Modulating factors for and consequences of cortical spreading depression

The research in this thesis was aimed at identifying and understanding mechanisms underlying modulating factors for and consequences of cortical spreading depression (CSD), the pathophysiological substrate for migraine aura that occurs in one-third of migraine patients. In this thesis, experimental studies on CSD were performed in wild-type (WT) and transgenic migraine mice, which express CaV2.1 Ca2+ channels with a mutated _1 subunit that contains the R192Q missense mutation. The R192Q mutation was previously identified in patients with familial hemiplegic migraine type 1 (FHM1) and causes gain-of-function effects in terms of neuronal Ca2+ influx, neurotransmission, and susceptibility to experimentally induced CSD. Using various experimental strategies, in this thesis, the FHM1 R192Q mouse model was used to study pathophysiological mechanisms of the initiation and modulation of CSD as well as of neurobiological and molecular changes that accompany CSD events.UBL - phd migration 201

Purkinje cell-specific ablation of CaV2.1 Channels is sufficient to cause cerebellar ataxia in mice

The Cacna1a gene encodes the α1A subunit of voltage-gated CaV2.1 Ca2+ channels that are involved in neurotransmission at central synapses. CaV2.1-α1- knockout (α1KO) mice, which lack CaV2.1 channels in all neurons, have a very severe phenotype of cerebellar ataxia and dystonia, and usually die around postnatal day 20. This early lethality, combined with the wide expression of CaV2.1 channels throughout the cerebellar cortex and nuclei, prohibited determination of the contribution of particular cerebellar cell types to the development of the severe neurobiological phenotype in Cacna1a mutant mice. Here, we crossed conditional Cacna1a mice with transgenic mice expressing Cre recombinase, driven by the Purkinje cell-specific Pcp2 promoter, to specifically ablate the CaV2.1- α1A subunit and thereby CaV2.1 channels in Purkinje cells. Purkinje cell CaV2.1-α1A-knockout (PCα1KO) mice aged without difficulties, rescuing the lethal phenotype seen in α1KO mice. PCα1KO mice exhibited cerebellar ataxia starting around P12, much earlier than the first signs of progressive Purkinje cell loss, which appears in these mice between P30 and P45. Secondary cell loss was observed in the granular and molecular layers of the cerebellum and the volume of all individual cerebellar nuclei was reduced. In this mouse model with a cell type-specific ablation of CaV2.1 channels, we show that ablation of CaV2.1 channels restricted to Purkinje cells is sufficient to cause cerebellar ataxia. We demonstrate that spatial ablation of CaV2.1 channels may help in unraveling mechanisms of human disease

GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders

Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23 microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development and behavioral hallmarks remains largely unknown.We integrated proteomic and transcriptomic profiling of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23 dosage–dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis as a molecular pathway amenable to therapeutic intervention in ASD

Stress hormone corticosterone enhances susceptibility to cortical spreading depression in familial hemiplegic migraine type 1 mutant mice

Stress is a putative migraine trigger, but the pathogenic mechanisms involved are unknown. Stress and stress hormones increase neuronal excitability by enhancing glutamatergic neurotransmission, but inhibitory effects have also been reported. We hypothesise that an acute rise in stress hormones, such as corticosteroids which are released after stress, increase neuronal excitability and thereby may increase susceptibility to cortical spreading depression (CSD), the mechanism underlying the migraine aura. Here we investigated effects of acute restraint stress and of the stress hormone corticosterone on CSD susceptibility as surrogate migraine marker, in a transgenic mouse model of familial hemiplegic migraine type 1 (FHM1), which displays increased glutamatergic cortical neurotransmission and increased propensity for CSD. We found that 20-min and 3-h restraint stress did not influence CSD susceptibility in mutant or wild-type mice, despite elevated levels of plasma corticosterone. By contrast, subcutaneous administration of 20. mg/kg corticosterone increased CSD frequency exclusively in mutant mice, while corticosterone plasma levels were similarly elevated in mutants and wild types. The effect of corticosterone on CSD frequency was normalised by pre-administration of the glucocorticoid receptor (GR) antagonist mifepristone. These findings suggest that corticosteroid-induced GR activation can enhance susceptibility to CSD in genetically susceptible individuals, and may predispose to attacks of migraine. Although corticosterone levels rise also during acute stress, the latter likely triggers a spatiotemporally more complex biological response with multiple positive and negative modulators which may not be adequately modeled by exogenous administration of corticosterone alone

Stress hormone corticosterone enhances susceptibility to cortical spreading depression in familial hemiplegic migraine type 1 mutant mice

