Ataxia in calcium channelopathies.

(A) Overall aim and key objectives. It is the long-term goal of our research to elucidate the major underlying mechanisms of cerebellar ataxia in calcium channelopathies. Over the past decade several new members of a family of voltage dependent calcium channels have been identified including the P, Q, N, R, L, and T subtypes. All these evolutionary related channels consist of an unique alpha (a1) unit containing the ion pore, and the accessory proteins ß, ? and a2d that modulate the electrophysiologica! properties of the ion pore. We recently demonstrated that the a1 unit of the P/Q subtype channel is encoded by the CACNA1A gene, and that mutations in this gene can lead to syndromes with ataxia such as episodic ataxia type 2, familial hemiplegic migraine, spinocerebellar ataxia type 6 and progressive ataxia (Ophoff et al., 1996; van den Maagdenberg et al., 2001). However, as this type of channel is widely distributed in the brain including several components of the olivocerebellar system, the exact pathophysiological mechanisms leading to ataxia as well as some of its variable features such as its episodic character remain to be elucidated. It is the overall aim of the present proposal to elucidate these mechanisms by investigating several spontaneous mouse mutants with mutations in the mouse ortholog Cacna1a (key objective 1) and by creating and testing cell-specific mouse mutants and rescues in which the various mutations of the CACNA1A gene are induced or rescued in a cell-specific and conditional manner (key objective 2). The natural mouse mutants carrying mutations in the gene encoding the a1a subunit include the tottering, rocker, rolling-Nagoya, and leaner, all these mice have different mutations and show various forms and levels of ataxia. The cell-specific mutants that will be created include both Purkinje cell-specific mutants and inferior olive cell-specific mutants as both major cell types carry the P-channel; the Purkinje cells form the sole output of the cerebellar cortex and the olivary neurons from the sole source of the climbing fibers, which constitute one of the two major inputs to the cerebellar cortex. The genes that will be manipulated and/or inserted do not only include the murine orthologs but also human constructs so as to optimally mimic the human syndromes and the underlying pathophysiological mechanisms. (B) Approach. To elucidate the mechanisms underlying the various forms of ataxia in human calcium channelopathies we will investigate the natural mouse mutants with mutations in the mouse ortholog Cacna1a (key objective 1) and we will create and test cell-specific mouse mutants and rescues in which the various mutations of the CACNA1A gene are induced or rescued in a conditional manner in the olivo-cerebellar system (key objective 2). Kev objective 1 (characterization of a1a natural mutants) The mouse mutants with mutations in their a1a subunit, i.e. the tottering, rolling-Nagoya, rocker and leaner, are already available in our laboratories and will be subjected to systems physiological, cell physiological and developmental investigations. All these investigations will be focused on the part of the olivo-cerebellar system that controls compensatory eye movements, i.e. the vestibulocerebellum, because in this control system one can determine and measure adequately both the sensory inputs (i.e. visual and vestibular stimuli) and the motor output (i.e. the eye movement behaviour), as well as the neuronal activities in between (De Zeeuw et al., 1998a; Goossens et al., 2001). Moreover, all the major behavioural and electrophysiological properties of this system as well as its major neuroanatomical circuitries are known, and the system lends itself well to both short-term and long-term manipulation so that both the acute dynamics and memory formation of cerebellar motor behaviour can be investigated (De Zeeuw et al., 1998ab; Stahl et al., 2000; Van Alphen et al., 2001). We have recently obtained behavioural and electrophysiological pilot-data in vivo from tottering mice, which reveal impairments of both gain and phase values. Based upon these recordings we postulate the hypothesis that an appropriate intracellular calcium homeostasis in Purkinje cells is essential for an optimal signal to noise ratio in their firing behaviour during modulation and adaptation. Thus, the cerebellar ataxia of natural mouse mutants with calcium channelopathies can be precisely measured and quantified leading to new hypotheses on cellular and systems, short-term and long-term processes, and equally important they can be directly related to eye movement recordings in patients with calcium channelopathy. Eye movement recordings of such patients take place both in Rotterdam and Cleveland (in collaboration with Dr. J. Stahl), but these studies