We determined that the Virtual Cell modeling environment [36
] can be used to study cellular dynamics associated with disease states, particularly spinocerebellar ataxias
with altered IP3R1 abundance or sensitivity or both. We modified a quantitative multicompartmental model recently developed in Virtual Cell to investigate the coupling of detailed biochemistry of IP3 and calcium signaling with membrane potential. Results show that computational modeling allows us to simulate the behavior of calcium dynamics and membrane potentials in neurons at the level of the spine and of the soma, in response to various stimuli. Our models include a myriad of ion channels and molecules important for normal Purkinje neuron function. They merge features of IP3 production, phosphoinositide and calcium signaling with conceptual representations of parallel fiber and climbing fiber stimulation, local PIP2 synthesis and sequestration, PIP2 hydrolysis, calcium influx, coincident detection at IP3R1, modulation of IP3R1 abundance and sensitivity, IP3R1-mediated supralinear calcium release, and components of membrane electrophysiology into comprehensive models that can have compartmental and spatial counterparts.
Results suggest that ICpeptides may be used to modulate calcium release in various IP3R1-associated ataxias (ataxias in which IP3R1 is less abundant, e.g., SCA15/SCA16 as well as SCA1 and SCA3, or more sensitive than in wild type mice or unaffected individuals, e.g., SCA2 and SCA3 and likely SCA1). Simulations predict that pathophysiology associated with decreases in IP3R1 abundance can be overcome by adjusting IP3R1 sensitivity to activation by IP3, and that targeting PP1α can restore normal calcium transients. Various concentrations of ICpeptide may be needed to increase IP3R1 sensitivity in SCA15/SCA16, based on the levels of reduced abundance. Similarly, various concentrations of ICpeptide could be needed to normalize IP3R1 sensitivity and consequently calcium release in SCA1, SCA2, and SCA3, based on the degrees to which IP3R1 sensitivity to IP3 is pathologically increased (dependent on the concentration of mutant protein and the relative balance with PP1α).
This project complements results by Hernjak et al. from a study on Purkinje spine calcium signaling that considered the importance of low sensitivity and high abundance of IP3R1 suggested by experiments [35
]. Findings from that study suggested that increasing IP3R1 sensitivity to IP3 could not rescue calcium release in the context of low IP3. However, our results show that increased sensitivity can
restore normal IP3 response if the abundance is not too low.
This provides further insight into the roles that IP3R1 abundance
play in normal cerebellar functioning and coincident detection at IP3R1. IP3R1 abundance and sensitivity can each function as the primary pathology in IP3R1-associated ataxias
or can serve to partially or wholly compensate the effects of pathology. Other important conclusions from the study by Hernjak et al. were that the low IP3R1 sensitivity observed in wild type Purkinje neurons relative to other cell types may be important to restrict biochemical signals and synaptic plasticity to one spine, and that the high IP3R1 abundance in Purkinje neurons is important for ensuring generation of a robust calcium signal in an individual spine. The current study confirms that the balance of IP3R1 abundance and sensitivity is critical for obtaining robust, but not hyperactive, calcium transients. It is possible that in ataxias
with reduced abundance of IP3R1, some spines will not generate robust calcium signals, while in ataxias
with supersensitive IP3R1, stimulation signals may not be confined to single spines. This could affect both compartmentalization of biochemical signals, and the influence of the biochemistry on membrane electrophysiology. Similarly, therapeutically increased IP3R1 sensitivity (by application of an appropriate ICpeptide) could potentially lead to spillover of calcium into adjacent spines, if sensitivity is not finely tuned. Modeling could thus be useful for helping to determine the adequate levels of ICpeptide to be administered experimentally.
