Stargazer—A Mouse to Seize!

Abstract

The goals of this short review are to familiarize readers with the stargazer mouse and to outline the major functional defects associated with this mutant. The roles of the stargazin protein in calcium channel function and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor trafficking are discussed; focus is placed on studies regarding the thalamus, whence absence seizures potentially originate, and the cerebellum, which is associated with the ataxic phenotype. Finally, two additional alleles of stargazer, waggler and stargazer 3Jackson (3J), illustrate the value of an allelic series for understanding stargazin function.

The Origin of the Stargazer Mouse

Stargazer arose on the A/J mouse inbred strain at The Jackson Laboratory in the 1980s. It was initially detected by its unsteady gait and unusual, repeated head elevations. Breeding studies revealed that stargazer was due to a single, recessive mutation on mouse chromosome 15 (1,2). The first published report on stargazer also described another very important phenomenon—that this mouse had frequent spike–wave discharges (SWDs), characteristic of absence seizures in humans (1).

The three mouse mutants, ducky, lethargic, and tottering, share phenotypic features with stargazer, including ataxic gait, paroxysmal dyskinesia (affecting limbs in ducky, lethargic, and tottering but seen as neck dystonia in stargazer), and absence seizures with SWDs between 5 and 7 Hz (3,4). These mouse mutants have defects in voltage-dependent calcium channel (VDCC) subunit genes: Cacna2d2 for ducky (5), Cacnb4 for lethargic (6), and Cacna1a for tottering (7).

The mutation in stargazer was identified as a retroviral-like, early transposon insertion in the second intron of the VDCC γ2 subunit gene, Cacng2. This insertion severely reduced normal Cacng2 expression (8–10). Several similarities were observed between the previously isolated VDCC γ protein found in muscle (11) and the Cacng2 product. The proteins shared 25% amino acid identity, and both had a similar secondary structure, including four transmembrane domains with both termini projecting into the cytoplasm. Furthermore, the Cacng2 protein caused a small hyperpolarizing shift in VDCC steady-state inactivation in vitro (8). The Cacng2 protein product is referred to interchangeably as stargazin, γ2, and CACNG2.

Absence Seizures with Stargazer Are Accompanied by VDCC and T-type Changes

Absence seizures arise from disturbances of the corticothalamic circuitry, including the cortex, thalamus, and thalamic reticular neurons and their interconnecting neuronal pathways (12–15). An oscillatory balance in the inhibitory and excitatory network activities between the cortex and thalamus is maintained in the normal state, but abnormal perturbations within this loop can result in SWDs. For instance, within the thalamus, low-voltage calcium channels (T-type or Cav3.1) act as critical pacemakers in a recurrent cycle with the hyperpolarization-activated cation I_h currents. Aberrant burst firing of the T-type channels results in rhythmic oscillations that generate SWDs (16–18). Alternatively, these SWDs can arise from abnormal neuronal discharges from the pyramidal neurons in the cortex (19). The SWDs exhibit widespread synchronization and rapid generalization, detected by EEG recordings from the cortical surface.

The role of stargazin as a regulator of VDCC activity has been questioned because the in vitro results show marginal changes, at best (20–25). However, the recordings from thalamocortical relay nuclei from slice preparations revealed that both VDCC and low-voltage T-type channels are altered in the stargazer mouse (26). These slices retained the integrity of the tissue and were taken directly from mutant and control mice. The stargazer mice showed both increased VDCC and low-voltage calcium channel activity, with a depolarizing shift in the steady-state inactivation of the T-type current. Furthermore, similar results were observed for the tottering mouse (26).

The α1G subunit is the major component of T-type calcium channels in the thalamocortical relay neurons (27). A mouse α1G knockout (Cacna1g^tm1Hssh) was generated to explore the functional consequences of deleting this particular channel. No burst-mode firing could be induced in the neurons from the targeted knockout, and the mice were resistant to baclofen-induced absence seizures (28). Moreover, when this α1G knockout mutation was combined with stargazer, lethargic, or tottering mutations, the incidence of absence seizures was severely reduced (29). In summary, the similarities between the phenotypes of these mice, the thalamic slice recordings, and the double-mutant studies provide strong evidence that stargazin plays a role in VDCC regulation. Changes in VDCC activity lead to an increase in low-voltage T-type activity within the thalamus that, in turn, initiates aberrant SWDs, the hallmarks of absence seizures.

