons accompanies reduced presynaptic Ca²⁺ influx, lower Ca_v2.1-mediated transients, lower vesicle release, and decreased synaptic levels of Ca_v2.1 pore-forming subunits [16••]. Co-transfection of Nrxn1α/α2δ-1, and not α2δ-1 alone or Nrxn1α/α2δ-3, rescued Ca_v2.1 transients in the triple KO, showing isoform specificity with αNrxns having opposite effects on α2δ-1/α2δ-3 surface mobility. The link between neurexins and α2δ is evolutionarily conserved, with postsynaptic NRX-1 binding presynaptic UNC-36/α2δ inCaenorhabditis elegans [17]. α2δ-1 surface mobility is also affected through its interaction with low density lipoprotein receptor-related protein 1 (LRP1), which is gated by the LRP chaperone receptor associated protein (RAP) [18]. By itself, LRP1 reduces α2δ-1 surface trafficking in tsA-201 cells, but LRP1/RAP co-expression increases α2δ-1 surface expression, N-linked glycosylation and proteolytic cleavage. Furthermore, interaction with LRP1 may prevent the inhibitory effects of GBP and pregabalin (another anti-epileptic drug with binding sites on α2δ-1 and α2δ-2), adding another level of regulatory control [18].

α2δ and the formation and function of synaptic connectivity

Though long known for regulating calcium channel expression in synaptic membranes, α2δ subunits have only recently been identified as important regulators of synaptic connectivity in the nervous system, particularly by serving as receptors for secreted proteins. Astrocytes, the most abundant glial cell in the brain, are essential participants in developmental synapse formation via the secretion of factors that either promote or inhibit this process [19–21]. The first of these prosynaptogenic factors secreted by astrocytes to be identified were the thrombospondin (TSP) family of matricellular proteins, which were subsequently found to induce their synaptogenic effects via α2δ-1 [22–24]. These newly formed synapses were characterized as postsynaptically silent, owing to a lack of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors on the postsynapse. Curiously, the involvement of α2δ-1 in promoting excitatory synapse formation was found to be independent of the subunit’s role in regulating L-type Ca²⁺ currents [23]. Instead, Risheret al. [25•] showed that the synaptogenic capabilities of α2δ-1 in the cortex were due to two specific events (Figure 2): in the initial step, α2δ-1 resides in the postsynaptic membrane of immature neuronal processes called dendritic filopodia that actively search for suitable presynaptic partners. TSP released from astrocytes binds to α2δ-1 on these filopodia, triggering the clustering of presynaptic and postsynaptic machinery to form structural synapses. In a subsequent step, the intracellular C-terminal domain of activated α2δ-1 triggers a downstream signaling cascade in the newly formed dendritic spine to activate the small Rho GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1). Activated Rac1 then promotes actin cytoskeletal reorganization and the subsequent growth and maturation of the spine [25•].

In the cochlea, α2δ-2 appears to play a similar role in the formation of inner hair cell synapses [26], promoting trans-synaptic alignment of presynaptic Ca²⁺ channels and postsynaptic PSD95 and AMPARs (the latter of which may be brought to the membrane via other methods, given their absence in previously described TSP/α2δ-induced synapses). Localization of α2δ for synaptogenesis may vary throughout the CNS, as Yuet al. [27] showed in the spinal cord that presynaptic, not postsynaptic, α2δ-1 interacts with TSP4 to induce synaptogenesis in DRGs. Furthermore, blocking the activity of T-type (i.e. low-voltage-activated) Ca²⁺ channels, which are highly expressed in primary afferents and involved in injury-induced hypersensitivity, prevents this TSP4/α2δ-1-mediated synaptogenesis. In hippocampal neurons, soluble Aβ peptide, which is associated with synaptic dysfunction in Alzheimer’s disease (AD) in an early pathogenic event in the disease, induced the downregulation of α2δ-1 and the synaptic adhesion molecule neuroligin-1 (NLGN1) [28]. This effect could be reversed by co-treatment with TSP1. Similarly, the authors used TSP1 treatment in the 5XFAD AD mouse model (which overexpresses several AD mutations and recapitulates many aspects of AD, including increased levels of Aβ) to decrease Aβ levels and restore α2δ-1 and NLGN1 expressionin vivo. It should be noted that, in this experiment, the source of TSP1 was human umbilical cord blood-derived-mesenchymal stem cells (hUCB-MSCs).