are part of a parallel project and are not included in the present proposal. In addition, we will investigate the intracellular consequences of calciurn channelopathy in both Purkinje cells and olivary neurons by investigating the processes of calcium influx and efflux in the natural mutants. These cell physiological in vitro investigations should provide insight into the intracellular pathophysiological mechanisms that may contribute to the in vivo phenotypes obtained with the extracellular recordings described above. As both the Purkinje cells and the olivary neurons, which provide the climbing fibers to the Purkinje cells, express the P-type calcium channel, the cell physiology experiments will initially focus on the climbing fiber-mediated complex spikes that can be measured in Purkinje cells using the patch-clamp technique. Complex spikes are composed of an initial Na spike that is followed by several slower components that are caused by activation of dendritic Ca conductances. The complex spike activity may not only underlie short-term control of the timing of movements (De Zeeuw et al., 1996 and 1998b), but it probably is also required for the induction of long-term depression (LTD) at the parallel fiber-Purkinje cell synapses (PFLTD), LTD at the climbing fiber input itself (CF-LTD; Hansel and Linden, 2000), and a longlasting enhancement of inhibitory drive ('rebound potentiation') resulting from the activation of GABAA receptors (for review see Hansel et al., 2001). Because the occurrence of complex spikes and associated dendritic Ca signals can be blocked by injection of Ca chelators into the postsynaptic Purkinje cells, the control of intracellular and extracellular calcium concentrations and fluxes are considered to be a key factor in triggering the above mentioned types of dynamic control and synaptic plasticity, and thereby the various forms of motor learning and ataxia (De Zeeuw et al., 1998ab). Part of key objective 1 will be to characterize the impact of the various naturally occurring mutations in the CACNA1A gene on the complex spike waveform and the complex spike-associated dendritic Ca signals. These Ca signals will be measured using microfluorometric Ca imaging techniques (see also Hansel et al., 1997). Key objective 2 (creation and analvsis of specific transgenic mutants). The experiments described above will determine the ultimate boundaries of the potential ataxic symptoms and they may reveal some of the pathophysiological mechanisms as they occur in patients. However, the natural mutants will provide the ultimate integration of a collection of individual aberrations, and as these individual effects are part of the same feedforward and feedback circuitries they won't allow us to pinpoint the exact impact of a mutated channel in one particular cell type. Even the cell physiological experiments in slices described above will unravel deficiencies that result from a cell developed in an organism with multiple defects at multiple locations. Thus, if we want to understand the impact of de novo channelopathy in individual cell types, and if we want to find out to what extent restoration of expression of the appropriate P-channel in an individual cell type can affect the various forms of ataxia in mice and humans, we also have to create and test cell-specific mutants. Therefore, for the present program, which is focused on the a1a-containing P-type calcium channel, we propose to create and test the following sets of mutants: a. Mutants that will allow us to determine the specific contribution of a mutated P-type channel in Purkinje cells ór olivary neurons to the generation of ataxia (L7/Cacna1a-T666M and Brn3a/Cacna1a-T666M). These mutants will be created in a conditional fashion so that the impact of different stages of development can be investigated. In conjunction with the Cre-system, we will employ the L7-vector and Brn3a-promotor to obtain inducible and cell-specific expression of the mutated channel in Purkinje cells and inferior olivary neurons, respectively (Oberdick et al., 1990; Xiang et al., 1996). Based upon our electrophysiological and behavioural recordings in the tottering mice we predict that expression of the mutated channels in the Purkinje cells, but not in the olivary neurons, will be sufficient to induce ataxia. As a control we will rescue a normal expression specifically in Purkinje cells ór olivary neurons of the tottering mutants that have a P6O1L mutation so as to find out to what extent the ataxia can be removed by over-expressing a normal channel in the respective cell types of the otherwise mutated animal. b. Mutants that will allow us to identify potential differences between the human and mouse channelopathies (L 7/humanCACNA1A-T666M re L 7/mouseCacna1 a-T666M). The mutants designed above will be created with mouse constructs