Transgenic mouse models in migraine
Ven, R.C.G. van de
Citation
Ven, R. C. G. van de. (2007, November 6). Transgenic mouse models in migraine. Retrieved from https://hdl.handle.net/1887/12473
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General Discussion
N I
N E
CHAPTER 9
Migraine is a frequent neurovascular paroxysmal disorder, characterised by recurrent headaches and associated neurological symptoms. Knowledge on how migraine attacks begin and why patients are more susceptible to attacks is lacking, but important for the development of novel treatments. However, recently three “migraine” gene disorders were identified in families with hemiplegic migraine (FHM), a monogenic form of migraine with aura. These genes, CACNA1A (FHM1), ATP1A2 (FHM2) and SCN1A (FHM3), all point to an important role for disturbed ion transport in migraine.
Existing experimental animal models of migraine mimic only certain components of the pathophysiology, such as the vascular or neuronal contribution (for review see1,2). The finding of the three FHM genes made it possible to generate transgenic mouse models for migraine. We generated and analysed various transgenic Cacna1a mouse models and discuss their usefulness to study the pathophysiology of migraine (Table 1).
9.1 Knockin FHM1 mouse models
Central in this thesis is the generation of the first transgenic knockin (KI) mouse models of migraine (Table 1). In these models, mutant Cav2.1 channels are expressed in their natural environment and at their endogenous level. Thus, the molecular basis of migraine susceptibility can be assessed. In this thesis, effects of FHM1 mutations on calcium channel function, neurotransmission and cortical spreading depression are investigated.
9.1.1 Generation of FHM1 R192Q and S218L knockin mice
KI mice were generated by introducing the mutations R192Q and S218L into the highly conserved orthologous mouse Cacna1a gene using gene targeting technology (Chapters 2, 4). The FHM1 mutation R192Q is associated with ‘pure’ hemiplegic migraine.9 The S218L mutation is linked to hemiplegic migraine and additional clinical features such as cerebellar ataxia, epilepsy and delayed cerebral edema upon mild head trauma, which may be fatal.10
The generation of two FHM1 models allows analysis of migraine pathophysiology in mice with phenotypes of different severity. FHM1 R192Q KI mice do not show any obvious behavioral abnormalities. In contrast, homozygous S218L KI mice have a phenotype of ataxia, epilepsy and increased mortality. In addition, these mice die more readily after mild head trauma. All these results are consistent with the phenotype in S218L patients, including fatal brain edema after a mild head injury. The wide range of neurological symptoms in the Cav.2.1-α1 KI mice make them useful models for various
Table 1 Findings in Cacna1a transgenic mice described in this thesis R192Q/+ R192Q/R192Q S218L/+ S218L/218LLeaner Cav2.1-α1 KO Conditional Ca
v2.1 Viability===↓† ~P20 † ~P20= Ataxianononoyesyesyesno Seizuresnonoabsencegeneralisedabsence [1]absence [5]no TH expressionnononoyesyes [2] Peak Cav2.1 current density130% 125%150% 30% [3]0% [6] Cav2.1
membrane density
===0% [6]
Upregulation other Ca
v typesnonoCav1Cav1 Cav2.2
Cav1 Cav2.2 Cav2.3no fMEPPa160%215%850%1250%50%50% EPP amplitudea====65%65% QCa====50%50%= QCb200%240%190%250%
Threshold of CSD
c30%60%30%1000% [4]
Propagation speed of CSD 150%150%234%60% [4] Duration of CSD===60% [4]
References from thesis
Chapters 2 & 3Chapters 2 & 3Chapter 4Chapter 4Chapter 5Chapter 5Chapter 6 =, equal to wild-type; ↓, lower than in wild-type; †, death at; % compared to wild-type; ameasured at 2 mM extracellular [Ca2+]; bmeasured at 0.2 mM extracellular [Ca2+]; cthreshold of CSD upon electrical stimulation. CSD, cortical spreading depression; EPP, endplate potential; fMEPP, miniature endplate potential frequency; KO, knockout; QC, quantal content; TH: tyrosine hydroxylase Data are in chapters unless indicated otherwise by [ ]; respective references are: 1. Noebels, 19843; 2. Austin et al., 19924; 3. Wakamori et al., 19985; 4. Ayata et al., 20006; 5. Song et al., 20047; 6. Jun et al., 19998.
neurological disease including migraine, ataxia, epilepsy and brain trauma.