Compared to excitatory synaptogenesis, the effects of TSP/α2δ signaling on inhibitory synaptic formation are not well-understood. However, a recent study from Geisleret al. [29••] showed that presynaptic overexpression of α2δ-2, but not α2δ-1 or α2δ-3, in cultured mouse neurons results in increased postsynaptic gamma-aminobutyric acid (GABA)-A receptor (GABA_AR) clustering. Curiously, this result held true in both inhibitory GABAergic synaptic populations (i.e. medium spiny neurons of the striatum), as expected, but also in excitatory glutamatergic synapses (i.e. hippocampal pyramidal neurons). The latter situation resulted in a mismatch between an excitatory glutamatergic presynaptic axon terminal and a GABA_AR-enriched postsynaptic dendrite. This mismatched synaptogenesis was further enhanced in the absence of αNrxn, suggesting that the balance between αNrxn and α2δ-2 can regulate GABA_AR abundance. Whether this aberrant synaptogenesis also occurs during normal physiological development or in disease remains to be investigated.

Axon growth is a vital process in synaptic network formation which, along with the related injury response of axon regeneration, may also be regulated by α2δ. In both the CNS and peripheral nervous system (PNS), α2δ-2 has been shown to limit the capacity for axon growth and regeneration. Deletion or silencing of Cacna2d2 creates a permissive environment for axon growthin vitro, while the administration of the inhibitory α2δ ligand pregabalin enhanced axon regeneration after spinal cord injury (SCI) in vivo [30]. This study showed specificity for α2δ-2 in these effects, as overexpressingCacna2d1 orCacna2d3 had minimal outcome on axon growth. However, α2δ-3 was implicated in the development of somatosensory cortex, where α2δ-3 global KO mouse neurons showed increase axon process growth with a concomitant decrease in dendrites [31].

Apart from brain and spinal cord, one CNS structure in which α2δ signaling has been shown to be critically important for development and function is the retina. In a retinal culture of elevated hydrostatic pressure, TSP2 from glial fibrillary acidic protein (GFAP)-positive macroglia induced presynaptic (synapsin) but not postsynaptic (PSD95/Homer/gephyrin) expression in an α2δ-1-dependent manner [32]. Furthermore, Kohet al. [33] recently demonstrated reduced levels of α2δ-1 in the retina in the Royal College of Surgeons (RCS) rat model of inherited retinal degeneration. Müller glia, the predominant source of secreted TSPs in the retina, were also observed with a reactive morphology and lower expression levels of these synaptogenic factors in the RCS rat. Transplantation of human umbilical tissue-derived cells (hUTCs), previously shown to induce synaptogenesis via TSP [34], was able to attenuate the Müller glia reactivity and preserve α2δ-1-expressing synapses [33]. At photoreceptor ribbon synapses, α2δ-4 was found to be necessary for clustering Ca_v1.4, and reduced PSD95, which is presynaptically located at these synapses, was observed in α2δ-4 KO mice [35]. Other findings from these mice included the presence of ectopic ribbons, bipolar cell sprouting, and structurally abnormal cone synapses; all of which were suggested to contribute to abnormal cone transmission through ON and OFF bipolar cell signaling. In the human retina, loss-of-function α2δ-4 mutations are associated with nonprogressive cone dysfunction, with minimal effect on rod photoreceptor cells [36]. However, another study using α2δ-4 KO mice did observe rod dysfunction, with disrupted rod localization and function of the calcium-activated chloride channel, TMEM16A [37]. TMEM16A associates with the α1 subunit of VGCCs and is therefore affected by the abolishment of α2δ-4-mediated VGCC trafficking.