of Cacna1a. However, although the human and mouse orthologs of the alA protein are well conserved, some differences in sequences do occur and some consequences in the dynamics of the channels cannot be excluded. Thus, to extrapolate the clinical implications of our mutant studies optimally, we will directly compare the effects of expression of mouse Cacna1a-T666M with that of its human ortholog. This investigation will initially be focused on the Purkinje cells, but if the investigations under key objectives 1 and 2A imply a dominant role for the olivary neurons, we will also express the mutated human a1a, unit specifically in olivary neurons. All the necessary promoters (L7 and Brn3a) as well as the human cDNA constructs are available in our laboratonies in Rotterdam and/or Leiden, and some of them have already been successfully applied (De Zeeuw et al., 1998; Kraus et al., 1998; Hans et al., 1999; Toru et al., 2000). c. Mutants that will allow us to compare the underlying mechanisms of episodic ataxia with those of non-episodic ataxia (L7/CACNA1A-EA-2truncation, and L7/CACNA1A-H253Y re L7/CACNA1A-CAG24, L7/CACNA1A-G293R and L7/CACNA1A-T666M). Another important specific aim of this proposal is to find out why some of the calcium channelopathies cause episodic ataxia (episodic ataxia / EA-2), while others induce permanent ataxia (spinocerebellar ataxia 6 / SCA6; familial hemiplegic migraine / FHM; progressive ataxia). Thus, once we have identified the different roles of the two major cell types in the olivocerebellar system (i.e. Purkinje cells and inferior olivary neurons; key objective 2A) and the major phenotypical differences between expression of the human and mouse constructs (key objective 2B), we will start to investigate the different impacts of the expression of the various human cDNA constructs that encode the mutated a1a units in the various syndromes of ataxia. These human cDNA constructs include L7/CACNA1A-EA~2truncation with a stop in exon 37 and L7/CACNA1A-H253Y with the H253Y mutation both of which cause episodic ataxia; they include L7/CACNA1A-CAG24 with an extended carboxyl-terminus and a translated CAG-repeat, which causes spinocerebellar ataxia 6; they include L7/CACNA1A-G293R with the G293R mutation, that causes progressive ataxia without episodic features; and they include the L7/CACNA1A-T666M with the T666M mutation that causes FHM and permanent ataxia and that will also be used for key objectives 2a and b. As indicated by the use of L7 these mutants will initially be made to investigate the expression of the mutated channel in Purkinje cells, but dependent on the outcome of key objective 2a some additional mutants may be created in which the olivary neurons are affected. The mutants that have been descnibed under key objectives 2a, b and c will in principle all be subjected to the major behavioural and systems physiological tests described under key objective 1. However, time wise it will be impossible to investigate all the mutants at the electrophysiological level; therefore we will restrict ourselves in this regard to the mutants with the most prominent and/or promising phenotypes. (C) Elements of innovation. Over the past decade an increasing number of mutations have been identified in the calcium channel gene CACNA1A in both mouse and man; yet, we still do not understand how the mutations lead to the various pathological syndromes. With the present multidisciplinary approach we may for the first time uncover the specific cells and their intracellular processes that are responsible for the various forms of ataxia. In addition, with the present rescue experiments we may start to get an impression how the ataxia and its related symptoms may be reduced or eliminated by a transgenic approach. These findings may set the stage for therapeutic measures in the future. (D) Relevance for health care. The calcium channelopathies form a unique group of diseases in that they include, as described above, a unique rich group of neurological human syndromes most of which are either mimicked by a natural mouse mutant or can be mimicked by the creation of a new cerebellar mutant. Thus, by investigating these diseases in conjunction with the wide variety of animal models of these diseases we open up a broad avenue of fundamental research that will probably have many and precise clinical implications. Morcover, it should be noted that at least some of the mutations described above also play a role in the occurrence of migraine (Ophoff et al., 1996). By unraveling the ins and outs of the consequences of the mutations in the calcium channels in a quantifiable fashion as can be done by investigating their role in the vestibulo-cerebellar system, we may also gain insight in how they may cause migraine, which is much harder to study quantitatively in an animal model.