Cacna1c: Protecting young hippocampal neurons in the adult brain

ABSTRACT

Neuropsychiatric disease is the leading cause of disability in the United States, and fourth worldwide.^1,2 Not surprisingly, human genetic studies have revealed a common genetic predisposition for many forms of neuropsychiatric disease, potentially explaining why overlapping symptoms are commonly observed across multiple diagnostic categories. For example, the CACNA1C gene was recently identified in the largest human genome-wide association study to date as a risk loci held in common across 5 major forms of neuropsychiatric disease: bipolar disorder, schizophrenia, major depressive disorder (MDD), autism spectrum disorder and attention deficit-hyperactivity disorder.³ This gene encodes for the Ca_v1.2 subunit of the L-type voltage-gated calcium channel (LTCC), accounting for 85% of LTCCs in the brain, while the Ca_v1.3 subunit comprises the remainder.⁴ In neurons, LTCCs mediate calcium influx in response to membrane depolarization,⁵ thereby regulating neurotransmission and gene expression. Here, we describe our recent finding that Ca_v1.2 also controls survival of young hippocampal neurons in the adult brain, which has been linked to the etiology and treatment of neuropsychiatric disease. We also describe the effective restoration of young hippocampal neuron survival in adult Ca_v1.2 forebrain-specific conditional knockout mice using the neuroprotective compound P7C3-A20.

In order to investigate the function of Ca_v1.2, genetic mouse models have been applied. Because global knockdown of cacna1c (KO) is embryonic-lethal,⁶ mice harboring forebrain-specific conditional knockout of cacna1c (forebrain- Ca_v1.2 cKO) were generated to specifically study the role of Ca_v1.2 in neuropsychiatric disease. These animals show reductions in spatial memory,^7,8 as well as high anxiety-like behavior,⁹ one of the core symptoms of all forms of neuropsychiatric disease in which CACNA1C has been implicated. Previous in vitro studies have shown that LTCCs mediate survival of cultured hippocampal neural precursor cells,¹⁰ and other investigators have shown that postnatal hippocampal neurogenesis is involved in memory, processing of fearful experiences, and anxiety.^11-16 We therefore wondered whether forebrain Ca_v1.2 cKO mice might display an altered pattern of hippocampal neurogenesis, which might contribute to the role of CACNA1C in neuropsychiatric disease.¹⁷

To assess the magnitude of hippocampal neurogenesis in forebrain- Ca_v1.2 cKO mice, we began with a standard week-long assay of daily injection of bromodeoxyuridine (BrdU),¹⁷ a thymidine analog that is incorporated into replicating DNA, and observed a 50% reduction in labeled cells in the subgranular zone (SGZ) of the dentate gyrus, compared to age/sex-matched wild type (WT) littermates (Fig. 1). This was accompanied by a similar reduction in expression of the immature neuronal marker doublecortin (DCX) in the SGZ. To differentiate between developmental versus adult roles of Ca_v1.2, we next used viral delivery of Cre-recombinase into the hippocampus of floxed-cacna1c adult mice. Here, we again observed a 50% decrease in the magnitude of hippocampal neurogenesis using the same assay. This finding supports a postnatal role for Ca_v1.2 in hippocampal neurogenesis.

Figure 1.

Role of Ca_v1.2 in the mouse forebrain. Forebrain-specific Ca_v1.2 deletion (fbCa_v1.2 KO) in mice results in anxiety-like behaviors and deficits in spatial memory. Heterozygous Ca_v1.2 mouse models also show deficits in acquisition of conditioned fear,44 as well as depression- and anxiety-like behaviors.45 Our recent findings show that fbCa_v1.2 KO mice exhibit 50% decrease in brain levels of BDNF, associated with a 50% decrease in survival of young hippocampal neurons in the adult brain. This deficit in neuron survival was overcome by treatment of mice with the neuroprotective compound P7C3-A20, without affecting levels of BDNF in the brain. Our findings identify a new role of the neuropsychiatric-associated gene CACNA1C in the brain, and future work will determine restoration of survival of young hippocampal neurons will help treat behavioral deficits associated with CACNA1C-deficiency. Our ultimate goal is to develop new treatment approaches for patients suffering from forms of neuropsychiatric disease associated with aberration in CACNA1C.