α2δ signaling can have varied effects on synaptic transmission. In cultured hippocampal neurons, presynaptic vesicle release is inhibited by uncleaved α2δ [38•]; maturation of the α2δ subunit requires proteolytic cleavage before permitting the trafficking of Ca_vs to presynaptic terminals, thereby facilitating synaptic transmission. ADrosophila study by Wanget al. [39] revealed that α2δ-3 is required for the phenomenon of presynaptic homeostatic potentiation (PHP), which is a change in presynaptic vesicle release that is initiated by disruption of receptors on the postsynaptic surface. Specifically, α2δ-3 is necessary for the homeostatic expansion of the readily releasable vesicle pool (RRP) required for PHP. Tissue-specific re-expression of presynaptic α2δ-3 is sufficient to rescue baseline transmission and permit the rapid induction of PHP [39]. Another synaptic plasticity mechanism affected by α2δ signaling is long-term potentiation (LTP), as shown by a recent study using theta-burst stimulation of the corticostriatal circuit [40•]. Pharmacological blockade of α2δ-1 (via GBP) or the interaction between α2δ-1 and NMDARs (with an α2δ-1 C-terminus peptide) abolishes corticostriatal LTP in this paradigm. In addition, ablation/inhibition of α2δ-1 in the striatum resulted in impaired alternation T-maze and rotarod performance [40•]. In sensory (DRG) neurons lacking α2δ-1, action potentials (AP) show reduced duration owing to a reduction in intracellular Ca²⁺ elevations [41]. Attenuating Ca²⁺ buffering in the WT resulted in decreased AP duration and frequency of AP firing, mimicking the α2δ-1 KO. Additionally, α2δ-1 showed specificity in its subcellular localization in DRGs, with the effect of the α2δ-1 KO being greater in the proximal segment of the main axon than in the cell body [41].

Circuit-specific investigations of α2δ have led to novel discoveries with regard to the generation and maintenance of ’normal’ network function throughout the CNS. In the hippocampus, chronic treatment with pregabalin, which is another small molecule drug that antagonizes α2δ signaling, reduces intrinsic excitability of dentate gyrus granule cells (DGGCs). In newborn DGGCs, pregabalin increases the expression of mature neuronal markers (NeuN, Calbindin) at the expense of immature ones (doublecortin/DCX), while also increasing the AMPA to NMDA ratio (typically indicative of functional maturation) [42]. Knockout of α2δ-3 affects auditory pathways, with impairments in audition/vocalization, pinna reflex, and object-based memory with the novel object recognition (NOR) task [43]. Intriguingly, despite no deficits in primary vision or olfaction, the α2δ-3 KO mice performed demonstrably better on the visible food pellet test, a multiple sensory task requiring integration of sight and smell; the authors interpreted this result as a possible mechanism for α2δ in the development of synesthesia (a condition of multisensory perception) in humans. Hypothalamus has also emerged as a significant area in α2δ research, with a 2014 study showing reduced α2δ-1 surface expression in the ventromedial hypothalamus (VMH) of brain-derived neurotrophic factor (BDNF) haploinsufficient mice [44]. Since then, α2δ-1 has been shown to regulate glucose and lipid balance in steroidogenic factor-1 (SF1) neurons in the VMH, with α2δ-1/SF1 conditional double KO neurons having deficient glucose and lipid homeostasis associated with decreased cholesterol content in white adipose tissue [45]. Reduced EPSCs were detected in the α2δ-1/SF1 double KO neurons without a change in normal Ca²⁺ currents. Evoked NMDAR-mediated currents have increased basal amplitude in spontaneously hypertensive rats, which also show higher protein expression of α2δ-1 and NMDARs in the paraventricular nucleus of hypothalamus than normotensive controls [46]. NMDAR currents were normalized with GBP-mediated inhibition of α2δ-1 signaling or by treatment with an α2δ-1 C-terminus peptide to inhibit the α2δ-1/NMDAR interaction.

