Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine.

Familial hemiplegic migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the α1A subunit of voltage-gated CaV2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation ('FHM1 R192Q mice') exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here, we analysed gene expression profiles from isolated cortical tissue of FHM1 R192Q mice 24 h after experimentally induced CSD in order to identify molecular pathways affected by CSD. Gene expression profiles were generated using deep serial analysis of gene expression sequencing. Our data reveal a signature of inflammatory signalling upon CSD in the cortex of both mutant and wild-type mice. However, only in the brains of FHM1 R192Q mice specific genes are up-regulated in response to CSD that are implicated in interferon-related inflammatory signalling. Our findings show that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after CSD in a relevant mouse model of migraine.

The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation.

INTRODUCTION: CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to toxic NOTCH3 protein accumulation in the small- to medium sized arterioles. The accumulation is systemic but most pronounced in the brain vasculature where it leads to clinical symptoms of recurrent stroke and dementia. There is no therapy for CADASIL, and therapeutic development is hampered by a lack of feasible clinical outcome measures and biomarkers, both in mouse models and in CADASIL patients. To facilitate pre-clinical therapeutic interventions for CADASIL, we aimed to develop a novel, translational CADASIL mouse model. RESULTS: We generated transgenic mice in which we overexpressed the full length human NOTCH3 gene from a genomic construct with the archetypal c.544C > T, p.Arg182Cys mutation. The four mutant strains we generated have respective human NOTCH3 RNA expression levels of 100, 150, 200 and 350 % relative to endogenous mouse Notch3 RNA expression. Immunohistochemistry on brain sections shows characteristic vascular human NOTCH3 accumulation in all four mutant strains, with human NOTCH3 RNA expression levels correlating with age at onset and progression of NOTCH3 accumulation. This finding was the basis for developing the 'NOTCH3 score', a quantitative measure for the NOTCH3 accumulation load. This score proved to be a robust and sensitive method to assess the progression of NOTCH3 accumulation, and a feasible biomarker for pre-clinical therapeutic testing. CONCLUSIONS: This novel, translational CADASIL mouse model is a suitable model for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation, using the NOTCH3 score as a biomarker.

RNA expression profiling in brains of familial hemiplegic migraine type 1 knock-in mice.

BACKGROUND: Various CACNA1A missense mutations cause familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of migraine with aura. FHM1 mutation R192Q is associated with pure hemiplegic migraine, whereas the S218L mutation causes hemiplegic migraine, cerebellar ataxia, seizures, and mild head trauma-induced brain edema. Transgenic knock-in (KI) migraine mouse models were generated that carried either the FHM1 R192Q or the S218L mutation and were shown to exhibit increased CaV2.1 channel activity. Here we investigated their cerebellar and caudal cortical transcriptome. METHODS: Caudal cortical and cerebellar RNA expression profiles from mutant and wild-type mice were studied using microarrays. Respective brain regions were selected based on their relevance to migraine aura and ataxia. Relevant expression changes were further investigated at RNA and protein level by quantitative polymerase chain reaction (qPCR) and/or immunohistochemistry, respectively. RESULTS: Expression differences in the cerebellum were most pronounced in S218L mice. Particularly, tyrosine hydroxylase, a marker of delayed cerebellar maturation, appeared strongly upregulated in S218L cerebella. In contrast, only minimal expression differences were observed in the caudal cortex of either mutant mice strain. CONCLUSION: Despite pronounced consequences of migraine gene mutations at the neurobiological level, changes in cortical RNA expression in FHM1 migraine mice compared to wild-type are modest. In contrast, pronounced RNA expression changes are seen in the cerebellum of S218L mice and may explain their cerebellar ataxia phenotype.

Severe and progressive neurotransmitter release aberrations in familial hemiplegic migraine type 1 Cacna1a S218L knock-in mice.

