Kalirin-7 is a Key Player in the Formation of Excitatory Synapses in Hippocampal Neurons

Kalirin-7 (Kal7), a major isoform of Kalirin in the adult rodent hippocampus, is exclusively localized to the postsynaptic side of mature excitatory synapses in hippocampal neurons. Kal7 interacts with multiple PDZ domain—containing proteins through its unique PDZ binding motif. Overexpression of Kal7 increases spine density and spine size, whereas reduction of endogenous Kal7 expression by small hairpin RNA (shRNA) causes a decrease in synapse number and spine density in cultured hippocampal neurons. Hippocampal CA1 pyramidal neurons of Kal7 knockout (Kal7) mice show decreased spine density, spine length, synapse number, and postsynaptic density (PSD) size in their apical dendrites; are deficient in long-term potentiation (LTP); and exhibit decreased frequency of spontaneous excitatory postsynaptic current (sEPSC). Kal7 plays a key role in estrogen-mediated spine/synapse formation in hippocampal neurons. Kal7 is also an essential determinant of dendritic spine formation following chronic cocaine treatment. Kal7 plays a key role in excitatory synapse formation and function.

 Cdk5 -Cdk5, a highly versatile kinase, plays a critical role in neurite extension, synaptic plasticity, the formation and function of dendritic spines, learning, and memory [56,57,58,59]. Cdk5 phosphorylates Kal7 at Thr1590, which is localized between the GEF domain and PDZ binding motif, and is the only Cdk5 phosphorylation site in Kal7 [33]. Expression of Kal7 with an Ala1590 mutation (T/A, blocking phosphorylation site) causes a decrease in spine size, whereas expression of Kal7 with an Asp1590 mutation (T/D, mimicking phosphorylation) results in an increase in spine size in cultured neurons in comparison to expression of wild-type Kal7[33]. Both Kal7 T/A and T/D increase spine density as well as wild-type Kal7 [33]. Alterations of spine size are implicated in learning and memory [12]. The levels of Cdk5 in the PSDs purified from the hippocampus in Kal7 KO mice decrease compared to their wild-type controls [60]. These data suggest that Cdk5 regulates Kal7 function during synaptogenesis.  CaMKII -CaMKII, the most abundant protein kinase in the mammalian brain, constitutes the major protein of the PSD in dendritic spines of excitatory neurons [61,62]. It is well established that CaMKII modulates synaptic plasticity, learning, and memory [62,63,64]. Phosphorylation of Kal7 by CaMKII may play an important role in synaptic activity-dependent enlargement of existing spines in cultured neurons[46].

FIGURE 2.
Kal7 is localized to the postsynaptic side of excitatory synapses in hippocampal neurons. The dendrites of mature hippocampal neurons (DIV30) were fixed with cold methanol and simultaneously stained for Kal7 (A, red), Vglut1 (B, green, a marker for excitatory presynaptic terminals), and MAP2 (C, blue, dendritic marker), showing that Kal7 is localized to the postsynaptic side of excitatory synapses (D, merge). Neuronal preparation and immunostaining were performed as described [44,45].
 N-Cadherin -The cadherin-catenin cell-adhesion complex plays a key role in both synapse formation and plasticity [65,66]. Kal7 interacts with N-cadherin [67]. In cultured neurons, Ncadherin and Kal7 colocalize at synapses, and Kal7 function is required for N-cadherindependent spine enlargement [67].  DISC1 -Recent studies show that DISC1, a strong candidate susceptibility gene for schizophrenia, plays an important role in the regulation of synaptic plasticity [52,68,69]. Schizophrenia patients show a reduction in spine density on their cortical pyramidal neurons [70,71]. DISC1 colocalizes with Kal7 at dendritic spines, and regulates spine structure and function via Kal7. Overexpression of DISC1 causes a decrease in both spine density and spine size, while overexpression of a mutated DISC1, which does not bind to Kal7, does not alter spine density and spine size in cortical neurons in comparison to control neurons expressing GFP only [52] .
 EphB -The receptor tyrosine kinase EphB and its membrane-bound ligand ephrinB play an important role in synapse formation and plasticity in the CNS [72,73]. EphB regulates Kal7 function through its phosphorylation of Kal7, and ephrinB-EphB receptor-induced activation of Kal7 causes an increase in the formation of dendritic spines in mature neurons [55].

