Academic literature on the topic 'Familial hemiplegic migraine type-1'

Background Familial hemiplegic migraine type 3 is a monogenic subtype of migraine caused by missense mutations in the neuronal voltage-gated sodium channel gene SCN1A, with 10 different mutations reported so far. In two familial hemiplegic migraine type 3 families, partial cosegregation with a rare eye phenotype (elicited repetitive daily blindness) was previously reported. Methods Two novel familial hemiplegic migraine pedigrees were subjected to genetic analysis and detailed work-up of associated clinical features. Results In both pedigrees, we identified SCN1A mutation p.F1499L, which has been previously associated with familial hemiplegic migraine type 3 and elicited repetitive daily blindness. Both families displayed a pure familial hemiplegic migraine phenotype without evidence of an episodic eye phenotype. Conclusion Like a substantial proportion of other familial hemiplegic migraine type 3 mutations, p.F1499L affects the intracellular linker between domains III and IV of SCN1A, which seems to be a mutational hot-spot. Our new data establish p.F1499L as a recurrent familial hemiplegic migraine type 3 mutation. Elicited repetitive daily blindness seems to be a rare phenomenon in familial hemiplegic migraine type 3, even in carriers of the same mutation.

Familial hemiplegic migraine type 1 (FHM-1) is a dominantly inherited subtype of migraine with aura and transient hemiplegia associated with mutations in the CACNA1A gene. FHM-1 shares many phenotypical similarities with common types of migraine, indicating common neurobiological pathways. Experimental studies have established that activation of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway plays a crucial role in migraine pathophysiology. Therefore, we tested the hypothesis that CACNA1A mutations in patients with FHM-1 are associated with hypersensitivity to NO-cGMP pathway. We included eight FHM-1 patients with R583Q and C1369Y mutations and nine healthy controls, who received intravenous infusions of 0.5 μg kg−1 min−1 glyceryl trinitrate (GTN) over 20 min. We recorded: headache intensity on a verbal rating scale; mean flow velocity in the middle cerebral artery (VmeanMCA) by transcranial Doppler; diameter of the superficial temporal artery (STA) by Dermascan. One patient reported migraine without aura 5 h after start of the GTN infusion. No aura was reported. The AUCheadache in the immediate phase was more pronounced in patients than in controls ( P = 0.01). In the 14 h following GTN infusion, there was no difference in the AUCheadache between patients and controls ( P = 0.17). We found no difference in the AUCVmeanMCA ( P = 0.12) or AUCSTA ( P = 0.71) between FHM-1 patients and controls. None of the control persons reported migraine-like headache. FHM-1 patients do not show hypersensitivity of the NO-cGMP pathway, as characteristically seen in migraine patients with and without aura. This indicates that the pathophysiological pathways underlying migraine headache in FHM-1 may be different from the common types of migraine.

Introduction: Familial hemiplegic migraine (FHM) is characterized by the familial occurrence of migraine attacks with fully reversible transient hemiplegia. Mutations in three different genes have been identified; CACNA1A (FHM1), ATP1A2 (FHM2) and SCN1A (FHM3). Besides hemiplegia, several other symptoms have been described in FHM 1–3 mutation carriers, including epilepsy and cerebellar symptoms. Case report: We describe two patients in whom hemiplegic attacks were not the presenting symptom, but in whom an otherwise unexplained head tremor led us to search for FHM mutations. Both patients carried a mutation in the CACNA1A gene. Discussion: CACNA1A mutations can give significant symptoms other than (hemiplegic) migraine as reason for presentation.

Objective: To compare clinical characteristics of patients with sporadic hemiplegic migraine (SHM) with those of patients with migraine with typical aura (MA) and patients with familial hemiplegic migraine (FHM).Methods: The authors used a computer search of Denmark’s National Patient Register to screen the population for patients with migraine with aura with motor weakness, and also examined case records from headache clinics and private practicing neurologists and placed advertisements. The authors screened patients and their relatives with a semi-structured validated telephone interview. All recruited patients were then interviewed by a physician and given a neurologic examination.Results: A total of 105 patients with SHM were identified. Seventy-two percent had four typical aura symptoms: visual, sensory, aphasic, and motor. All had at least two symptoms present during SHM attacks. A gradual progression and sequential appearance of aura symptoms was typical; compared with MA, the duration of each aura symptom was usually prolonged and bilateral motor symptoms were more frequent. Of the patients with SHM, 72% fulfilled the criteria for basilar migraine during SHM attacks. The aura was usually followed by headache, as is common in FHM but not MA.Conclusions: Patients with sporadic hemiplegic migraine had clinical symptoms identical to familial hemiplegic migraine and significantly different from migraine with typical aura. Sporadic hemiplegic migraine is a separate entity, and should be classified with familial hemiplegic migraine.