Stress is a putative migraine trigger, but the pathogenic mechanisms involved are unknown. Stress and stress hormones increase neuronal excitability by enhancing glutamatergic neurotransmission, but inhibitory effects have also been reported. We hypothesise that an acute rise in stress hormones, such as corticosteroids which are released after stress, increase neuronal excitability and thereby may increase susceptibility to cortical spreading depression (CSD), the mechanism underlying the migraine aura. Here we investigated effects of acute restraint stress and of the stress hormone corticosterone on CSD susceptibility as surrogate migraine marker, in a transgenic mouse model of familial hemiplegic migraine type 1 (FHM1), which displays increased glutamatergic cortical neurotransmission and increased propensity for CSD. We found that 20-min and 3-h restraint stress did not influence CSD susceptibility in mutant or wild-type mice, despite elevated levels of plasma corticosterone. By contrast, subcutaneous administration of 20. mg/kg corticosterone increased CSD frequency exclusively in mutant mice, while corticosterone plasma levels were similarly elevated in mutants and wild types. The effect of corticosterone on CSD frequency was normalised by pre-administration of the glucocorticoid receptor (GR) antagonist mifepristone. These findings suggest that corticosteroid-induced GR activation can enhance susceptibility to CSD in genetically susceptible individuals, and may predispose to attacks of migraine. Although corticosterone levels rise also during acute stress, the latter likely triggers a spatiotemporally more complex biological response with multiple positive and negative modulators which may not be adequately modeled by exogenous administration of corticosterone alone

High-throughput screening identifies histone deacetylase inhibitors that modulate GTF2I expression in 7q11.23 microduplication autism spectrum disorder patient-derived cortical neurons

Background: Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental condition affecting almost 1% of children, and represents a major unmet medical need with no effective drug treatment available. Duplication at 7q11.23 (7Dup), encompassing 26-28 genes, is one of the best characterized ASD-causing copy number variations and offers unique translational opportunities, because the hemideletion of the same interval causes Williams-Beuren syndrome (WBS), a condition defined by hypersociability and language strengths, thereby providing a unique reference to validate treatments for the ASD symptoms. In the above-indicated interval at 7q11.23, defined as WBS critical region, several genes, such as GTF2I, BAZ1B, CLIP2 and EIF4H, emerged as critical for their role in the pathogenesis of WBS and 7Dup both from mouse models and human studies. Methods: We performed a high-throughput screening of 1478 compounds, including central nervous system agents, epigenetic modulators and experimental substances, on patient-derived cortical glutamatergic neurons differentiated from our cohort of induced pluripotent stem cell lines (iPSCs), monitoring the transcriptional modulation of WBS interval genes, with a special focus on GTF2I, in light of its overriding pathogenic role. The hits identified were validated by measuring gene expression by qRT-PCR and the results were confirmed by western blotting. Results: We identified and selected three histone deacetylase inhibitors (HDACi) that decreased the abnormal expression level of GTF2I in 7Dup cortical glutamatergic neurons differentiated from four genetically different iPSC lines. We confirmed this effect also at the protein level. Limitations: In this study, we did not address the molecular mechanisms whereby HDAC inhibitors act on GTF2I. The lead compounds identified will now need to be advanced to further testing in additional models, including patient-derived brain organoids and mouse models recapitulating the gene imbalances of the 7q11.23 microduplication, in order to validate their efficacy in rescuing phenotypes across multiple functional layers within a translational pipeline towards clinical use. Conclusions: These results represent a unique opportunity for the development of a specific class of compounds for treating 7Dup and other forms of intellectual disability and autismPublished versio

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.Pattern Recognition and Bioinformatic

Automatic Registration of Mass Spectrometry Imaging Data Sets to the Allen Brain Atlas

Mass spectrometry imaging holds great potential for understanding the molecular basis of neurological disease. Several key studies have demonstrated its ability to uncover disease-related biomolecular changes in rodent models of disease, even if highly localized or invisible to established histological methods. The high analytical reproducibility necessary for the biomedical application of mass spectrometry imaging means it is widely developed in mass spectrometry laboratories. However, many lack the expertise to correctly annotate the complex anatomy of brain tissue, or have the capacity to analyze the number of animals required in preclinical studies, especially considering the significant variability in sizes of brain regions. To address this issue, we have developed a pipeline to automatically map mass spectrometry imaging data sets of mouse brains to the Allen Brain Reference Atlas, which contains publically available data combining gene expression with brain anatomical locations. Our pipeline enables facile and rapid interanimal comparisons by first testing if each animal’s tissue section was sampled at a similar location and enabling the extraction of the biomolecular signatures from specific brain regions