Ca
2+ serves as a critical second messenger in neuronal signaling and can exert both positive and negative effects on neurite regeneration, involving a variety of proteins that transduce Ca
2+ signals for neurite growth. Several identified Ca
2+-binding proteins play a crucial role in controlling Ca
2+ concentrations and may be essential for locally manipulating cellular calcium to facilitate neuronal repair. However, their neuron-specific expression and processes triggered following traumatic damage can lead to variations in their neuroregenerative properties and neurite growth dynamics. Within the category of calcium-binding proteins, there is a subset of EF-hand proteins related to calmodulin (CaM), which are highly expressed in the neuronal cell types of the brain. The EF-hand (helix-loop-helix motif) is one of the most frequently observed domains, and over 1000 have been identified based on their unique sequence signatures
[93][66].
4.1. Oncomodulin
Oncomodulin (OCM) is a small Ca
2+-binding protein (CaBP) belonging to the parvalbumins family, which are classical, small, mostly cytosolic and EF-hand-containing motifs molecules
[94][67]. The EF-hand motif is characterized by an α-helix-loop-α-helix arrangement spanning approximately 30 amino acids, responsible for Ca
2+ binding and triggering a conformational change leading to the activation or inactivation of target proteins
[95][68]. OCM, as a member of the parvalbumin family, displays distinct characteristics in its ability to bind metal ions and sense calcium, resulting in functional differences when compared to other family members. Of the three domains in oncomodulin, EF1 cannot bind calcium ions due to the absence of key residues, but plays a vital role in OCM’s structural stability, EF2can bind a single Ca
2+ and is characterized by a higher specificity for calcium ions, while EF3 accommodates both Ca
2+and Mg
2+. Upon exposure to calcium ions, OCM undergoes distinctive structural changes not observed in other parvalbumins. OCM features a cation binding site that accommodates both calcium and magnesium, with one being more Ca
2+-specific. It is proposed that OCM might function as a calcium sensor or modulator under specific physiological conditions, rather than solely acting as a Ca
2+ buffer
[95][68].
OCM’s function appears ambiguous and cell-type-dependent when distinguishing it from other EF-hand CaBPs. Mammalian OCM expression is unique and confined to specific inner ear hair cells and specific immune cells
[96,97,98][69][70][71]. In adult mammals, OCM can act as a Ca
2+ buffer and likely influence outer hair cell (OHC) motility mechanisms, contributing to sensory hair cell function and playing a crucial role in maintaining hearing function and health
[99,100][72][73].
4.2. Caldendrin
Among the interesting Ca
2+-binding proteins is CaBP1, which, due to alternative splicing, forms three variants: caldendrin, CaBP1-S, and CaBP1-L
[132,133][74][75]. Caldendrin has been shown to be highly expressed in different neuronal cell types, including the brain, retina, and inner ear
[134,135][76][77]. Specifically, it is present in the synapses of cerebral cortical neurons, the cerebellum, hippocampus, and thalamus, as well as in the postsynaptic density of spine synapses
[136,137][78][79]. Caldendrin has a unique bipartite structure with high homology to calmodulin, a ubiquitous EF-hand calcium sensor protein. However, in contrast to calmodulin, other Ca
2+ sensor proteins have more specialized functions. Caldendrin possesses only two functional domains (EF3 and EF4) and two atypical domains (EF1 and EF2)
[138][80]. The EF1 hand can bind Mg
2+ with high affinity, whereas EF2 is non-functional. Two shorter isoforms of caldendrin detected in rats and humans differ in their sequences and are N-terminally myristoylated
[133,137,139][75][79][81]. These proteins are mainly localized in the cytosol but are also present in the Golgi structures
[140][82]. It should be underlined that caldendrin exhibits a high degree of conservation in humans, rats, and mice
[141][83].
Caldendrin has been suggested to be involved in the processing of synaptic Ca
2+ signaling
[142][84]. A long-lasting increase in intracellular Ca
2+ levels, either due to an influx through Ca
2+ channels or a release from Ca
2+ stores, is neurotoxic. However, some channels can be rapidly inactivated by the Ca
2+-dependent inactivation process (CDI), which is a negative feedback mechanism important for regulating Ca
2+ entry under both physiological and pathological conditions
[143,144,145][85][86][87]. Physiologically, caldendrin increases the Ca
2+influx throughL-type voltage-gated Ca
2+ channels—Ca
v1.2 and Ca
v1.3—by interacting with their C-terminal domain
[146,147,148,149,150][88][89][90][91][92]. In contrast, CaBP1 enhances the inactivation of Ca
v2.1 channels (P/Q-type) by interacting with the CaM-binding domain of the channel
[151][93].
