Hippocalcin was originally cloned from a rat brain cDNA library based on the partial amino acid sequences from a calcium-binding protein with a molecular mass of 23,000 Da (P23k), which was purified during a survey of recoverin-like immunoreactivity in the brain (Kobayashi et al. 1992). During the 1990s, a number of calcium-binding proteins structurally related to hippocalcin were identified in the nervous system and named NCS (neuronal calcium sensor) proteins. There are 14 protein-coding genes in the NCS family in the human genome (Burgoyne 2007). Hippocalcin is a highly conserved protein and has an identical amino acid sequence in rat, mouse, bovine, and human (Kobayashi and Takamatsu 2009).
Protein Chemical Characteristics
Hippocalcin, a 23 kDa protein of 193 amino acid residues containing 10 α-helices and 2 antiparallel β-sheets, has three functional EF-hand calcium-binding domains and an N-terminal glycine residue covalently linked to myristic acid (Kobayashi and Takamatsu 2009; Li and Ames 2014). In hippocampal pyramidal cells, hippocalcin is located in the cytoplasm and associated with the plasma membrane of the cell body, dendrites, and axon. The hippocalcin concentration in hippocampal pyramidal cells is estimated to be higher than 30 μM. Calcium binding to the EF-hands of hippocalcin induces a conformational change that results in exposure of the myristoyl group and association of hippocalcin with the cellular membranes in response to free calcium in the 200–800 nM range. This process is referred to as a calcium myristoyl switch and is a prominent characteristic of some members of the NCS family, including recoverin, VILIP1, VILIP2, and VILIP3 (Burgoyne 2007). Hippocalcin reversibly translocates from the cytosol to the plasma membrane and the trans-Golgi network of the perinuclear region in transfected HeLa cells and in hippocampal cells via the calcium myristoyl switch mechanism (Kobayashi and Takamatsu 2009). The maximal rate of translocation to the membrane is approximately 1 s. Half-maximal translocation occurred at approximately 300 nM free calcium. In hippocampal neurons, spontaneous and action potential-dependent translocation of hippocalcin-YFP fluorescence was observed in different parts of neuronal processes, reaching peak translocation within 1–5 s (Markova et al. 2008). Local α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptor activation resulted in fast hippocalcin-YFP translocation to specific sites within a dendritic tree, and short local N-methyl-D-aspartate (NMDA) receptor activation induced fast hippocalcin-YFP translocation in a dendritic shaft at the application site, indicating that hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site- and time-specific translocation to its targets (Dovgan et al. 2010).
These results suggest that hippocalcin would be able to interact with target proteins on membranes when free calcium levels are only slightly elevated above resting levels. N-myristoylated hippocalcin, as well as an N-myristoylated hippocalcin peptide (1–14), interacts with liposomes containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) with high affinity (Kd = 50 nM) in a Ca2+-dependent manner (O’Callaghan et al. 2005). Expression of hippocalcin (1–14)-ECFP (enhanced cyan fluorescent protein) partially displaced the pleckstrin homology domain of phospholipase δ1, a PtdIns(4,5)P2-specific binding partner, from the plasma membrane in living cells. These results suggest that the interaction with PtdIns(4,5)P2 is likely to be a physiologically important event contributing to the targeting of hippocalcin.
Hippocalcin is expressed in various regions of the rat hippocampus, as well as other regions of the brain (Kobayashi et al. 1992; Kobayashi and Takamatsu 2009). In the hippocampus, hippocalcin mRNA and immunoreactivity are detected at high levels in the pyramidal cells of Ammon’s horn and at moderate levels in the granule cells of the dentate gyrus. Hippocalcin is also found at high levels in other regions of the brain, including the pyramidal cells of cerebral cortex layers II–VI and the caudate-putamen, taenia tecti, claustrum, olfactory tubercle, anterior olfactory nucleus, and olfactory bulb, as well as in the ganglion cell layer and amacrine cell layer in the retina and in the apical layer in the olfactory epithelium. No hippocalcin protein expression has been detected outside of the central nervous system, although expressed sequence tag sequences have been found in a few peripheral tissues (Kobayashi and Takamatsu 2009).