Experimental observations in SCA1 mice appear to be paradoxical [30
] (Table ). The expression of various molecules (for example, IP3R1, Homer, and SERCA) involved in glutamatergic calcium signaling and IP3R1-mediated calcium release is reduced in mice and humans with SCA1 [9
]. However, IP3-induced calcium release is hyperactive in SCA1 mice [8
]. Insight may be gained by examining the pathology of SCA2 and SCA3. Liu et al. [10
] and Chen et al. [6
] found that mutant Ataxin-2 and Ataxin-3, respectively, bind the C-terminal of IP3R1 and increase the receptor’s sensitivity to activation by IP3. This corresponds to hyperactive IP3-induced calcium release in both SCA2 and SCA3. Above normal numbers of CAG repeats in SCA1, SCA2, and SCA3 give each respective mutant ataxin protein a toxic gain of function that disrupts calcium homeostasis in neurons. Thus, there may be a common mechanism underlying some of the pathology of the three polyQ ataxias
. This mechanism could be binding of the polyQ-expanded protein to the C-terminal of IP3R1, thereby increasing the receptor’s sensitivity to IP3. This mechanism may be extendable to a large variety of polyglutamine diseases. In fact, Liu et al. reported that the mutant Ataxin-1 protein and polyQ-expanded atrophin-1, the protein mutated in Dentatorubral-pallidoluysian atrophy (DRPLA), both associate with the C-terminal of IP3R1 [10
], though IP3R1 supersensitivity has not yet been assessed experimentally in either disease. Of note, a prominent feature of DRPLA is cerebellar ataxia
The pathological gain of function of IP3R1 sensitivity that destabilizes calcium signaling [84
] is also observed in a mouse model of Huntington’s disease (HD), another polyQ disease [18
]. Lentiviral and adenoviral infection of affected cultured medial striatal neurons or in the striatum of the ataxic HD mice themselves with the IC10 peptide lead to reduced brain atrophy and improved motor coordination, respectively [53
]. Perhaps similar genetic targeting of Purkinje neurons in the cerebellum of existing mouse models with IC-G2736X could mitigate the pathology of SCA1, SCA2, and SCA3. This could first be tested in in situ
experiments using cerebellar slices from existing mouse models, in which mutant ataxin1 binding IP3R1 directly upstream of the PP1αlpha binding site, would be close enough to preclude binding of PP1αlpha.
Reduced gene expression of IP3R1, Homer/MyoVa, mGluR and other molecules in SCA1 may be due to direct pathology due to the effect of the mutant proteins on transcription [7
]; it could also be due to the tight regulation of calcium homeostasis in the cerebellar Purkinje neuron [30
]. Lin et al. suggest that reduced gene expression is a part of the pathology [9
], but our model indicates that with exception of the calcium buffer proteins the effect on IP3R1-mediated calcium release is partially compensatory. Using the reduced fold expression observed in SCA1, normal calcium release was restored to varying degrees, depending on how much mutant Ataxin protein was placed in the model. Reduced expression of several calcium signaling genes is also observed in some Purkinje neuron subtypes in plasma membrane calcium ATP-ase (PMCA) knockout mice [20
]. This could also have the effect of partial compensation in that mouse model, since PMCA contributes to expelling calcium from the Purkinje neuron cytoplasm. ‘Compensatory pathology’ is thus an interesting characteristic of various ataxias
, and provides a level of complexity to the study of these neurological disorders. Disruption of calcium signaling and homeostasis, whether due to reduced IP3R1 levels or supersensitive IP3R1, whether in IP3R1-associated ataxias
or in other ataxias,
can lead to dysfunction of Purkinje cells, and impaired long-term depression and synaptic plasticity that are involved in learning and memory.
Calcium homeostasis is critical for normal function of Purkinje neurons and is thought to be tightly regulated [30
]. Downregulation of parvalbumin and calbindin could therefore lead to a loss of compensation, by destabilizing supralinear calcium release and disrupting any homeostasis achieved by downregulation of other molecules. Furthermore, it has been postulated that normal or high concentration of calcium buffer proteins have a protective role in certain neurons [63
]. Vig et al. suspect that the decrease in parvalbumin expression in Purkinje cells from SCA1 patients may reflect alterations in a regulatory biochemical pathway that may be important for neuronal survival [63
]. Further, our model suggests that these changes (downregulation of various calcium channels and buffers) alone may not be sufficient to reproduce elevated calcium response as observed experimentally in SCA1 mice, without assuming concurrent supersensitive IP3R1.