Caldendrin can also support the cytoskeleton network by stabilizing F-actin within the network’s spines through its interaction with cortactin, a protein implicated in actin filament nucleation and branching. This regulation contributes to spine stability and participates in the development of long-term memory and its storage
[142][84]. Interestingly, cortactin activation appears to be caldendrin-specific and CaM-independent. The dynamics of spine morphology are attributed to the actin cytoskeleton that is highly concentrated in spines. However, the transport of organelles and their anchoring at dendritic spines also depend on F-actin-linked myosins
[156][94].
A number of studies using caldendrin knockout animals have shown a more intensive regenerative growth of neurites, confirming the inhibitory action of this protein in the regeneration and elongation of neurites
[158,159][95][96]. It is suggested that caldendrin may influence an initial phase of regeneration by altering the transcription of genes that promote axon outgrowth
[159,160][96][97].
A crucial role in coordinating neuronal processes involves the formation of protein complexes named signalosomes, which are formed by scaffold proteins at specific subcellular locations. A notable example is a family of A-kinase anchoring proteins (AKAPs) that bind to protein kinase A (PKA) and other secondary messenger-regulated enzymes
[161][98]. One of the essential elements for signal transduction is the protein AKAP79/150, which links receptors, channels, and other signaling proteins to physiological substrates and has been associated with the regulation of neurite growth
[162][99]. At postsynaptic sites in neurons, AKAP79/150 can form complexes with protein kinases A and C, calcineurin, calmodulin, phosphatidylinositol 4,5-bisphosphate, and also with caldendrin
[163][100].
One of the signaling pathways in neurons involves interactions between initial activity-dependent molecular changes at the synapse and the subsequent regulation of gene transcription in the nucleus. An interesting example of such communication is the contribution of Jacob, a protein messenger abundantly expressed in the brain, whichconnects NMDA-receptor-derived signalosomes to the transcription factor CREB
[165,166,167][101][102][103]. The activation of synaptic NMDARs induces the expression of pro-survival genes, but the activation of extrasynaptic NMDARs initiates the expression of cell death genes
[168][104]. The overexpression of Jacob triggers the expression of genes that induce neurodegeneration, whereas the nuclear knockdown of Jacob increases the phosphorylation of CREB and protected neurons from an extrasynaptic NMDA receptor-induced loss of synaptic contacts and neuronal cell death
[169][105]. Caldendrin was shown to bind to Jacob’s nuclear localization signal in a Ca
2+-dependent manner, and this interaction enabled the proper formation of the signalosome, representing a powerful regulatory mechanism of synapse-to-nucleus communication. Ultimately, significant changes in the morphology of the dendritic tree were generated
[169][105].
4.3. Calneurons
Within the CaBP family, there also exists an interesting calciumsensor subfamily of proteins consisting of calneuron 1 (CaBP8) and calneuron 2 (CaBP7), which are abundant in the brain and exhibit developmental changes in expression
[135,141,171][77][83][106]. They have 64% similarity and are present at high levels in distinct regions of the adult mammalian brain
[172,173][107][108].
It is noteworthy that calneurons are highly conserved among different species, exhibiting 100% identity at the amino acid level between mice, rats, monkeys, and humans. This underscores their crucial cellular function. Both calneurons are distributed across various subcellular fractions, primarily localized in Golgi membranes, cytoplasm, and vesicles. In the mouse brain, calneuron 1 was found in significant amounts in the cerebellum, hippocampus, and cortex
[174][109].
Additionally, calneurons exhibit sequence homology with calmodulin. In contrast to other calcium-binding proteins (CaBPs), calneurons display a distinct pattern of EF-hand inactivation, featuring active EF-hands 1 and 2 and inactive EF-hands 3 and 4
[171][106].
Less is known about functions of calneuron-1 and calneuron-2
[138][80]. As of publication, they have been found to regulate the activity of phosphatidylinositol 4-OH kinase IIIβ (PI4KIIIβ), which catalyzes the local synthesis of the phosphoinositides necessary for vesicle assembly in the trans-Golgi network (TGN)
[178][110]. The activity of PI4KIIIβ at the Golgi membrane is a crucial initial step in trans-Golgi network-to-plasma-membrane trafficking. A primary and central regulator of PI4KIIIβ activity is NCS-1, a neuronal calcium sensor protein responsible for the rapid transduction of Ca
2+ signals. NCS-1 is involved in numerous physiological neuronal functions, including exocytosis, the regulation of calcium channels, nuclear Ca
2+ regulation, neurite outgrowth, and neuroprotection, as well as axonal regeneration in response to neuronal damage
[155,179,180][111][112][113]. Therefore, it was interesting to find that both calneurons can directly associate with PI4KIIIβ both in vitro and in vivo in a Ca
2+-independent manner
[173][108].