9.1.2 Consequences of FHM1 R192Q and S218L mutations on channel function
Electrophysiological studies have been done for both R192Q and S218L mutated Cav2.1 channels in transfected HEK293 cells. The conductance of single mutant channels remains unchanged compared to wild-type. However, both mutant channels have an increased open probability, and therefore increased Ca2+ influx.11-13 In fact, this is a common trait of all FHM1 mutations analyzed thus far, with the activation curve of all channels shifted to more negative voltages.11-13 Mutation S218L shows the largest gain-of-function effect, with the channel opening at voltages close to the resting potential of many neurons.13 On top of this, the S218L mutant channel also inactivates more slowly, and recovers faster from inactivation.13 In conclusion, both Cav2.1 channel mutations cause an increased Ca2+ influx, the effect being stronger for the S218L mutation.
On the whole cell level, HEK293 cells or neurons from Cav2.1-α1 knockout (KO) mice (see §9.2) transfected with different FHM1 mutant cDNAs produce inconsistent results for P/Q current density, which strongly depends on membrane density of functional channels.11-17 The latter varies from increased to decreased, depending on the mutation and transfected cell-type. Therefore, it is important to study neurons, isolated from transgenic mice, in which the mutant channels are expressed in their natural environment and at endogenous expression levels.
Functional Cav2.1 channel density in the membrane of isolated cerebellar granule cells of both R192Q and S218L KI mice is identical to that of neurons isolated from wild-type mice (Chapters 2 and 4). Electrophysiology in cerebellar granule cells reveals that Cav2.1 current density at low voltages of -40 to -30 mV , was 4 and 6-7 times larger than in wild-type, for R192Q and S218L respectively (Table 1). Consequently, small depolarizations (e.g. minor changes in extracellular ion concentrations), may be enough to open mutant Cav2.1 channels and thus result in neurotransmitter release. This may explain why triggers that normally have no consequences trigger attacks in migraine patients.
9.1.3 Synaptic consequences of FHM1 mutations at neuromuscular junctions
The consequences of Cav2.1 channel mutations on neurotransmission have been studied in single synapses of diaphragm neuromuscular junctions (NMJs) of FHM1 mouse models.
At this synapse, release of the neurotransmitter acetylcholine (ACh) is almost entirely
determined by Cav2.1 channels.18 ACh release can be either spontaneous or evoked.
Spontaneous release of single ACh quanta is defined by the miniature endplate potential (MEPP). Evoked release is defined as the number of ACh quanta that is released after a single action potential, i.e. quantal content (QC).
R192Q and S218L KI mice show a gain-of-function for both evoked and spontaneous neurotransmitter release at the NMJ, in agreement with the found increased Ca2+ influx through mutated Cav2.1 channels (Table 1)(Chapters 2, 3 and 4). Spontaneous release is about two-fold higher at R192Q NMJs compared to wild-type. At S218L NMJs, it is increased far more dramatically by ~12-fold. Notably, while evoked release in the KI models is unaltered under normal extracellular [Ca2+] and [K+]. However, evoked neurotransmitter release is more than two-fold higher compared to wild-type under conditions that also occur during CSD (i.e. at low extracellular Ca2+ and high K+ levels)23 or after a trigger (e.g. mild head trauma). This might help to explain the paroxysmal nature of the disorders.24 In both models, the increase in spontaneous release and quantal content are gene-dosage dependent. In S218L mice, these effects also showed progression with age; this was not the case for R192Q mice (S. Kaja, J. Plomp, personal communication).
