HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/7/1288 00-0562fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by FLETCHER, C. F. Articles by JENKINS, N. A. Search for Related Content PubMed PubMed Citation Articles by FLETCHER, C. F. Articles by JENKINS, N. A.

Dystonia and cerebellar atrophy in Cacna1a null mice lacking P/Q calcium channel activity¹

COLIN F. FLETCHER^*2,3, ANGELITA TOTTENE^,3, VANDA A. LENNON, SCOTT M. WILSON^*, STEFAN J. DUBEL, RICHARD PAYLOR^¶, DAVID A. HOSFORD, LINO TESSAROLLO^*, MAUREEN W. McENERY, DANIELA PIETROBON, NEAL G. COPELAND^* and NANCY A. JENKINS^*

^* Mouse Cancer Genetics Program, National Cancer Institute-FCRDC, Frederick, Maryland 21702, USA;
Department of Biomedical Sciences and National Research Council (Consiglio Nationale delle Richerche) Center of Biomembranes, University of Padova, 35121 Padova, Italy;
Departments of Immunology and Neurology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, Minnesota 55905, USA;
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA;
^¶ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA; and
Clinical Genetics/Disease Sciences, GlaxoWellcome, Stevenage, Herts, SG12NY, U.K.

²Correspondence: GNF, 3115 Merryfield Row, San Diego, CA 92121, USA. E-mail: Fletcher{at}gnf.org

SPECIFIC AIMS

P/Q-type voltage-dependent calcium channel CACNA1A mutations cause dominantly inherited migraine, episodic ataxia, and cerebellar atrophy in humans and recessively inherited ataxia, episodic dyskinesia, cerebellar atrophy, and absence epilepsy in the mouse. To investigate the disease mechanism and identify required P/Q function in vivo, we created and characterized a Cacna1a null mutation (designated Cacna1a^Fcrtm1).

PRINCIPAL FINDINGS

1. Targeted disruption of the Cacna1a gene produces severe ataxia and late-onset neurodegeneration
To construct a null allele of the _1A gene, we created a targeting vector in which exons encoding most of domain II (DII) of the _1A protein were replaced by a neomycin resistance selection cassette. Cerebellar and forebrain homogenates from control, Cacna1a^Fcrtm1/+, and Cacna1a^Fcrtm1/ Cacna1a^Fcrtm1 animals were examined by Western blot for the presence of _1A protein. No _1A protein was detected in Cacna1a^Fcrtm1/ Cacna1a^Fcrtm1 animals. Cacna1a^Fcrtm1/+ animals expressed 47% of wild-type levels of _1A protein.

Homozygous mutant mice could be identified reliably by visual inspection 10 days after birth. Mutant mice were initially detected by difficulty in righting themselves and later by smaller size and dystonia. By 20 days of age, mutant mice lie on their side or back, with limbs extended, in a stiff, hunched posture and sometimes make slow movements of their limbs, appearing to attempt to right themselves. Mice placed upright maintain a stiff posture with extended limb(s) and rigid muscle tone.

To determine whether morphogenetic abnormalities could explain the early lethality, brains from 20-day-old mice were examined. Histological analysis revealed no obvious cytoarchitectural differences between Cacna1a^Fcrtm1/ Cacna1a^Fcrtm1 and control animals. To detect progressive neurodegeneration, brains of mice aged 15 wk were examined. There is an obvious reduction in the size of the cerebellum in in Cacna1a^Fcrtm1/ Cacna1a^Fcrtm1 mice (Fig. 1C , F ), whereas Cacna1a^Fcrtm1/+ sections (Fig. 1B , E ) are similar to normal controls (Fig. 1A , C ). Neuronal loss was detected in hematoxylin- and eosin- stained sections. Staining of Purkinje cells with calbindin antibodies reveals a striped pattern of cell loss in Cacna1a^Fcrtm1/ Cacna1a^Fcrtm1 mice (Fig. 1C ). Cresyl violet staining revealed a graded loss of granule neurons, more severe in the anterior lobes (Fig. 1F ). Frank neuronal loss of Purkinje cells was not apparent before postnatal day 40 (data not shown). Examination of brains from three aged mice did not reveal abnormalities in other regions.

