T-type Ca(2+) channels: New players in the aging brain.

Perhaps one of the most remarkable features of T-type calcium channels is their low-threshold of activation that makes these channels important candidates for calcium entry near the resting membrane potential of neurons. Hence, they mediate low-threshold burst discharges that occur during different forms of neuronal rhythmogenesis, but play also important roles in sensory transmission, as well as hormone and neurotransmitter release. Additionally, they have been implicated in an increasing number of neuronal pathologies including neuropathy, autism spectrum disorders and some forms of epilepsy. More recently, an implication of T-type calcium channel in the processing of Amyloid Precursor Protein was documented, with possible implication in the pathogenesis of Alzheimer's disease.

Neuronal calcium (Ca2+) signaling is of critical importance as it participates in a plethora of cellular processes including membrane excitability, gene expression, synaptic transmission, synaptogenesis, cell death and survival, but also neuronal processes underlying learning and memory. In order to make use and regulate the amplitude, duration and subcellular localization of the Ca2+ signal, cells have developed a complex machinery, the so-called “Ca2+ signaling toolkit,” comprised of ion channels, pumps, and exchangers both in the plasma membrane and in the membranes of intercellular organelles (endoplasmic reticulum, mitochondria, Golgi apparatus and nucleus), but also Ca2+ binding proteins and transcriptional factors that altogether coordinate neuronal Ca2+ signaling and homeostasis. It is thus not surprising that alteration of the Ca2+ signaling machinery, causing either a loss or a gain of function, can lead to serious chronic neuronal disorders. Therefore, despite major intrinsic differences in the pathogenesis of neurodegenerative diseases, alteration in Ca2+ homeostasis has emerged as a common underlying mechanism in Parkinson’s disease, Huntington’s disease, spinocerebellar ataxias, amyotrophic lateral sclerosis or Alzheimer’s disease (AD). Whereas the molecular mechanisms responsible for alterations in neuronal Ca2+ signaling in the aging brain are not clearly understood, numerous studies have reported age-related changes in the expression and/or activity of some of the key players of the Ca2+ machinery, often associated with decreased synaptic plasticity, and in some cases with progressive neuronal loss. Hence, an increased activity of L-type voltage-gated Ca2+ channels has been reported in aged hippocampal neurons., In parallel, elevated intracellular Ca2+ release from the endoplasmic reticulum through ryanodine receptors (RyRs) has been observed, possibly via enhanced Ca2+-induced Ca2+ release (CICR) as consequence of increased Ca2+ influx through L-type Ca2+ channels. Additionally, decreased plasma membrane Ca2+-ATPase activity and Ca2+ extrusion, as well as diminished Ca2+ buffering capacity of the aged neuron (decreased SERCA pump activity and mitochondrial Ca2+ sink have been reported, leading to an overall intracellular free Ca2+ overload. Consistent with the Ca2+ theory of the aging brain, pharmacological regulation of Ca2+ homeostasis using either Ca2+ chelators or Ca2+ channel inhibitors have shown beneficial effects on age-related cognitive declines. In a recent study published in Neurobiology of Aging, Rice et al., have examined the possible implication of new Ca2+-related proteins in the aging brain. The results of this study indicate an age-dependent alteration in the expression of Cav3.1 T-type Ca2+ channel. More importantly, the authors demonstrate that T-type Ca2+ channels are functionally coupled to the proteolytic processing of Amyloid Precursor Protein (APP), one of the key suspects in the pathogenesis of Alzheimer’s disease.