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.

Introduction Familial hemiplegic migraine (FHM) is a rare subtype of migraine with transient hemiplegic aura. Patients and methods We describe three unrelated families with familial hemiplegic migraine type II (FHM2). Retrospectively, information on 47 family members could be obtained, 15 by personal examination and 32 by indirect anamnesis from relatives. Genetic analyses were performed in 13 patients. Results One family had a novel missense mutation in the ATP1A2 gene (c.659C>T, p.Ser220Leu) that segregated with the phenotype in three generations. Two further unrelated families with different ethnic backgrounds (one from Germany and one from Russia) had a missense mutation that has not been described as yet in FHM, but occurred in only a single patient with sporadic hemiplegic migraine (c.2723G>A, p.Arg908Gln). Clinically the patients had severe attacks lasting up to several weeks as well as epileptic seizures. Three patients with a proven mutation in the ATP1A2 gene clinically presented without hemiparesis. Furthermore, there was a possible relation of FHM2 to mental retardation in another two patients. Conclusion Clinical symptoms may last for several weeks in some patients. Patients with FHM2 may also present without hemiplegia. Therefore, the full family history has to be taken into account to establish the diagnosis of FHM.

Objective: To study the link between nonverbal learning disorder and right cerebral hemisphere dysfunction due to migraine attack in a case of Familial Hemiplegic Migraine. Background: Familial Hemiplegic Migraine can cause neuropsychological deficits besides the motor ones. The nonverbal learning disorder is thought to be caused by a right hemisphere dysfunction. Methods: We describe a child with Familial Hemiplegic Migraine type 2 who showed a transient neuropsychological impairment featuring a nonverbal learning disorder during and after a Hemiplegic migraine attack. Results: Clinical and neuropsychological data showed a nonverbal learning disorder. A mutation in the ATP1A2 gene on chromosome 1q23 was found. Symptoms of nonverbal learning disorder outlasted the left hemiparesis. Two months later he showed a full recovery. Neurophysiological and neuroradiological evaluations were congruent with clinical course and with right hemisphere involvement. Conclusion: The link between nonverbal learning disorder and right cerebral hemisphere dysfunction due to migraine attack is confirmed. Familial Hemiplegic Migraine can cause transient complex neuropsychological syndromes that can be overlooked if not appropriately investigated.

Familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6) are allelic disorders caused by mutations in the CACNA1A gene on chromosome 19p13. It is well described that FHM1 can present with cerebellar signs, but parkinsonism has not previously been reported in FHM1 or EA2 even though parkinsonism has been described in SCA6. We report a 63-year-old woman with FHM1 caused by an R583Q mutation in the CACNA1A gene, clinically presenting with migraine and permanent cerebellar ataxia. Since the age of 60 years, the patient also developed parkinsonism with rigidity, bradykinesia and a resting tremor. An MRI showed a normal substantia nigra, but a bilateral loss of substance in the basal ganglia, which is in contrast to the typically normal MRI in idiopathic Parkinson's disease. Dopamine transporter (DAT) imaging with single-photon emission computed tomography demonstrated a decreased DAT-binding potential in the putamen. We wish to draw attention to FHM1 associated with parkinsonism; however, whether the reported case is a consequence of FHM1 being allelic to SCA6, unknown modifiers to the specific R583Q CACNA1A mutation or idiopathic Parkinson's disease remains unanswered.