Spontaneous release may result in activation of the postsynaptic neuron.21,22 It is therefore imaginable that an increased spontaneous release causes ectopic activation of neurons that are involved in the pathogenesis of migraine. Release of single quanta also plays a role in synaptic development.19,20 Indeed, homozygous S218L KI mice show abnormal Purkinje cell dendritic morphology in Purkinje cells. The found increase in ACh release is not accompanied by morphological abnormalities of the endplates in either R192Q or S218L KI mice (S. Kaja, J. Plomp, personal communication).25
Dr. Daniela Pietrobon and co-workers were able to demonstrate that release of glutamate was increased in autaptic cortical neurons from FHM1 mice compared to wild- type (D. Pietrobon, Univ. Padova, unpublished; comment in26). Therefore, our findings of enhanced spontaneous and evoked neurotransmitter release in the NMJ of FHM1 models suggest that similar consequences of the mutations also occur in the central nervous system. Based on these results, one can hypothesize that extracellular concentrations of K+ and glutamate will be increased in the migraine brain.
9.1.4 Hyperexcitability and Cortical Spreading Depression in FHM1 mice
On the molecular level, FHM mutations are predicted to result in increased levels of extracellular K+ and neurotransmitters (such as glutamate) due to increased synaptic vesicle release (FHM1 and FHM3) or decreased clearance of neurotransmitters in the
synapse by glial cells (FHM2) (Figure 1). Glutamate and K+ levels are important for the initiation, duration and propagation of CSD.23,27 Therefore, FHM mutations likely lead to increased neuronal excitability and susceptibility for CSD.28 Our FHM1 mouse models provided the opportunity to test these hypotheses (for review on the rationale see Chapter 8).
We already showed that activation of Cav2.1 channels in FHM1 mouse models occurs at lower voltages and neurotransmitter release is increased. The mouse models show a lower threshold for CSD, and a higher propagation speed (Chapters 2 and 4). FHM1 S218L mice also have a tendency to develop multiple CSD events upon electrical stimulation of the cortical surface. These findings indeed indicate neuronal hyperexcitability in the FHM1 mouse models. One of the main hypotheses of migraine pathophysiology is that CSD can activate the trigeminovascular system (TGVS), and thereby not only cause the aura, but also the migraine pain. In rats it was shown that CSD can indeed activate the TGVS and thus trigger headache mechanisms.29 At this moment it is unknown whether increased susceptibility to CSD will also result in increased
Ca2+
Presynaptic
Postsynaptic
Astrocyte
C
xx x
Ca2+ Ca2+ FHM2 Ca2+
Presynaptic
Postsynaptic
Astrocyte
A
Ca2+ Ca2+ Ca2+
Presynaptic
Postsynaptic
Astrocyte
B
Ca2+ Ca2+
FHM1
Ca2+
Presynaptic
Postsynaptic
Astrocyte
D
Ca2+ Ca2+
FHM3
Normal Hyperactive calcium channels
Increased NT release
Defective Na+.K+pump
Decreased NT clearance Faster recovery sodium channels
More action potentials � increased NT release
Figure 1
Figure 1. FHM1, FHM2 and FHM3 mutations increase neuronal sensitivity and susceptibility to CSD by an increase of extracellular K+ and neurotransmitter levels. (A) Influx of Ca2+ through Cav2.1 channels ( ) at presynaptic terminal occurs after an action potential (red arrow). Synaptic vesicles fuse with the plasma membrane. Neurotransmitters ( ) are released and can bind to their receptors ( ) on the postsynaptic membrane or are cleared from the synaptic cleft by Na+ gradient-driven glutamate transporters ( ) of the astrocyte (light gray arrows). (B) FHM1 mutations in Cav2.1 calcium channels result in increased influx of Ca2+ and subsequent increase in neurotransmitter release. (C) FHM2 mutations result in a reduced activity of the ATP-driven Na+,K+ pump, disturbing the Na+ gradient required for glutamate transporter function. (D) FHM3 mutations result in a faster recovery from Na+ channel inactivation. As a result, neuronal firing rate is increased (dark gray arrows), enhancing excitability.
sensitivity for TGVS activation.
It would be important to demonstrate whether drug intervention (e.g. by modulating Cav2.1 channels or associated pathways) will prevent TGVS activation in FHM1 models.
Of note, unlike FHM1 models, the natural Cacna1a mouse mutants tottering and leaner have a decreased susceptibility for CSD, suggesting that these models will have opposite effects in drug studies.