In the week-long assay of daily BrdU labeling, the magnitude of BrdU signal is due to the combined effects of proliferation and survival of newborn neurons in the hippocampus. These aspects of hippocampal neurogenesis are distinct physiologic processes that can be distinguished by pulse-chase BrdU labeling.¹⁷ Quantification of signal 1 hour after BrdU labeling showed equal rates of neural precursor cell proliferation in forebrain Ca_v1.2 cKO mice and age / sex-matched WT littermates. Thirty days after labeling, however, forebrain Ca_v1.2 cKO mice showed significantly fewer surviving BrdU+ cells, indicating that Ca_v1.2 mediates survival, and not proliferation, of young hippocampal neurons in the adult brain. This suggests that survival of these young neurons may be important in warding off neuropsychiatric disease. Since increased cell death in some cases may result in decreased hippocampal volume,¹⁸ we next performed morphometric analysis of the dentate gyrus, CA1 and CA3 hippocampal regions. We observed no differences in size of these regions between forebrain Ca_v1.2 cKO mice and age / sex-matched WT littermates, further supporting a more prominent role of Ca_v1.2 in adulthood rather than development.

Because the survival component of postnatal hippocampal neurogenesis is regulated in part by the neurotrophic agent brain-derived neurotrophic factor (BDNF),¹⁹ and LTCC-signaling controls BDNF expression in in vitro systems,^20,21 we measured BDNF protein levels in forebrain Ca_v1.2 cKO mice. Here, we observed a 50% reduction relative to age / sex-matched WT littermates (Fig. 1), providing the first in vivo evidence that BDNF expression in the adult brain is controlled by Ca_v1.2. We next wondered whether we could restore normal hippocampal neurogenesis in forebrain Ca_v1.2 cKO mice without intervening in the underlying BDNF-deficit, as there are no current drug therapies that specifically target BDNF levels in the brain. To address this problem, we turned to the neuroprotective aminopropyl carbazole compound P7C3-A20, a small molecule that blocks neuronal cell death,^22-28 thereby increasing the magnitude of hippocampal neurogenesis.^29-31 Treatment of forebrain Ca_v1.2 cKO mice with P7C3-A20 restored normal survival of young hippocampal neurons (Fig. 1), as measured by BrdU-labeling and DCX expression. Restoration of normal hippocampal neurogenesis was not accompanied by an increase in BDNF, however, demonstrating that deficits in hippocampal neurogenesis associated with BDNF deficiency in the adult brain can be corrected with therapeutic agents that do not restore BDNF.

Anxiety-like behavior, which is prominently displayed by forebrain-conditional Ca_v1.2 KO mice,⁹ can occur as a consequence of over-activation of the hypothalamic-pituitary-adrenal (HPA) axis, leading to elevated levels of circulating corticosterone.³² Moreover, environmental stress, either chronic or acute, is known to augment the development and progression of mental illness.^33-37 Since forebrain Ca_v1.2 cKO mice show elevated anxiety-like behavior, we tested whether they also displayed altered levels of plasma corticosterone. We found no difference in either basal or acute stress-induced corticosterone levels in forebrain Ca_v1.2 cKO mice compared to age / sex-matched WT littermates. Thus, the anxiety-like behavior in this preclinical model of neuropsychiatric disease is independent of the HPA axis, further underscoring the importance of CACNA1C in precipitating common symptoms across multiple forms of neuropsychiatric disease.