Roles of α2δ in injury, disease, and addiction

As the importance of α2δ has become increasingly apparent for normal synaptic and network function, the implications of dysfunctional α2δ signaling in disorders of the nervous system continue to expand (seeTable 1 for summary). Many of the investigations into α2δ and disease up to this point have involved the conditions of chronic or neuropathic pain, with a recent review article [47] highlighting the importance of the α2δ-1 subunit in the pathology and treatment of pain. New evidence continues to come to light for α2δ in pain mechanisms, including the importance of the α2δ-1/NMDAR heteromeric interaction for the development of pain hypersensitivity [48]. Furthermore, recent therapeutic advances continue to probe the benefits afforded by blockade of α2δ signaling, as the novel gabapentinoid medication mirogabalin has shown promise in enhancing potency and analgesic duration following sciatic nerve ligation (SNL) [49] and in the attenuation of tactile allodynia and anxiety after chronic constriction injury [50].

α2δ signaling has also been linked to numerous neurodevelopmental and neurodegenerative conditions, including epilepsy, stroke, and various psychiatric disorders. Increased epileptiform activity is accompanied by behavioral arrests in α2δ-1 overexpressing mice, with prolonged bursts of polysynaptic EPSCs recorded from Layer V somatosensory pyramidal neurons [51]. Maladaptive circuit reorganization in this cortical layer is also associated with epileptogenesis in a rat model of neocortical trauma [52]; a brief postinjury treatment with GBP was able to suppress excitatory synaptogenesis/epileptogenesis for up to two weeks, but later time points have yet to be assessed. Linkages for genes encoding each of the four α2δ subunits, as well as their associated L-type VGCCs, have been found for most major psychiatric disorders including bipolar, schizophrenia, autism, and major depressive disorder [53,54]. In stroke studies, the α2δ-1/NMDAR interaction is once again cited as a major contributing factor to injury and recovery [55•]. Systemic treatment with GBP, α2δ-1 C-terminus interfering peptide, or genetic α2δ-1 ablation all attenuated infarct volume and improved neurological deficit scores at 24 hours after injury. A longer-term study in BDNF mutant mice (Val66Met) showed increased TSP2 and α2δ-1 mRNA (and α2δ-1 protein) expression in the contralateral hemisphere at six months post-ischemic stroke [56]. These changes were accompanied by increased axospinous excitatory synapses in the contralateral dorsal striatum, implicating aberrant TSP/α2δ-induced synaptic rewiring.

Recent years have seen a growing emphasis on the possible involvement of α2δ in the development of addiction and consequences of substance use. Recent genome-wide association studies (GWAS) for nicotine dependence have implicated bothCacna2d1 andCacna2d3 [57,58], with a genotype-tissue expression study in human brain finding protection against nicotine dependence associated with reducedCacna2d3 mRNA [57]. Alcohol studies suggest that the long-lasting maladaptive synaptic remodeling observed in hippocampus following binge ethanol consumption during adolescence [59] may be attributable to aberrant TSP/α2δ-1 signaling [60]. Opioid studies show similar α2δ involvement in synaptic changes, as α2δ-1 is required for the increased presynaptic NMDAR activity associated with hyperalgesia following chronic morphine [61]. Genetic or pharmacological inhibition of α2δ-1 was shown to reduce hyperalgesia and opioid tolerance. Finally, in a recent meta-analysis investigating misuse of gabapentin, up to 65% of patients currently prescribed GBP reported misuse of the drug (defined as ‘use of [the] drug in a manner or for a purpose other than indicated’, including non-recommended routes of administration or dosages), while 15–22% of opioid users reported co-abuse of GBP [62]. As the rates continue to climb (as of this writing, 5 U.S. states have classified GBP as a Schedule V controlled substance, with several others listing it as a ‘substance of concern’), so too does the incidence of neonatal abstinence syndrome (NAS) amongst children born to mothers who used GBP during pregnancy. Pediatricians have identified a unique clinical presentation of NAS in infants whose mothers co-abused opioids and GBP, with average length of stay for these infants nearly doubled compared to those who were not exposed to GBP [63]. Given the extensive functions of α2δ subunits in development of the nervous system, the prospect of inhibiting them perinatally via maternal GBP abuse is alarming and may indicate significant lifelong clinical challenges for children who are exposed to GBP at such a critical period of nervous system development.