View larger version (60K): [in this window] [in a new window]   Figure 1. Sections from postnatal day 100 animals. Sections were stained with anticalbindin antibody to visualize Purkinje cells (A–C) and cresyl violet to visualize granule cells (D–F). A, C) Control, B, D) Cacna1a Fcrtm1 heterozygous, and C, E) Cacna1aFcrtm1 homozygous animals. Loss of Purkinje cells in a pattern of parasagittal stripes and loss of granule cells in an anterior to posterior gradient are evident in the Cacna1aFcrtm1 homozygous sections.

2. Obliteration of P/Q-type calcium channel current and increase of L- and N-type currents in Cacna1a null mice
We measured the P/Q-type calcium current of mouse cerebellar granule cells in primary culture using -CTx-MVIIC, a toxin that inhibits reversibly N-type channels and irreversibly both P- and Q-type channels. -CTx-MVIIC was applied after subsequent additions of saturating concentrations of nimodipine and the specific blocker of N-type channels -CgTx-GVIA. This protocol enabled us to measure the L-, N-, and R-type calcium current components in addition to the P/Q type. A large fraction of current was inhibited slowly and irreversibly by -CTx-MVIIC in neurons from wild-type control mice (32±2%, 15.5±1.2 pA/pF, n=23), whereas neurons from homozygous mutant Cacna1a^Fcrtm1 mice had no detectable current inhibited irreversibly by -CTx-MVIIC, indicating that disruption of the _1A gene obliterates P/Q-type channel activity in these cells (Fig. 2) . Obliteration of the P/Q-type calcium channel current in homozygous knockout mice was partially compensated by an increase of the L- and N-type current densities. The contribution of L-type channels increased from 9.1 ± 0.9 to 14.7 ± 1.1 pA/pF (P<0.001) and that of N-type channels from 6.1 ± 0.4 to 7.6 ± 0.5 pA/pF (P<0.05). The R-type current densities were similar in control and mutant mice (18.2±1.1 and 17.8±1.3 pA/pF, respectively). The total current density decreased from 48.9 ± 2.6 to 40.1 ± 2.2 pA/pF (P0.05).

View larger version (15K): [in this window] [in a new window]   Figure 2. P/Q-type current density in cerebellar granule cells from targeted mutant mice. Whole-cell recordings with 5 mM Ba2+ as charge carrier. A) Control mouse, cell U226A, 7 DIV. Plot of peak current vs. time for a representative experiment in which nimodipine (5 µM), -CgTx-GVIA (1 µM), and -CTx-MVIIC (3 µM) were sequentially applied. Examples of traces from the same experiment taken at the times indicated by a, b, c, and d are shown in the inset. Depolarizations at Vt = -10 mV were delivered every 10 s from Vh = -80 mV. Scale bars: 100 pA, 20 ms. B) Homozygous Cacna1aFcrtm1/ Cacna1aFcrtm1 mice. Cacna1aFcrtm1 is referred to here as ‘ko’ for clarity; cell U203E, 7DIV. Plot of peak current vs. time for a representative experiment performed as in panel A. C) Average density of current inhibited irreversibly by -CTx-MVIIC in 23 cells of control mice, 24 cells of Cacna1aFcrtm1/+ mice, and 24 cells of Cacna1aFcrtm1/Cacna1aFcrtm1 mice from 5, 5, and 4 different neuronal cultures, respectively.

3. Reduction of P/Q-type calcium channel current without compensation by other calcium current components in heterozygous knockout mice
The P/Q current density in cerebellar granule neurons from heterozygous mutant animals was reduced 50% (from 15.5±1.2 to 6.8±0.5 pA/pF, P<0.001) compared to wild-type control animals. The contributions of L-, N-, and R-type channels were similar in control and heterozygous mice. The total current density decreased from 48.9 ± 2.6 to 39.2 ± 1.3 pA/pF (P<0.05).