In addition to the HPA axis, a substantial body of literature supports a role for hippocampal neurogenesis in depression and anxiety, including evidence that increasing postnatal hippocampal neurogenesis reduces anxiety and depression-like behaviors in mice.^12,38-40 Furthermore, a recent small Phase 1B, randomized, double blind, placebo controlled, multiple-dose escalation study using a new drug that stimulates hippocampal neurogenesis, known as NSI-189 phosphate, has shown favorable outcome in treatment for MDD in humans, with significant improvement shown in the Symptoms of Depression Questionnaire and Cognitive and Physical Functioning Questionnaire.⁴¹ Future work in forebrain Ca_v1.2 cKO mice will determine whether restoration of normal hippocampal neurogenesis in adulthood will ameliorate anxiety-like behavior.

Taken together, our recent work demonstrates that Ca_v1.2 serves a critical role in protecting young hippocampal neurons from death in the adult brain. This is in contrast to the known role of the other predominant LTCC in the hippocampus, Ca_v1.3, which is involved in both proliferation and maturation of newborn hippocampal neurons.⁴² Mice harboring genetic deficiency in Ca_v1.3 also show smaller dentate gyrus volume, underscoring the likelihood of a developmental role for Ca_v1.3 as well.⁴² Future delineation of the distinct and overlapping physiologic roles of Ca_v1.2 and Ca_v1.3 in hippocampal neurogenesis will provide insight into new therapeutic strategies to augment neurogenesis in ways that may benefit patients.

Another critical future direction is to determine whether the survival deficit in young hippocampal neurons of forebrain Ca_v1.2 cKO mice is a cell autonomous or non-autonomous effect. Since BDNF is released from neurons and promotes neuronal survival,⁴³ and we observe a 50% BDNF reduction in these mice, our results suggest a non-autonomous effect. However, this does not exclude the possibility that Ca_v1.2 may also play a role in neuronal cell death by regulating calcium signaling in a cell-autonomous manner.

If the latter possibility proved to be true in young hippocampal neurons, this would raise the possibility that Ca_v1.2 might also play a role in regulating death of mature nerve cells as well. If so, this could point to new therapeutic opportunities to mitigate neuronal cell death in a broad array of neuropsychiatric conditions, including neurodegenerative disease and normal aging, while also providing evidence for a role of neurodegenerative processes in progression of multiple forms of mental illness. Indeed, the progressive nature of many forms of neuropsychiatric disease, including those linked to CACNA1C, is consistent with neurodegenerative processes that would be amenable to treatment with neuroprotective agents.⁴⁶ In this light, it is useful to note that P7C3 compounds have demonstrated protective efficacy in preclinical models of Parkinson disease,^24,28-29 cognitive decline with aging,³¹ amyotrophic lateral sclerosis,²³ traumatic brain injury,^25-27 peripheral nerve injury,²² neurodegeneration-associated depression,⁴⁶ and now cell death associated with genetic susceptibility to mental illness.¹⁷ It has recently been reported that P7C3 compounds enhance flux of the nicotinamide adenine dinucleotide (NAD) salvage pathway in normal mammalian cells, and also facilitate NAD recovery following doxorubicin exposure.³⁶ NAD is important in regulating essential substrates for mitochondrial metabolism, and is also a major substrate for ATP production important to neuronal survival.³⁷ Thus, P7C3 compounds, or other pharmacologic interventions with neuroprotective or NAD-augmenting capability, might be useful for mitigating disease progression in many forms of mental illness, for which only symptomatic treatment is currently available.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by funds from the University of Iowa Carver College of Medicine to A.A.P, funds from an anonymous donor to the Mary Alice Smith Fund for Neuropsychiatry Research to A.A.P, a National Science Foundation fellowship to H.DJ-C. Funding from The Hartwell Foundation to A.M.R and A.A.P., and Weill Cornell Autism Research Program funding to A.M.R. A.A.P. holds patents on the P7C3 family of neuroprotective compounds.