4.4. Neuronal Calcium Sensor-1
Neuronal calcium sensor-1 (NCS-1) is a member of the NCS superfamily, characterized by four EF-hand domains. As with other members, the first EF-hand is unable to bind Ca
2+, EF-2 serves as a Mg
2+/Ca
2+-binding domain, and the C-terminal domain contains Ca
2+-binding EF-3 and EF-4
[187][114]. NCS-1 features an N-terminal myristoylation site facilitating its binding to cell membranes
[188][115]. Ca
2+ binding to NCS1 causes a structural rearrangement, modulating its affinity for target molecules
[189][116]. In the brain, NCS-1 is widely expressed, with the highest abundance observed in neuronal tissues, particularly in the cortex, as well as in the hippocampus and dorsal root ganglion cells
[190,191][117][118]. The specific expression patterns of NCS-1 in various brain regions, cell types, and subcellular areas can influence the availability of its unique target proteins.
To date, NCS-1 has been demonstrated to regulate various cellular functions, encompassing exocytosis, neurite outgrowth, neuroprotection, and axonal regeneration
[195,198][119][120]. The functional diversity of NCS-1 arises from its interaction with numerous downstream targets. These include the binding to and regulation of the Ca
V2.1 of VGCCs, the enhancement of inositol 1,4,5-trisphosphate receptor activity, and the spatial and temporal control of phosphatidylinositol 4-phosphate levels through the activation of phosphatidylinositol 4-kinase III-β (PI-4Kβ). Additionally, NCS-1 contributes to the desensitization of dopamine type-2 receptors, among other pathways
[199,200,201,202,203,204,205,206,207][121][122][123][124][125][126][127][128][129]. An intriguing relationship has been proposed regarding a molecular switch in the Ca
2+ regulation of PI-4Kβ activity by calneurons and NCS-1 in Golgi membranes. Calneurons at low Ca
2+ levels suppress PI-4Kβ activity and dominate in the regulation of this enzyme. As Ca
2+ increases, the formation of the NCS-1/PI-4Kβ complex is promoted, potentially allowing for the override of the inhibition of PI-4Kβ activity imposed by calneurons
[173][108].
5. Conclusions
The highly diverse nature of the Ca
2+ signaling in the nerve system is well established. At every stage of life, the functional adaptation of the molecular pathways in individual neurons is required to properly integrate external and internal factors. Disturbances in calcium homeostasis, resulting from a dysregulation of the ability to handle, store, and transfer information, contribute to the neuropathological processes in neurons, particularly after brain injury. The coordinated actions of all systems responsible for the maintenance and restoration of calcium homeostasis involve a complex interplay between functional proteins, including oncomodulin, caldendrin, calneuron, NCS-1, GAP-43, and CaM kinase II (
Figure 32).
Figure 32. Schematic presentation of the regulatory function of Ca
2+-associated proteins in neuron repair. A subtle regulatory control of Ca
2+ signals plays a crucial role in integrating cellular mechanisms, and the cooperation of all potentially interacting partners is essential for specific neuronal functions. These functions encompass neurotransmitter release, proper neuronal transmission, activity-dependent gene transcription, endoplasmic reticulum (ER) targeting, Golgi-to-plasma-membrane trafficking, axonal growth, and cytokinesis. Arrows within the figure indicate potential direct sites and/or processes where specific Ca
2+-associated proteins may be engaged. The activation or inhibition of neuron repair depends on the presence of partner proteins and the Ca
2+ level. CaMKII actively participates in axon regeneration, colocalizes, and interacts with F-actin, activating the cAMP-response element-binding (CREB) protein. Phosphorylated GAP-43 stimulates F-actin polymerization and stability, leading to F-actin accumulation. GAP-43 also plays a pivotal role in axon outgrowth, neurotransmission, and synaptic plasticity. Oncomodulin (OCM) facilitates axon regeneration by operating within cellular signaling pathways, notably the CaMKII pathway, and is linked to the phosphorylation and activation of CREB. OCM contributes to the upregulation of selected RAGs, including Gap-43. Caldendrin (Cald) and two shorter CaBP1 forms regulate the Ca
2+ influx through Ca
2+ channels, including L-type and P/Q-type VGCC, and also TRPC5, which is implicated in neurite extension and growth cone morphology. Furthermore, Caldendrin, in a Ca
2+-independent manner, could inhibit IP
3 binding to inositol 1,4,5 trisphosphate receptors (IP3Rs), thereby blocking Ca
2+ release from the ER. It also supports the cytoskeleton network by stabilizing F-actin within the spines.Calneuron-1 and Calneuron-2 (Caln) regulate the activity of phosphatidylinositol 4-OH kinase IIIβ, catalyzing the local synthesis of phosphoinositides necessary for vesicle assembly in the trans-Golgi network (TGN). Calneuron-1 can modulate G-protein-coupled receptor (GPCR) heteromers, such as adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) and, as recently shown, cannabinoid CB1 receptor. Neuronal calcium sensor-1 (NCS-1) binds to and regulates the Ca
V2.1 of VGCCs and TRPC5, enhancing the activity of IP3Rs and PI-4Kβ in Golgi membranes. Additionally, it contributes to the desensitization of D2Rs. The NCS-1-induced activation of cytosolic CaMKII-α can regulate CREB.