Stargazin and AMPA-Receptor Trafficking

The ataxic phenotype is most commonly associated with cerebellar defects (30–35). Although the cellular organization within the stargazer cerebellum was grossly normal (1), numerous neurophysiological defects associated with the granule cell layer have been described (36). Granule cells from the cerebellum of stargazer and its allelic partner, waggler, were found to be missing functional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (37,38). These receptors mediate fast excitatory synaptic transmission in the brain in response to glutamate. They recycle rapidly at the plasma membrane and contribute to the overall synaptic plasticity of the neuronal circuitry.

Following up on this loss of AMPA function, Chen and colleagues (39) determined that the stargazin protein was essential for the trafficking of AMPA receptors from the Golgi complex to the plasma membrane and, furthermore, was required for the ultimate targeting of the receptors to the postsynaptic membrane. Stargazin's critical binding and trafficking domains involved in the migration of the AMPA receptor to the plasma membrane include the first extracellular loop and the intracellular carboxy terminus (40). The final amino acids, trp-trp-pro-val (TTPV), at the carboxy tail of stargazin, are essential for the subsequent binding of the postsynaptic proteins, such as PSD-95, to target the entire complex to its active site at the postsynaptic membrane (39,41,42). Additionally, in vitro studies revealed that stargazin binding enhanced glutamate-induced currents at the synapse (43) and ultrastructural changes in the excitatory synapses within the cerebellum reflect the loss of normal AMPA-receptor trafficking in stargazer (44).

Stargazin and other closely related members of the γ subunit family, including γ3, γ4 and γ8, share a high degree of amino acid conservation, including the TTPV motif. These four proteins are referred to as transmembrane AMPA-receptor regulatory proteins, or TARPs, and all can promote expression of functional AMPA receptors at the postsynaptic membrane (45). TARP members associate independently with AMPA receptors and cocluster with the receptors at the postsynaptic sites. However, in the cerebellar granule cells, only stargazin is expressed significantly (20), explaining why AMPA-receptor localization is noticeably defective in these cells.

Stargazer also lacked brain-derived neurotrophic factor message in its granule cells (46). A reduction in the inhibitory neurotransmitter GABA was observed, and fewer GABAergic synapses were present (47). The GABA_A-receptor α6 (an indicator of mature granule cells) and β3 subunits were reduced in granule cells (48). Finally, stargazer mice showed an impaired cerebellum-dependent eye-blink conditioning response (49). The outcome of all these impairments appears to be that, although stargazer cerebellar granule cells retain the ability to migrate correctly, they lack the neurotransmitter and neurotrophic innervations required for their full maturation.

Additional Mutants for Studying Stargazin Function

Waggler, Stargazer 3Jackson (3J), and a γ4 Targeted Mutant

The stargazer mouse has proved to be a complex model with pleiotrophic defects. It can survive despite its severe phenotype, with very reduced Cacng2 message expression and no detectable protein (9,10). After stargazer, two spontaneous mutants have been found with defects in the same Cacng2 gene: waggler and stargazer 3J. Surprisingly, all three mutations are caused by similar insertions into Cacng2 introns (10).

Waggler mice are ataxic but lack the head elevation of stargazer. They also showed no stargazin protein expression but had a variable SWD profile (10). Additionally, wagglers were missing functional AMPA receptors in granule cells and showed similar impairments in granule cell maturation to stargazer (38,38). The waggler mutation arose from an early transposon insertion in the first intron, whereas stargazer 3J, like stargazer, had an early transposon insertion in the second intron at a more distal 3′ position (10).

Stargazer 3J has the mildest phenotype of the three alleles. The mice are ataxic but retain approximately 25% of the normal Cacng2 message. In contrast to stargazer and waggler, stargazin protein also was detected in this allele (10). Stargazer 3J consistently showed no SWD activity, suggesting that sufficient stargazin exists to overcome the seizure phenotype and head tossing, but not the ataxia.