KAL7 IS REQUIRED FOR NORMAL SPINE/SYNAPSE FORMATION IN HIPPOCAMPAL PYRAMIDAL NEURONS IN VITRO AND IN VIVO
Overexpression of Kal7 increases spine density and spine size in hippocampal pyramidal neurons[45] (Fig. 3). Reducing endogenous Kal7 expression by expressing a Kal7 shRNA causes a decrease in synapse number and spine density in cultured hippocampal pyramidal neurons [44,45]. Kal7 shRNAmediated reduction in spine density was accompanied by a decrease in the frequency of sEPSCs in these neurons (Lemtiri-Chlieh and Ma et al., unpublished). The Kal7 shRNA-induced decrease in spine density is rescued by simultaneous expression of exogenous Kal7 in cultured hippocampal neurons [45]. In addition, a recent interesting study shows that Kal7 is required for microRNA-stimulated spine formation in hippocampal neurons [74]. To evaluate the role of Kal7 in vivo, Kal7 KO mice were created; these mice grow and reproduce normally [60]. Spine density, spine length, PSD thickness, PSD length, and synapse number decrease in the apical dendrites of Kal7 KO hippocampal CA1 pyramidal neurons, which are deficient in LTP and exhibit a decrease in sEPSC frequency. These decreases may be due to decreased levels of NR2B and Cdk5 in the PSDs purified from the hippocampus of Kal7 KO mice. Importantly, deficits in spine formation in Kal7 KO neurons are rescued by exogenous Kal7 [60]. Similarly, a recent study reports that Kal7 plays a key role in spine formation in cortical neurons [75]. These studies show that Kal7 is essential for normal excitatory synapse formation [60,76].

KAL7 PLAYS A KEY ROLE IN EXCITATORY SYNAPSE FORMATION IN HIPPOCAMPAL INTERNEURONS
Hippocampal interneurons, which are free of dendritic spines, play an essential role in maintaining normal circuits in the CNS, which requires a delicate balance between synaptic excitation and inhibition [77,78]. Low levels of Kal7 are detected at the postsynaptic side of excitatory synapses on the dendritic shaft of hippocampal interneurons [44]. Overexpression of Kal7 increases dendritic branching, and induces the formation of dendritic spines along the dendrites and on the soma of normally aspiny hippocampal interneurons, whereas reducing endogenous Kal7 by shRNA results in a decrease in the number of excitatory synapses on the dendritic shafts of these interneurons [44]. Disruption of interneuron development may contribute to development of psychiatric and neurological diseases [79,80,81]. These studies suggest a role of Kal7 in the development of these diseases.

KAL7 PLAYS A KEY ROLE IN ESTROGEN-MEDIATED SPINE/SYNAPSE FORMATION IN HIPPOCAMPAL NEURONS
Estrogen increases spine density and excitatory synapse number in both hippocampal CA1 pyramidal neurons of ovariectomized rats in vivo and cultured hippocampal neurons in vitro [45,82,83]. Spine density [84] and spine size [85] in CA1 pyramidal neurons change across the estrous cycle, peaking during proestrous with its high estrogen levels. However, the underlying mechanisms of estrogen-mediated spine formation are not fully understood. Estrogen replacement increases the intensity of Kal7 staining in both