The hyperexcitability of the S218L model is further demonstrated by the fact that mild head trauma results in enhanced cortical edema and increased mortality compared to wild-type (N. Plesnila, Univ. Munich, unpublished). One explanation is that edema is caused by disruption of the blood-brain barrier through MMP-9 upregulation, which is known to occur after CSD.30 This makes FHM1 models valuable tools to study the consequences of CSD with respect to blood-brain barrier permeability and edema formation.
In conclusion, FHM1 mouse models indicate that neuronal hyperexcitability and CSD are important in migraine and support clinical findings of altered brain excitability in migraine patients (for review see reference 31). In addition, neuronal hyperexcitabilty and CSD may also be important in the pathogenesis of lethal brain edema upon mild head trauma in children.10,32
9.2 Other transgenic Ca
v2.1 mouse models:
Ca
v2.1-α
1KO and conditional Ca
v2.1-α
1mice
9.2.1 Cav2.1-α1 KO mice
We and two other groups have generated Cav2.1-α1 knockout (KO) mice by introducing the neomycine cassette into the endogenous Cacna1a gene. Thereby either exon 4 (Cacna1aex4KO)8 (Chapter 5) or exons 14 to 17 (Cacna1aex14-17KO) were disrupted.33 As a consequence, these mice do not express Cav2.1 calcium channels. Although Cav2.1- α1 expression normally starts during embryonic development34,35, Cav2.1 channels are developmentally upregulated between postnatal days 7 and 10 at the expense of Cav2.2 channels at many presynaptic terminals (including synapses of Purkinje cells and NMJs).36,37 At that time, Cav2.1 channel activity is important for neurite growth and neuronal development.38-42
The onset of symptoms in Cav2.1-α1 KO mice corresponds well with the normal developmental expression of Cav2.1-α1 protein. The phenotype is very similar between
strains: around postnatal day 10 the animals develop difficulties in walking and righting themselves. If left unaided, Cav2.1-α1 KO mice die between postnatal day 20 and 28.
In addition, Cav2.1-α1 KO mice are smaller, develop ataxia and exhibit dystonia and absence epilepsy. In line with the importance of Cav2.1 during neuronal development, Cav2.1-α1 KO mice have persistent granule cell layer and Purkinje cell abnormalities.8,42 Most likely, ataxia in these mice is due to a combination of abnormalities in cerebellar developmental and neuronal signalling, instead of mere loss of cerebellar cells, since cerebellar cell loss at young age has not been reported.
The total calcium current density in cerebellar granule cells of homozygous Cacna1aex4KO and Cacna1aex14-17KO mice is decreased by ~70% and ~20%, respectively.
This difference is due to compensation by other types of voltage-gated calcium in these cells: L-type (Cav1) and N-type (Cav2.2) calcium channels are upregulated in Cacna1aex14-
17KO mice only. Comparison of neurotransmission at the NMJ of Cacna1aex4KO and leaner mice revealed a ~50% decrease in MEPP frequency and QC for both strains (Table 1). At leaner NMJs, Cav2.3 channels partly compensate for the reduced Cav2.1 channel activity.
At Cacna1aex4KO NMJs the Cav2.1 activity is lost completely, but compensated by up- regulation of Cav2.3 as well as Cav2.2 and Cav1 channels at the transmitter release site.
These findings can be explained as follows: Transmitter release sites have type-specific
‘slots’ that are preferentially filled with Cav2.1 channels.14 There is a hypothesis that in the absence of Cav2.1, these slots become occupied by Cav2.3 channels, which would explain the larger functional compensation by Cav2.3 in Cacna1aex4KO mice.47 Cav2.2 channels do not contribute to neurotransmission in wild-type and leaner NMJs, from which we conclude that these channels do not occupy slots at the transmitter release site.
In Cacna1aex4KO mice, Cav2.2 compensation does occur. Combined, these observations indicate that Cav2.2 channel expression at the transmitter release site is inhibited by Cav2.1 channel-mediated Ca2+ influx, even if this influx is reduced.48 Apparently, compensatory recruitment of Cav2.2 channels at these synapses is only possible in the case of complete absence of Cav2.1 channels.