Roles in Neural Plasticity
Moreover, hippocalcin has been implicated in hippocampal NMDA receptor-dependent long-term depression (LTD) (Palmer et al. 2005; Fig. 1). Hippocalcin binds directly to the β2-adaptin subunit of the AP2 adaptor complex, which couples clathrin to the cytosolic domains of membrane-bound proteins destined to be internalized. In hippocampal neurons, this hippocalcin-AP2 complex bound only to the GluR1 subunit of the AMPA receptor. The calcium-dependent endocytosis of the AMPA receptor is a key event in NMDA receptor-dependent LTD. Expression of a mutated hippocalcin lacking all EF-hand structures in CA1 pyramidal neurons blocked synaptically evoked LTD without affecting basal AMPA receptor-mediated transmission or long-term potentiation.
In addition, hippocalcin is the key intermediate between calcium influx in response to a train of action potentials and potassium channel activation, which mediates the slow after-hyperpolarization current (IsAHP) in hippocampal neurons (Tzingounis et al. 2007; Fig. 1). Brief depolarizations, which are sufficient to activate IsAHP in wild-type mice, did not elicit IsAHP in hippocalcin−/− mice. Introduction of hippocalcin into cultured rat hippocampal neurons led to a pronounced IsAHP, while neurons expressing a mutant hippocalcin lacking N-terminal myristoylation exhibited a small IsAHP similar to that recorded in uninfected neurons, confirming that hippocalcin gates the potassium channel that mediates IsAHP (IsAHP channel). Application of a KCNQ-type potassium channel activator accelerates the rise of IsAHP, indicating that slow after-hyperpolarization (sAHP) activation is critically dependent on KCNQ channel kinetics. The decay of sAHP is prolonged in hippocalcin−/− mice, suggesting that hippocalcin determines the composition of the IsAHP channels. The role of hippocalcin in activation of the IsAHP channel is mediated by the generation of PtdIns(4,5)P2 (Kim et al. 2016).
Protection Against Neuronal Damage
Roles in Energy Shuttle
Hippocalcin specifically binds to the creatine kinase B (CKB) subunit, which constitutes brain-type creatine kinase (BB-CK), in a calcium-dependent manner (Kobayashi et al. 2012). Hippocalcin mediates calcium-dependent partial translocation of CKB to membranes. N-myristoylation of hippocalcin is critical for membrane translocation but not for binding to CKB. In cultured hippocampal neurons, ionomycin treatment leads to colocalization of hippocalcin and CKB adjacent to the plasma membrane. These results indicate that hippocalcin associates with BB-CK and that together they translocate to membrane compartments in a calcium-dependent manner.
Roles in Olfactory Signaling
Hippocalcin interacts with MLK (mixed-lineage kinase) 2 (Kobayashi and Takamatsu 2009). Hippocalcin and MLK2 colocalize in the halo surrounding Lewy bodies in patients with Parkinson’s disease (PD) (Nagao and Hayashi 2009). This suggests that both proteins are associated with the pathogenesis of PD; however, the molecular basis has not yet been determined. Hippocalcin also interacts with microsomal cytochrome b5, calmodulin-dependent cyclic nucleotide 3′,5′-phosphodiesterase, CAPS1 (a calcium-dependent activator protein involved in secretion), and the large conductance calcium-activated potassium channel (BK channel). However, the functional consequences of these interactions have not yet been determined (Kobayashi and Takamatsu 2009; Kathiresan et al. 2009).