Familial hemiplegic migraine type 1 (FHM1) is caused by mutations in the CACNA1A gene, encoding neuronal presynaptic Ca(V)2.1 (P/Q-type) Ca(2+) channels. These channels mediate neurotransmitter release at many central synapses and at the neuromuscular junction (NMJ). Mutation S218L causes a severe neurological phenotype of FHM and, additionally, ataxia and susceptibility to seizures, delayed brain edema, and fatal coma after minor head trauma. Recently, we generated a Cacna1a S218L knock-in mutant mouse, displaying these features and reduced survival. A first electrophysiological study showed high susceptibility for cortical spreading depression, enhanced neuronal soma Ca(2+) influx, and at diaphragm NMJs, a considerable increase of neurotransmitter release. We here assessed the function of S218L knock-in NMJs at several muscle types in great detail. Pharmacological analyses using specific Ca(V) subtype-blocking toxins excluded compensatory contribution of non-Ca(V)2.1 channels. Endplate potentials were considerably broadened at many NMJs. High rate (40 Hz)-evoked acetylcholine release was slightly reduced; however, it was not associated with block of neurotransmission causing weakness, as assessed with grip strength measurements and in vitro muscle contraction experiments. The synaptopathy clearly progressed with age, including development of an increased acetylcholine release at low-rate nerve stimulation at physiological extracellular Ca(2+) concentration and further endplate potential broadening. Our results suggest enhanced Ca(2+) influx into motor nerve terminals through S218L-mutated presynaptic Ca(V)2.1 channels, likely because of the earlier reported negative shift of activation potential and reduced inactivation. Similar severe aberrations at central synapses of S218L mutant mice and humans may underlie or contribute to the drastic neurological phenotype.

Quantitative cortical synapse proteomics of a transgenic migraine mouse model with mutated Ca(V)2.1 calcium channels.

Familial hemiplegic migraine type 1 (FHM1) is caused by missense mutations in the CACNA1A gene that encodes the alpha1A pore-forming subunit of Ca(V)2.1 Ca(2+) channels. Knock-in (KI) transgenic mice expressing Ca(V)2.1 Ca(2+) channels with a human pathogenic FHM1 mutation reveal enhanced glutamatergic neurotransmission in the cortex. In this study, we employed an iTRAQ-based LC-LC MS/MS approach to identify differentially expressed proteins in cortical synapse proteomes of Cacna1a R192Q KI and wild-type mice. All expression differences determined were subtle and in the range of 10-30%. Observed upregulated proteins in the mutant mice are involved in processes, such as neurite outgrowth and actin dynamics, vesicle turnover, and glutamate transporters. Our data support the view that in Cacna1a R192Q KI mice, several compensatory mechanisms counterbalancing a dysregulated glutamatergic signaling have come into effect. We propose that such adaptation mechanisms at the synapse level may play a role in the pathophysiology of FHM and possibly in the common forms of migraine.

High cortical spreading depression susceptibility and migraine-associated symptoms in Ca(v)2.1 S218L mice.

OBJECTIVE: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. METHODS: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. RESULTS: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. INTERPRETATION: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema.

Conditional inactivation of the Cacna1a gene in transgenic mice.

Ca(v)2.1 (P/Q-type) voltage-gated calcium channels play an important role in neurotransmitter release at many brain synapses and at the neuromuscular junction. Mutations in the CACNA1A gene, encoding the pore forming alpha(1) subunit of Ca(v)2.1 channels, are associated with a wide spectrum of neurological disorders. Here we generated mice with a conditional, floxed, Cacna1a allele without any overt phenotype. Deletion of the floxed Cacna1a allele resulted in ataxia, dystonia, and lethality during the fourth week, a severe phenotype similar to conventional Ca(v)2.1 knockout mice. Although neurotransmitter release at the neuromuscular junction was not affected in the conditional mice, homozygous deletion of the floxed allele caused an ablation of Ca(v)2.1 channel-mediated neurotransmission that was accompanied by a compensatory upregulation of Ca(v)2.3 (R-type) channels at this synapse. Pharmacological inhibition of Ca(v)2.1 channels is possible, but the contributing cell-types and time windows relevant to the different Ca(v)2.1-related neurological disorders can only be reliably determined using Cacna1a conditional mice.

Two de novo mutations in the Na,K-ATPase gene ATP1A2 associated with pure familial hemiplegic migraine.