In a first set of experiments, the authors investigated age-related changes in the expression of some of the Ca2+-related genes. Microarray analysis from human brain samples revealed a significant decrease of about 45% in the expression of CACNA1G, that encodes the low-voltage-activated Cav3.1 T-type Ca2+ channel, in aged non-diseased (74–95) compared with young non-diseased (20–59) individuals. This drop was observed in various brain regions supporting cognitive functions, including the entorhinal cortex, the hippocampus, the posterior cingulate gyrus as well as the superior frontal gyrus. Interestingly enough, a further decrease in CACNA1G expression was observed in AD aged-patients compared with control aged-individuals in the hippocampus, a region well known to play an important role in the formation of new memories, but also one of the first regions in the brain to suffer damage in AD. Consistent with a drop in CACNA1G expression in the aging brain, western blot analysis of Cav3.1 channel performed on 3xTg-AD mouse brain (a genetic AD mouse model) showed a significant decrease in channel expression with aging. However, a similar decrease in Cav3.1 channel expression was also observed in wild-type animals, suggesting that alteration in Cav3.1 channel expression is mostly related to aging, regardless of the development of AD. In order to evaluate if alteration in Cav3.1 channel expression observed in the aging brain contributes to the development of AD, the authors analyzed the functional implication of the channel in the processing of that is believed to play a critical role in the pathogenesis of AD. It is well established that proteolytic cleavage of APP by the β-secretase (also known as β-site APP cleaving enzyme-1 BACE1) produces a soluble form of APP (sAPPβ) and a 99-amino acid C-terminal fragment (C99) that remains anchored in the plasma membrane. Subsequent processing of C99 by a γ-secretase generates the so-called amyloid-β peptide (Aβ), mainly consisting of a 40-amino acid peptide (Aβ40) and the hydrophobe 42-amino acid product (Aβ42) that tends to form fibrils, and constitutes the main component of extracellular senile plaques observed in AD brains. In contrast, proteolytic cleavage of APP by the α-secretase produces a soluble non-amyloidogenic product sAPPα with neutrophic effects and promoting synaptogenesis, and an 83 amino acid C-terminal peptide (C83), precluding the production of Aβ. Interestingly, in vivo pharmacological inhibition of T-type Ca2+ channels by intraperitoneal injection of NNC-55–0396 (NNC, a specific T-type channel inhibitor) in 14–16 mo old 3xTg-AD mice produced an increase in soluble Aβ (Aβ40 and Aβ42). However, increased levels of soluble Aβ40 and Aβ42 were not accompanied by any changes in the number and density of typical senile plaques formed by Aβ. It is possible that the relatively short-term application (2 wk) of NNC precludes the formation of new fibrils. Consistent with this idea, a trend to an increased insoluble Aβ42 was noticed, suggesting that long-term treatment of 3xTg-AD mice with T-type channel inhibitors could not only produce an increase in soluble Aβ but may also be accompanied by the formation of new senile plaques. Moreover, active α-secretase ADAM10 and its proteolytic product C83 were found significantly decreased upon in vivo inhibition of T-type Ca2+ channels. In contrast, immature ADAM10 was found unchanged, suggesting that blocking T-type Ca2+ channels in vivo possibly prevents the maturation of ADAM10 rather than its genic expression. Consistent with in vivo observations, in vitro application of NNC on neuroblaoma cells N2a reduced expression of active ADAM10 and C83 peptide while soluble Aβ40 and Aβ42 levels were found increased. In contrast, transient overexpression of Cav3.1 channel in HEK cells stably expressing APP increased the formation of soluble α-APPs suggesting activation of the α-secretase pathway. However, in contrast to in vivo observations, only a non-significant trend to an increased level of active ADAM10 was observed, questioning the molecular mechanisms by which T-type Ca2+ channels contribute to the processing of APP. Because of the rather depolarized membrane potential of HEK cells (around -30mV depending on the culture conditions), T-type Ca2+ channels are mostly inactivated and their contribution in any Ca2+-dependent processes at rest is rather unlikely. The same applies to neuroblastoma cells. Besides the fact that it remains unclear if T-type Ca2+ channels are functionally expressed in N2a cells,, it is unlikely that undifferentiated cells can generate electrical activity required for T-type Ca2+ channel activation. Further investigations will certainly identify the precise molecular mechanisms by which T-type Ca2+ channels contribute to the processing of APP. (Fig. 1)

Figure 1.A putative model of functional coupling between T-type Ca The amyloidogenic pathway is characterized by the proteolytic cleavage of APP by β-secretase (BACE1) and the resulting production of a soluble APP fragment (sAPPβ) and a 99-amino acid C-terminal fragment (C99) that is subsequently processed by γ-secretase to generate neurotoxic Aβ (Αβ40 and Αβ42) peptides that tend to aggregate and form extracellular senile plaques. In contrast, the non-amyloidogenic processing of APP by α-secretase (ADAM10) produces an 83-amino acid C-terminal fragment (C83) precluding the formation of APP, and a soluble sAPPα product with neurotrophic effects that counteract apoptotic signaling and promote synaptogenesis. In the young developing brain, activation of T-type Ca2+ channels stimulates non-amyloidogenic processing of APP, possibly by facilitating the maturation of ADAM10, which supports neuronal growth and synaptogenesis. In contrast, alteration of T-type Ca2+ channel expression in the aging brain favors amyloidogenic processing of APP and associated neuronal damages.

In summary, Rice et al. documented for the first time an age-dependent decrease in Cav3.1 T-type Ca2+ channel expression, extending the growing list of Ca2+-dependent proteins possibly involved in the aging brain. More importantly, they provided evidence for a functional implication of T-type Ca2+ channels in the proteolytic processing of APP where proper channel activity is required for non-amyloidogenic processing of APP, precluding the production of Aβ and possibly associated senile plaques. Interestingly, a recent study documented an increase in insoluble Aβ42 production in the aging brain. The age-dependent alteration in Cav3.1 T-type Ca2+ channel expression observed by Rice et al. could certainly contribute to the changes in APP processing with aging and possibly contribute to the pathogenesis of AD. Moreover, considering the critical role of T-type Ca2+ channels in supporting physiological functions as learning and memory, it is conceivable that alteration in channel activity could participate in the cognitive declines associated with the aging brain. It thus appears that pharmacological activation of T-type Ca2+ channels could represent a promising therapeutic strategy to prevent and/or reverse age-dependent cognitive declines, and possibly to preclude the formation of Aβ senile plaques that is still considered as one of the main hypothesis of AD., Consistent with this idea, it was recently shown that T-type Ca2+ channels are the pharmacological targets of the cognitive enhancer ST101. In contrast, pharmacological inhibition of T-type Ca2+ channels has recently emerged as a promising therapeutic strategy for the treatment of some forms of epilepsy, as well as for the management of neuropathic pain., Hence, if the development of new T-type Ca2+ channel modulators is emerging as especially attractive therapeutic strategy for the treatment of various neurological disorders, their general use in clinic has to be cautious to not face the Jekyll and Hide syndrome of T-type channels like it has been shown for Aβ.