Associations with Diseases
Using a combination of homozygosity mapping and whole-exome sequencing in a consanguineous kindred affected by autosomal-recessive (AR) isolated dystonia, homozygous mutations in the hippocalcin gene were identified as the cause of an AR primary isolated dystonia. Compound heterozygous mutations of the gene were also identified in a second independent kindred affected by AR isolated dystonia, suggesting a role for perturbed calcium signaling in the pathogenesis of isolated dystonia (Charlesworth et al. 2015).
Hippocalcin levels are increased in human Alzheimer’s disease (AD) brain and in amyloid-β (Aβ) plaque-forming APP23 transgenic mice compared with controls. The primary cultures derived from hippocalcin−/− mice are more susceptible to Aβ toxicity than controls. Aβ treatment induces high levels of toxicity on hippocampal mitochondria from hippocalcin−/− mice already at 3 months of age, indicating a stronger aging phenotype. These data suggest that hippocalcin has a neuroprotective role in AD, presenting it as a putative biomarker (Lim et al. 2012).
A recent report demonstrates that hippocalcin is associated with cancer development. Overexpression of the hippocalcin gene is observed in serrated adenocarcinoma (SAC) and is highly suggestive of SAC diagnosis (sensitivity = 100%) (Conesa-Zamora et al. 2013). This suggests that hippocalcin might be a potential new diagnostic biomarker for SAC; however, the pathological consequence of overexpression of hippocalcin has not yet been determined.
Hippocalcin, a member of the NCS protein family that is predominantly expressed in the hippocampus, is a highly conserved protein that has an identical amino acid sequence in rat, mouse, bovine, and human. Hippocalcin has three functional EF-hand calcium-binding domains and its N-terminal glycine residue is myristoylated. Hippocalcin is regulated by a calcium myristoyl switch mechanism within cells, allowing it to translocate from the cytosol to intracellular membranes of the trans-Golgi network and to the plasma membrane in response to an increase in free calcium. Synaptic AMPA receptor and NMDA receptor activation produces different spatiotemporal patterns of hippocalcin translocation, indicating that hippocalcin may differentially decode various calcium signaling induced via glutamate receptor activation. Hippocalcin is involved in activity-dependent activation of the MAP kinase pathway via ERK signaling and activation of CREB, key gene expression events mediating the long-lasting synaptic plasticity underlying learning and memory. Spatial and associative learning abilities are impaired in hippocalcin-null mutant (hippocalcin−/−) mice, as determined by the probe test, the Morris water maze, and visual discrimination learning tasks. Hippocalcin is implicated in hippocampal NMDA receptor-dependent LTD via binding to the β2-adaptin subunit of the AP2 adaptor complex, which mediates internalization of the GluR1 subunit of the AMPA receptor. IsAHP, which is activated by brief depolarizations, was not elicited in hippocalcin−/− hippocampal neurons. The decay of sAHP is prolonged in hippocalcin−/− mice. This observation indicates that hippocalcin mediates potassium channel activation in response to calcium influx. Hippocalcin exhibits a protective effect against calcium-induced cell death by interacting with NAIP, diminishing ER stress, and upregulating calcium extrusion. Hippocalcin associates with BB-CK, and together they translocate to membrane compartments in a calcium-dependent manner. Hippocalcin is also expressed in olfactory receptor neurons and regulates the activities of ciliary adenylyl cyclase and particulate guanylyl cyclase in a calcium-dependent manner. Hippocalcin is associated with the pathological processes of several diseases. Homozygous mutations in the hippocalcin gene are the cause of an AR primary isolated dystonia. Aβ treatment induces high levels of toxicity on the primary cultures and hippocampal mitochondria from hippocalcin−/− mice, suggesting that hippocalcin has a neuroprotective role in AD. Overexpression of hippocalcin has been demonstrated in SAC (sensitivity = 100%), providing hippocalcin as a diagnostic biomarker for SAC. Thus, hippocalcin may act as a multifunctional modulator in calcium signaling pathways, such as those underlying neuronal plasticity, neuronal excitability, neuronal cell death, olfaction, and disease progression.
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