FHM1 mutations are all missense mutations and result in gain-of-function effects on Ca2+ influx, which appears to be part of the mechanism of migraine. In contrast, the Cav2.1-α1 KO and leaner mice show loss-of-function effects. Combined with the severe phenotype in Cav2.1-α1 KO mice, one can conclude that these mice are not suitable as models for migraine. However, since patients with episodic ataxia type 2 (EA2) that have truncation or missense mutations that do result in a loss-of-function of Cav2.1 channels, the Cav2.1 KO-α1 and leaner mice seem better suited as models for EA2.
9.2.2 Conditional Cav2.1-α1 mice
Early postnatal lethality in conventional Cav2.1-α1 KO mice and widespread expression of Cav2.1 channels in brain complicates the identification of specific cell types and time windows involved in the pathophysiology of ataxia, epilepsy or migraine. Therefore, we generated a conditional mouse model that allows spatial and temporal ablation of Cav2.1 channels. We could show that the conditional allele does not cause obvious disturbances in phenotype, Cav2.1-α1 expression, or dysfunction of neurotransmission at the NMJ (Chapter 6). Experiments are ongoing to determine the relative contribution of cerebellar Purkinje and granule cells with respect to the pathophysiology of ataxia. We want to achieve this by crossing the conditional mouse with transgenic mice overexpressing Cre-recombinase, driven by the L749,50 or GABAA receptor α6 promoter, for Purkinje or granule cells respectively.51 In future experiments, cortical and hippocampal ablation of Cav2.1 channels can be achieved by crossing conditional Cav2.1-α1 mice with NSE-CreCK2 deleter mice.52 Alternatively, targeted Cre-recombination can be obtained for any region or time point of interest with viral delivery of Cre recombinase, as was demonstrated for the dorsal raphe nucleus.53
9.3 Magnetic resonance imaging in migraine
Magnetic resonance imaging (MRI) is a valuable method to study neurological diseases such as migraine. MRI is a non-invasive, in vivo technique that enables functional and prospective studies of anatomy, metabolic changes, glutamate turnover (13C-MR spectroscopy) and consequences of CSD.57 Also, screening of drugs (e.g. by evaluation of the effects on metabolism or CSD), can be combined with MRI techniques.58 For instance, in migraine patients, structural MRI revealed an increased risk of subclinical cerebellar lesions, especially in migraine with aura patients with a high attack frequency.54 Functional neuroimaging has been used to identify neurovascular events during the migraine attack.55 Striking similarities were observed between CSD and the cortical haemodynamic events that occur during the aura.56 Moreover, imaging studies show involvement of brainstem regions in migraine.55
MRI can also be used for the analysis of the transgenic migraine mouse models that were generated in this thesis. To compensate for the large difference in size between humans and mice, MRI measurements in small animals are done on high magnetic field systems. The high field causes an increase in signal, but also changes MR parameters such as T1 and T2.
T1 and T2 relaxation times are important tissue-specific parameters in MR and often
change with pathology or during processes like CSD or edema.59-61 Knowledge of relaxation times is necessary for successful implementation of (functional) brain-imaging studies, but such information is lacking for high magnetic field strengths. Therefore, to enable MR studies in migraine mouse models, we determined T1 relaxation times of the mouse brain in Chapter 7. With this knowledge, proper T1-weighted imaging protocols were developed that will in the future enable in vivo visualization of migraine- related brain regions, with techniques such as manganese-enhanced MRI (MEMRI), blood oxygen level-dependent (BOLD) functional MRI and MRI of blood brain barrier integrity using contrast agents (for Review see62).
In Chapter 2 we presented the first MRI results for the FHM1 R192Q KI mice and could show shape changes in the cerebellum, but we still have to determine whether this finding is relevant to the disease.