4. Reduced P/Q current density is not associated with a discernible neurological deficit
Upon visual inspection, the Cacna1a^Fcrtm1 heterozygous mice had no abnormal phenotype and could perform simple tasks as well as wild-type littermates, such as descending from a pole or crossing a beam. To detect subtler deficits, age- and sex-matched mice were evaluated with an accelerating rotarod. No difference in performance between Cacna1a^Fcrtm1 heterozygous and control mice was detected (P0.05) in 12 trials. Electroencephalograms were recorded from Cacna1a^Fcrtm1 heterozygous mice. The null allele, therefore, behaves as a strictly recessive mutation.

CONCLUSIONS

We have constructed and characterized a targeted mutation of the mouse Cacna1a gene that encodes a pore-forming subunit of the P/Q-type calcium channel. Mice homozygous for this mutation exhibit dystonia and most do not survive past weaning. We have successfully aged some mice and found that they exhibit progressive cerebellar degeneration in a specific pattern, indicating a requirement for P/Q function for survival of a subpopulation of neuronal cells. The pattern of neurodegeneration in the hull mutant is identical to that seen in another mouse Cacna1a mutant allele, leaner (Cacna1a^tg-rol), and resembles the cerebellar atrophy seen in human patients with CACNA1A mutations. The leaner mutation is consistent with severe loss of function, since the P/Q current density is 70–80% reduced in Purkinje cells. Our data suggest a critical threshold of P/Q function necessary for neuronal survival in the cerebellum. Partial reduction in P/Q current density, seen in tottering (Cacna1a^tg) and the heterozygous knockout mice, does not cause degeneration, but more severe reduction, seen in Cacna1a^tg-ro and homozygous null, causes degeneration. Since the overall calcium current density is similar in heterozygous and homozygous null (due to compensation), it would appear that the degeneration is specifically related to the P/Q channel current level.

Our novel observation of reduced P/Q current density in heterozygous mice presented an opportunity to explore the significance of reduced P/Q current density in the absence of other mutation-associated alterations of Cacna1a function. Even though no alterations in biophysical properties of P/Q channels have been reported in Cacna1a^tg other than 50% reduced current density, those mice exhibit ataxia and seizures. Our examination of the Cacna1a^Fcrtm1 heterozygous mice indicates no detectable phenotype in terms of motor performance, neuroanatomical lesion, or EEG due to a 50% loss of P/Q channels. We conclude that reduced current density is not itself sufficient to cause the pathophysiology seen in the tottering mutant. This observation suggests that subtle functional alterations of P/Q channels or neuron-specific changes in VDCC expression must exist in tottering mice. For example, the tottering mutation may selectively affect different neuronal compartments if membrane targeting, transport, or assembly is specifically affected. It is important to note that, in general, relatively small changes in the absolute magnitude of channel activity may tip the delicate balance between excitatory and inhibitory signals and lead to abnormal excitability and neurological symptoms.

A most intriguing observation is the highly selective pattern of neuronal degeneration, with a graded loss of granule cells more severe in the anterior lobe and loss of Purkinje cells in parasagittal stripes in the cerebellum. It implies the existence of two types of cerebellar neurons: one with absolute dependence on P/Q channel function for survival, and another that can tolerate a lack of P/Q channels. It remains to be determined whether this dichotomy reflects 1) different intrinsic requirements for P/Q channels or 2) different extrinsic loads/inputs on cells that must be buffered by P/Q channel function. The first hypothesis suggests a functional specialization of P/Q channels, possibly due to isoform specific expression. Alternatively, our finding of an increase in the density of functional L- and N-type channels in cerebellar granule cells, and the similar finding reported by others in Purkinje cells, suggest the possibility that the two populations of cerebellar neurons may have a different ability to compensate for loss of P/Q channels. This is consistent with primary culture of granule cells selecting for neurons able to compensate and survive; therefore, differences in compensation need to be investigated further in intact preparations in order to identify vulnerable and resistant neurons. The second hypothesis posits different network functions between the stripes. Such a functional grouping cannot be completely related to inputs, since the current descriptions of the striped topography of afferent fibers are not completely concordant with this pattern.SCHEME

View larger version (30K): [in this window] [in a new window]   Scheme 1. No caption available.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0562fje ; to cite this article, use FASEB J. (March 5, 2001) 10.1096/fj.00-0562fje

³ Both authors contributed equally to this work.