KAL7 PLAYS AN ESSENTIAL ROLE IN SPINE FORMATION FOLLOWING CHRONIC COCAINE TREATMENT
Kal7-mediated spine formation is not specific to hippocampal neurons. Kal7 is expressed in medium spiny neurons of the nucleus accumbens, a brain region involved in drug addiction. Overexpression of Kal7 caused an increase in spine density and spine size, whereas reducing endogenous Kal7 levels caused a decrease in spine density in medium spiny neurons in slice cultures of the nucleus accumbens (Ma et al., unpublished). Chronic cocaine treatment results in a long-lasting increase in dendritic spine density in the nucleus accumbens, but the molecular mechanisms underlying this morphological change are not understood [86]. Repeated cocaine treatment, which increases Kal7 levels in the nucleus accumbens, causes a robust increase in dendritic spine density in medium spiny neurons of the nucleus accumbens core in wild-type mice, but not in Kal7 KO mice. In behavioral tests, Kal7 KO mice show increased locomotor sensitization in comparison to wild-type controls [87]. Enhanced sensitivity to cocaine in Kal7 KO mice suggests that the increase in dendritic spines, normally seen after repeated cocaine, is protective and helps to prevent even more locomotor activity if the spines had not increased. This may also correlate with the decrease in Cdk5 and NR2B seen in Kal7 KO mice [60], since lowered Cdk5 and NR2B both enhance locomotor sensitization to stimulant [88,89]. In conclusion, Kal7 plays an essential role in chronic cocaine treatment-induced spine formation, and is necessary for the normal behavioral response to cocaine.

KAL7 MAY BE RELATED TO PSYCHIATRIC AND NEUROLOGICAL DISEASES
Dendritic spine density and/or spine morphology are altered in psychiatric and neurological diseases, and recent studies suggest a role for Kal7 and its interactors in specific diseases [20,90,91,92,93,94,95]. Kalirin expression increases in the rat hippocampus after electroconvulsive shock, an animal model for electroconvulsive treatment (ECT), an effective therapy for drug-refractory depression [96,97]. Spine density decreases in the hippocampus in animal models of depression [98] and ECT treatment causes an increase in synapse number in hippocampal neurons [99]. These studies suggest that Kal7 may play a role in the ECT-induced increase in synapse number and, thus, in ECT therapy for depression. Several clinical studies suggest that alterations in Kalirin expression may lead to disease. Neuropsychiatric disease is associated with reduced excitatory synaptic connectivity [100,101]. Decreased Kalirin levels are accompanied by a decrease in spine density in the cortex of schizophrenic patients [70,71,102]. Kal7 KO mice show decreased anxiety-like behavior and impaired acquisition of a passive avoidance task [60]. Kalirin KO (deletion of major Kalirin isoforms) mice exhibit locomotor hyperactivity and deficits in working memory, sociability, and prepulse inhibition [60,75]. These behaviors are similar to those described for neuropsychiatric disease [103,104,105]. Disturbances in the DISC1-Kal-7 complexmediated synaptic maintenance may contribute to the onset of neuropsychiatric disease [52]. Kalirin levels are decreased in the hippocampus of Alzheimer's disease patients [106,107]. The interaction of Kalirin and huntingtin-associated protein 1 (HAP1) suggests that Kalirin is relevant to Huntington's disease [108]. High levels of Kal7 are found in the CA2-3 hippocampal areas, which are relatively ischemia resistant, suggesting a role of Kal7 in the regulation of ischemic signal transduction [109]. A more recent study indicates that Kalirin is a novel genetic risk factor for ischemic stroke [110].

CONCLUSIONS AND FUTURE DIRECTIONS
Kal7 is required for synapse formation and function in hippocampal neurons in vitro and in vivo. Kal7 plays a key role in the mechanisms of spine/synapse formation induced by estrogen and chronic cocaine exposure. Future studies will investigate the underlying molecular mechanisms of Kal7-mediated spine/synapse formation and its contributions to learning and memory, and determine the role of Kal7 in the development of psychiatric and neurological diseases in which normal dendritic spines are altered. Conditional gene deletion approaches will be used to alter Kal7 expression in specific areas or neuronal subpopulations, such as hippocampal CA1 region and nucleus accumbens core.