9.4 Future Perspectives
This thesis describes the generation of four transgenic Cav2.1-α1 mouse models. Main focus of this thesis is the generation and analysis of two KI models with pathogenic FHM1 mutations. These models are the first genetically sensitized models of migraine and show strikingly similar phenotypes as FHM1 patients. Functional analyses of FHM1 mice show that increased Ca2+ influx, increased neurotransmitter release, and enhanced susceptibility to CSD are important for migraine pathophysiology. Our results indicate that the migraine brain is hyperexcitable. Although KI models provide further evidence that CSD indeed is the underlying cause of migraine aura, a lot of questions remain to be answered:
Firstly, does the enhanced susceptibility for CSD result in increased activation of the TGVS and headache? Single and multiple waves of CSD can activate the TGVS as was demonstrated in rats by increased neuronal activity in the brainstem trigeminal nucleus caudalis (TNC).29 It will be interesting to see in FHM1 KI mice whether activation of TNC neurons is more pronounced and can facilitate mechanisms leading to headache.
Secondly, does the brainstem have an initiating or modulating role in the pathogenesis of migraine? Knight and colleagues63 studied nociceptive transmission of TNC neurons in rats after stimulation of the parietal dura mater. They demonstrate that Cav2.1 calcium channels in the periaqueductal gray (PAG) play a role in modulating trigeminal nociception. It is still unknown whether nociceptive neurotransmission is also changed
in FHM1 KI mice.
Thirdly, do migraine mice experience headache attacks? It will be important to evaluate whether FHM1 KI mice have migraine attacks. Although FHM1 R192Q mice do not reveal an obvious behavioural phenotype, it is possible that subtle paroxysmal changes in behaviour are missed. Preliminary results from Dr. Mogil (Montreal, Canada) indicate that spontaneous head grooming, not in the context of body grooming, is increased in FHM1 R192Q mice. Indication for headache in our mice can hopefully be shown by activation of the TGVS in these tests.
Fourthly, can migraine attacks be triggered? Most importantly we need to define a reliable trigger that causes migraine relevant behavioural changes in the mutant vs wild- type animals. Nitroglycerin64 can induce migraine attacks that fulfil the International Headache Society criteria, only in migraineurs, 4 to 6 hours after infusion. Similar experiments need to be performed in FHM1 KI mice to provoke migraine attacks so that we can study how migraine attacks actually begin. We know there are many modulators of migraine sensitivity (e.g. sensory stimuli, menstrual cycle, circadian rhythm, sleep deprivation, stress). A possible mechanism for these modulators may be activation of the TGVS via the parasympathetic superior salivatory nucleus and sphenopalatine ganglion.65 Via a feedback loop, the TGVS then signals to brain regions that are involved in migraine-related symptoms (e.g. nausea, loss of appetite).65 Our mouse models will be instrumental to put such hypotheses to the test.
Comparison of FHM1 KI mice and patients with the same mutations using transcriptomics, metabolomics and proteomics, under both unprovoked and provoked conditions, will hopefully yield biomarkers for migraine that are currently lacking.
Such biomarkers not only will improve diagnosis of migraine patients, but also help to understand migraine pathophysiology and guide drug target identification needed for development of novel, prophylactic, drugs. This leads us to our final question:
Can we reverse or prevent pathology by drugs that directly act on Cav2.1 channels or indirectly change susceptibility for neurotransmission and/or CSD? Several drugs that are used as migraine prophylaxis were shown to increase the threshold for CSD.66 Similar and novel migraine drugs can be evaluated in FHM1 KI mice to see whether they can reverse or prevent the pathophysiology of migraine (e.g. increased activation of TGVS, enhanced neurotransmitter release and susceptibility for CSD).
In conclusion, results in FHM1 KI mice indicate that neuronal hyperexcitability and increased susceptibility for CSD underlie migraine pathophysiology. In addition, the Cacna1a mouse models presented in this thesis are valuable models to study other neurological diseases such as ataxia, trauma, and epilepsy (FHM1 S218L mice) or episodic ataxia (leaner and Cav2.1-α1 KO). Of special note, the FHM1 S218L mouse model is an interesting model to study head trauma resulting in coma and lethality, which is a devastating neurological condition that is known to occur in children.32 Last but not least, we presented conditional Cav2.1-α1 mice that can be used to identify the most relevant cell types and time windows for these neurological diseases.
We envisage that investigation of the episodic components of migraine can be studied using non-invasive MRI techniques that enable in vivo visualization of neurovascular events during brain activation, e.g. during CSD or after a migraine trigger. It will be a challenge to make full use of this powerful methodology in the near future.
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