This article has been cited by other articles:

				A. Mezghrani, A. Monteil, K. Watschinger, M. J. Sinnegger-Brauns, C. Barrere, E. Bourinet, J. Nargeot, J. Striessnig, and P. Lory A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels J. Neurosci., April 23, 2008; 28(17): 4501 - 4511. [Abstract] [Full Text] [PDF]

				C. G. Inchauspe, I. D. Forsythe, and O. D. Uchitel Changes in synaptic transmission properties due to the expression of N-type calcium channels at the calyx of Held synapse of mice lacking P/Q-type calcium channels J. Physiol., November 1, 2007; 584(3): 835 - 851. [Abstract] [Full Text] [PDF]

				A. Koschak, G. J. Obermair, F. Pivotto, M. J. Sinnegger-Brauns, J. Striessnig, and D. Pietrobon Molecular Nature of Anomalous L-Type Calcium Channels in Mouse Cerebellar Granule Cells J. Neurosci., April 4, 2007; 27(14): 3855 - 3863. [Abstract] [Full Text] [PDF]

				A. Katoh, J. A. Jindal, and J. L. Raymond Motor Deficits in Homozygous and Heterozygous P/Q-Type Calcium Channel Mutants J Neurophysiol, February 1, 2007; 97(2): 1280 - 1287. [Abstract] [Full Text] [PDF]

				N. E. Pardo, R. K. Hajela, and W. D. Atchison Acetylcholine Release at Neuromuscular Junctions of Adult Tottering Mice Is Controlled by N-(Cav2.2) and R-Type (Cav2.3) but Not L-Type (Cav1.2) Ca2+ Channels J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1009 - 1020. [Abstract] [Full Text] [PDF]

				H. B. Kordasiewicz, R. M. Thompson, H. B. Clark, and C. M. Gomez C-termini of P/Q-type Ca2+ channel {alpha}1A subunits translocate to nuclei and promote polyglutamine-mediated toxicity Hum. Mol. Genet., May 15, 2006; 15(10): 1587 - 1599. [Abstract] [Full Text] [PDF]

				S. Kaja, R. C. G. Van de Ven, M. D. Ferrari, R. R. Frants, A. M.J.M. Van den Maagdenberg, and J. J. Plomp Compensatory Contribution of Cav2.3 Channels to Acetylcholine Release at the Neuromuscular Junction of Tottering Mice J Neurophysiol, April 1, 2006; 95(4): 2698 - 2704. [Abstract] [Full Text] [PDF]

				C.-J. Jeng, Y.-T. Chen, Y.-W. Chen, and C.-Y. Tang Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2 Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1209 - C1220. [Abstract] [Full Text] [PDF]

				S.-M. Pulst, N. Santos, D. Wang, H. Yang, D. Huynh, L. Velazquez, and K. P. Figueroa Spinocerebellar ataxia type 2: polyQ repeat variation in the CACNA1A calcium channel modifies age of onset Brain, October 1, 2005; 128(10): 2297 - 2303. [Abstract] [Full Text] [PDF]

				D. Chaudhuri, B. A. Alseikhan, S. Y. Chang, T. W. Soong, and D. T. Yue Developmental Activation of Calmodulin-Dependent Facilitation of Cerebellar P-Type Ca2+ Current J. Neurosci., September 7, 2005; 25(36): 8282 - 8294. [Abstract] [Full Text] [PDF]