Purkinje neurons are largely spared in SCA3 [88
], while nerve cells in the pons and substantia nigra are substantially damaged. Chou et al. suggest that although prominent neuronal loss was not found in the cerebellum, the SCA3 mice displayed pronounced ataxic symptoms, suggesting that instead of neuronal demise, mutant Ataxin3 causes neuronal dysfunction and resulting ataxia
]. While Ataxin1 is found in the nucleus, Ataxin 2 and Ataxin3 are cytoplasmic proteins, under normal conditions. However, in brains from patients with SCA3, mutant Ataxin3 accumulates in the nucleus [28
], as does mutant Ataxin1 [90
]. Hence, results from our study of SCA1 could be extended to SCA3 modeling.
Long term feeding of SCA2 and SCA3 mice with dantrolene improved motor coordination and slowed brain atrophy [6
]. Dantrolene is thought of as a ‘calcium stabilizer’ [6
] and has been shown to inhibit the ryanodine receptor (RYR), which is another calcium channel on the smooth endoplasmic reticulum [92
]. However, all the targets of dantrolene are not known [94
]. The details of the mechanism of action of dantrolene are incomplete [94
], though it has been proposed as a possible therapeutic drug for SCA2 [10
] and SCA3 [6
]. The drug is currently approved to treat malignant hypothermia as a one-time application in response to adverse reaction due to anesthesia [94
]. It is also used to reduce muscle spasticity in patients with neurological incidents or disorders. However, dantrolene leads to fluid buildup in the lungs, among other adverse effects [94
]. Yet, considering the disturbed neuronal calcium signaling observed in polyQ ataxias
, it is likely that inhibiting or downregulating either or both of the sER intracellular calcium release channels (IP3R1 and RYR) should attenuate IP3R1-mediated supralinear calcium release into the cytosol and thereby alleviate SCA2 and SCA3. This is particularly so, since both the RYR and IP3R1 channels increase in function when directly bound by cytosolic calcium. Therefore the supralinear calcium release cascade initiated by IP3 binding IP3R1 may involve cytosolic calcium also binding RYR to increase calcium release from the sER. Adding RYR to the model in the future will facilitate further study of the signaling interactions among RYR, IP3R1, other calcium channels, and the calcium-activated potassium channels. However, it should be noted that there is evidence that RYR is localized to the dendritic shaft and is excluded from spines [97
]. Yet, there is also evidence that these physically separate calcium release sites functionally interact [99
Predictions from our computer model involving SCA15/16 could be compared with novel experiments in an existing mouse model using GST-IC4 to dissociate PP1α from IP3R1 in Purkinje neurons in cerebellar slices from ataxic mice. This could restore normal calcium and membrane potential response in ataxic mice. For such experiments, it would be useful to select a mouse model that: (i) does not completely knock out IP3R1 expression as in the IP3R1−/−
knockout mice, but has reduced expression of IP3R1 protein, (ii) shows motor discoordination, (iii) does not
possess a mutation in neuronal IP3R1 at the preferred site of PKA phosphorylation (Ser-1755) [100
], and (iv) does not
possess an IP3R1 mutation in the carboxyl terminal PP1α-binding site (2731–2749) [43
]. For assessing IP3R1 haploinsufficiency in particular, as found in SCA15 and SCA16 patients [3
], it would also be useful to select a mouse model with a large heterozygous deletion mutation in IP3R1. The IP3R1 +/−
] fulfill all of these desired features. The IP3R1delta18/delta18
mice have also been suggested as a mouse model for SCA15/16 [3
]. The ITPR1opt/opt
mouse model (see Figure b) shows reduced IP3R1 protein levels on Western blot, but has a mutation that deletes the preferred PKA phosphorylation site (Ser-1755) [4
] in neuronal IP3R1, and therefore violates (iv). Given that a very small region of the gene is deleted and that it occurs at such an important regulation site, we suspect that IP3R1 is dysregulated in these mice. As such, we expect that these mice possess cellular pathophysiology resulting from dysregulation in addition to effects of IP3R1 insufficiency. Consequently, it is yet unclear whether results from this study will be easily extendable to calcium signaling and membrane excitability in ITPR1opt/opt
Results from our models suggest that IP3R1-mediated calcium release can activate voltage-gated KCa channels and thereby alter membrane excitability in the Purkinje neuron. This can have implications for ataxias that involve disruption of intracellular calcium homeostasis. It is possible that pathological alterations in calcium transients result in pathological activation of BK channels. This could lead to variations in the timing of action potentials and other electrophysiological events in the Purkinje neuron, which controls modulation of neurons downstream of the cerebellum.