Recently a targeted mutation was introduced into the VDCC γ4-subunit gene, Cacng4 (51). The targeted homozygous Cacng4^tm1Frk mouse had no discernible phenotypic abnormalities, including no spontaneous absence seizure activity.

Absence Seizures in Double Cacng2;Cacng4 Mutants

Double-mutant studies can provide insight into functional interactions, as illustrated by the studies of Song et al. (29), in which the α1G knockout was combined with VDCC mutants. However, early death can confound full phenotypic analysis—in particular, EEG recording and ataxia, for which the mice must age to about 2 weeks. For example, the double mutant between stargazer and Cacng4^tm1Frk rarely survived. However, double mutants between waggler or stargazer 3J and Cacng4^tm1Frk were viable. Although neither single mutants had SWDs, the stargazer 3J; Cacng4^tm1Frk double homozygotes proved to be most informative, as the double mutants showed absence seizure activity (51). In summary, the depletion of γ4 alone appears to have no effect on the mouse. However, both the duration and recurrence of seizure episodes increased in the double homozygotes, exacerbating the seizures compared with the waggler mutant and introducing seizures into the previously seizure-free stargazer 3J. These results suggest that γ4 has a role in seizure susceptibility, but this role is revealed only when expression of stargazin also is compromised.

Are the Functions of Stargazin Interconnected?

One study was performed of VDCC activity in the cerebellum of stargazer mice. Whole-cell measurements from stargazer granule cells showed no differences in VDCC currents compared with wildtype (39), although individual VDCC, including P/Q, N, R, and L types, were not assessed. Notably, increased N- and L-type channels in the cerebellum of the tottering mouse (with a mutation in the P/Q-type channel) are proposed to contribute to tottering's ataxic and dystonic phenotypes (52–54).

Conversely, no reports exist of AMPA-receptor localization or activity in the stargazer mouse cortex and thalamus, although stargazer hippocampal studies indicated that AMPA receptor function is normal (37). Is there less AMPA-receptor activity in the thalamus of stargazer, waggler, or stargazer 3J mice? The double mutants between stargazer 3J and Cacng4^tm1Frk may be particularly informative for thalamic studies, as stargazin and γ4 constitute the major TARP expression in this region (20,51). If both these functions are indeed altered in the thalamus, the challenge will be to assess the relative contributions of VDCC activation and AMPA-receptor mislocalization to the seizure phenotype.

AMPA-receptor–knockout Mutants and Thalamic Defects

Studies of mouse mutants with knockouts of the AMPA-receptor subunits could reveal an association between AMPA receptors and absence seizures without the conflicting VDCC mutational effects. Four subunits, GLUR1-4, form the heterotetrameric receptor (55), and knockout mice exist (Gria1-3) of GLUR 1, GLUR 2, and GLUR 3, respectively (56–60). These mutants have generally been studied for long-term potentiation disorders; only one absence seizure study has been reported on the Gria2 knockout (61). This mutant, despite having multiple behavioral abnormalities (58,59), proved to be more resistant to absence-seizure induction than were controls (61). Similar studies may be worth pursuing with the Gria2; Gria3 double knockout (60). This particular mutant combination showed a striking reduction in AMPA-receptor activity; more closely resembling the AMPA-receptor defect in stargazer than the single Gria2 mutant.

Last, But Not Least, Stargazin Is Also a Cell-adhesion Molecule

The structural similarities between stargazin and the claudin family led to a recent article describing yet another role for stargazin as a cell-adhesion molecule (62). Furthermore, Cacng2 message is expressed as early as embryonic day 12 in mouse (51). Thus, stargazin may be involved in forming close cell–cell contacts during neuronal development. Future studies comparing stargazer with control mice should reveal more about stargazin's cell-adhesion function in vivo.

Conclusion

The stargazer mouse has proved to be an exceptionally informative mutant. With the removal of one small stargazin protein from the brain, numerous disorders, including spontaneous absence seizures, ataxia, and head tossing, have materialized. The breadth of research generated from this one mutant underscores the power of mouse genetics. Spontaneous and genetically engineered mouse mutants give researchers insight into individual protein function within the context of the whole animal and provide the mechanistic tools to reveal the pathways that underlie the affected phenotype.