				D. Pietrobon Migraine: New Molecular Mechanisms Neuroscientist, August 1, 2005; 11(4): 373 - 386. [Abstract] [PDF]

				A. Tottene, F. Pivotto, T. Fellin, T. Cesetti, A. M. J. M. van den Maagdenberg, and D. Pietrobon Specific Kinetic Alterations of Human CaV2.1 Calcium Channels Produced by Mutation S218L Causing Familial Hemiplegic Migraine and Delayed Cerebral Edema and Coma after Minor Head Trauma J. Biol. Chem., May 6, 2005; 280(18): 17678 - 17686. [Abstract] [Full Text] [PDF]

				Y.-Q. Cao and R. W. Tsien Effects of familial hemiplegic migraine type 1 mutations on neuronal P/Q-type Ca2+ channel activity and inhibitory synaptic transmission PNAS, February 15, 2005; 102(7): 2590 - 2595. [Abstract] [Full Text] [PDF]

				S. D. Spacey, L. A. Materek, B. I. Szczygielski, and T. D. Bird Two Novel CACNA1A Gene Mutations Associated With Episodic Ataxia Type 2 and Interictal Dystonia Arch Neurol, February 1, 2005; 62(2): 314 - 316. [Abstract] [Full Text] [PDF]

				P. Imbrici, S. L. Jaffe, L. H. Eunson, N. P. Davies, C. Herd, R. Robertson, D. M. Kullmann, and M. G. Hanna Dysfunction of the brain calcium channel CaV2.1 in absence epilepsy and episodic ataxia Brain, December 1, 2004; 127(12): 2682 - 2692. [Abstract] [Full Text] [PDF]

				S. Luvisetto, T. Fellin, M. Spagnolo, B. Hivert, P. F. Brust, M. M. Harpold, K. A. Stauderman, M. E. Williams, and D. Pietrobon Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels: I. The Slow and the Fast Gating Modes and their Modulation by {beta} Subunits J. Gen. Physiol., October 25, 2004; 124(5): 445 - 461. [Abstract] [Full Text] [PDF]

				K. M. Page, F. Heblich, A. Davies, A. J. Butcher, J. Leroy, F. Bertaso, W. S. Pratt, and A. C. Dolphin Dominant-Negative Calcium Channel Suppression by Truncated Constructs Involves a Kinase Implicated in the Unfolded Protein Response J. Neurosci., June 9, 2004; 24(23): 5400 - 5409. [Abstract] [Full Text] [PDF]

				Q. Li, A. Lau, T. J. Morris, L. Guo, C. B. Fordyce, and E. F. Stanley A Syntaxin 1, G{alpha}o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization J. Neurosci., April 21, 2004; 24(16): 4070 - 4081. [Abstract] [Full Text] [PDF]

				R.A. Maselli, J. Wan, V. Dunne, M. Graves, R.W. Baloh, R.L. Wollmann, and J. Jen Presynaptic failure of neuromuscular transmission and synaptic remodeling in EA2 Neurology, December 23, 2003; 61(12): 1743 - 1748. [Abstract] [Full Text] [PDF]

				F. J. Urbano, E. S. Piedras-Renteria, K. Jun, H.-S. Shin, O. D. Uchitel, and R. W. Tsien Altered properties of quantal neurotransmitter release at endplates of mice lacking P/Q-type Ca2+ channels PNAS, March 18, 2003; 100(6): 3491 - 3496. [Abstract] [Full Text] [PDF]

				A. Tottene, T. Fellin, S. Pagnutti, S. Luvisetto, J. Striessnig, C. Fletcher, and D. Pietrobon Familial hemiplegic migraine mutations increase Ca2+ influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons PNAS, October 1, 2002; 99(20): 13284 - 13289. [Abstract] [Full Text] [PDF]

				Z. Khan and H. A. Jinnah Paroxysmal Dyskinesias in the Lethargic Mouse Mutant J. Neurosci., September 15, 2002; 22(18): 8193 - 8200. [Abstract] [Full Text] [PDF]

				D. M. Kullmann The neuronal channelopathies Brain, June 1, 2002; 125(6): 1177 - 1195. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/7/1288 00-0562fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by FLETCHER, C. F. Articles by JENKINS, N. A. Search for Related Content PubMed PubMed Citation Articles by FLETCHER, C. F. Articles by JENKINS, N. A.