Familial hemiplegic migraine (FHM) is a rare autosomal dominantly inherited subtype of migraine, in which hemiparesis occurs during the aura. The majority of the families carry mutations in the CACNA1A gene on chromosome 19p13 (FHM1). About 20% of FHM families is linked to chromosome 1q23 (FHM2), and has mutations in the ATP1A2 gene, encoding the alpha2-subunit of the Na,K-ATPase. Mutation analysis in a Dutch and a Turkish family with pure FHM revealed two novel de novo missense mutations, R593W and V628M, respectively. Cellular survival assays support the hypothesis that both mutations are disease-causative. The identification of the first de novo mutations underscores beyond any doubt the involvement of the ATP1A2 gene in FHM2.

Familial hemiplegic migraine type 1 mutations K1336E, W1684R, and V1696I alter Cav2.1 Ca2+ channel gating: evidence for beta-subunit isoform-specific effects.

Mutations in the Cav2.1 alpha1-subunit of P/Q-type Ca2+ channels cause human diseases, including familial hemiplegic migraine type-1 (FHM1). FHM1 mutations alter channel gating and enhanced channel activity at negative potentials appears to be a common pathogenetic mechanism. Different beta-subunit isoforms (primarily beta4 and beta3) participate in the formation of Cav2.1 channel complexes in mammalian brain. Here we investigated not only whether FHM1 mutations K1336E (KE), W1684R (WR), and V1696I (VI) can affect Cav2.1 channel function but focused on the important question whether mutation-induced changes on channel gating depend on the beta-subunit isoform. Mutants were co-expressed in Xenopus oocytes together with beta1, beta3, or beta4 and alpha2delta1 subunits, and channel function was analyzed using the two-electrode voltage-clamp technique. WR shifted the voltage dependence for steady-state inactivation of Ba2+ inward currents (IBa) to more negative voltages with all beta-subunits tested. In contrast, a similar shift was observed for KE only when expressed with beta3. All mutations promoted IBa decay during pulse trains only when expressed with beta1 or beta3 but not with beta4. Enhanced decay could be explained by delayed recovery from inactivation. KE accelerated IBa inactivation only when co-expressed with beta3, and VI slowed inactivation only with beta1 or beta3. KE and WR shifted channel activation of IBa to more negative voltages. As the beta-subunit composition of Cav2.1 channels varies in different brain regions, our data predict that the functional FHM1 phenotype also varies between different neurons or even within different neuronal compartments.

Cell autonomy of the mouse claw paw mutation.

Mice homozygous for the autosomal recessive mutation claw paw (clp) are characterized by limb posture abnormalities and congenital hypomyelination, with delayed onset of myelination of the peripheral nervous system but not the central nervous system. Although this combination of limb and peripheral nerve abnormalities in clp/clp mice might suggest a common neurogenic origin of the syndrome, it is not clear whether the clp gene acts primarily in the neurone, the Schwann cell or both. In the work described here, we address this question of cell autonomy of the clp mutation through reciprocal nerve grafting experiments between wild-type and clp/clp animals. Our results demonstrate that the clp mutation affects the Schwann cell compartment and possibly also the neuronal compartment. These data suggest that the clp gene product is expressed in Schwann cells as well as neurones and is likely to be involved in direct axon--Schwann cell interactions. Within the Schwann cell, clp affects a myelin-related signaling pathway that regulates periaxin and Krox-20 expression, but not Oct-6.

A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression.

Migraine is a common, disabling, multifactorial, episodic neurovascular disorder of unknown etiology. Familial hemiplegic migraine type 1 (FHM-1) is a Mendelian subtype of migraine with aura that is caused by missense mutations in the CACNA1A gene that encodes the alpha(1) subunit of neuronal Ca(v)2.1 Ca(2+) channels. We generated a knockin mouse model carrying the human pure FHM-1 R192Q mutation and found multiple gain-of-function effects. These include increased Ca(v)2.1 current density in cerebellar neurons, enhanced neurotransmission at the neuromuscular junction, and, in the intact animal, a reduced threshold and increased velocity of cortical spreading depression (CSD; the likely mechanism for the migraine aura). Our data show that the increased susceptibility for CSD and aura in migraine may be due to cortical hyperexcitability. The R192Q FHM-1 mouse is a promising animal model to study migraine mechanisms and treatments.