Introduction. Familial hemiplegic migraine (FHM) is a rare disorder characterized by migraine attacks with motor weakness during the aura phase. Mutations in CACNA1A, ATP1A2, SCN1A, and PRRT2 genes have been described.Methods. To describe a mutation in ATP1A2 gene in a FHM case with especially severe and prolonged symptomatology.Results. 22-year-old woman was admitted due to migraine-type headache and sudden onset of right-sided weakness and aphasia; she had similar episodes in her childhood. Her mother was diagnosed with hemiplegic migraine without genetic confirmation. She presented with fever, decreased consciousness, left gaze preference, mixed aphasia, right facial palsy, right hemiplegia, and left crural paresis. Computed tomography (CT) showed no lesion and CT perfusion study evidenced oligohemia in left hemisphere. A normal brain magnetic resonance (MR) was obtained. Impaired consciousness and dysphasia began to improve three days after admission and mild dysphasia and right hemiparesis lasted for 10 days. No recurrences were reported during a follow-up of two years. We identified a variant in heterozygous state in ATP1A2 gene (p.Thr364Met), pathogenic according to different prediction algorithms (SIFT, PolyPhen2, MutationTaster, and Condel).Conclusion. Prolonged and severe attacks with diffuse hypoperfusion in a FHM seemed to be specially related to ATP1A2 mutations, and p.T364M should be considered.

Missense mutations in the gene that encodes the pore-forming α1 subunit of voltage-gated CaV2.1 (P/Q-type) Ca2+ channels cause a rare autosomal dominant subtype of migraine with aura: Familial Hemiplegic Migraine type 1 (FHM1). Knock-in (KI) mice carrying FHM1 mutations show increased P/Q-type Ca2+ current density in central neurons including cortical pyramidal (PYR) cells (Tottene et al., 2009; van den Maagdenberg et al., 2004). The investigation of neurotransmission in neuronal microcultures and in acute cortical slices of R192Q FHM1 knock-in (KI) mice revealed increased strength of excitatory synaptic transmission due to increased action potential-evoked Ca2+ influx through presynaptic P/Q-type Ca2+ channels and increased probability of glutamate release at cortical PYR cell synapses of mutant mice; in striking contrast, inhibitory neurotransmission at connected pairs of fast-spiking interneurons and PYR cells was unaltered in FHM1 KI mice, despite being initiated by P/Q-type Ca2+ channels (Tottene et al., 2009). The finding of different effects of FHM1 mutation at different synapses suggests that episodic disruption of the excitation-inhibition balance may underlie the increased susceptibility to cortical spreading depression (CSD), the phenomenon underlying migraine aura and a likely trigger of the migraine headache mechanisms. To further test this hypothesis I focused on the effect of FHM1 mutations at other cortical synapses. To study the synaptic connections involving somatostatin-positive (SOM+) interneurons we took advantage of GIN (GFP-expressing Inhibitory Neurons) mice which express an enhanced GFP restricted to a subpopulation somatostatin-positive (SOM+) interneurons (GIN interneurons)(Oliva et al., 2000). I performed double patch-clamp experiments on acute slices of somatosensory cortex to study excitatory and inhibitory synaptic transmission between layer 2/3 PYR cells and GIN interneurons in GIN mice. I have found that the excitatory synapse onto SOM+ interneurons shows short-term facilitation during trains of action potentials at 25 Hz suggesting that these interneurons can be recruited during sustained activity; in contrast, the inhibitory synapse between GIN interneurons and PYR cells displayed a relatively weak short-term depression during trains of action potentials at 20 Hz. Since voltage-gated Ca2+ channels controlling neurotransmitter release at these synapses are not known I performed pharmacological experiments in the presence of the specific inhibitors of P/Q- (ω-Agatoxin IVA) and N-type (ω-Conotoxin GVIA) Ca2+ channels to determine the contribution of these Ca2+ channels in controlling neurotransmitter release. I found that both P/Q- and N-type Ca2+ channels contribute to control GABA release at the GIN interneuron synapses and glutamate release at the PYR cell synapses, with a predominant role of the P/Q-type Ca2+ channels. Given the important role of P/Q-type Ca2+ channels in controlling neurotransmitter release at these synapses it will be interesting to study excitatory and inhibitory neurotransmission between PYR cells and GIN interneurons in GIN mice carrying the R192Q FHM1 mutation. To test the general hypothesis that FHM1 mutations might lead to an unbalance between inhibition and excitation towards excitation, I collaborated with Alessandra Fabbro in studying the total excitatory and inhibitory synaptic charge received by a layer 2/3 PYR cell during ongoing network activity without any type of stimulation. We performed voltage clamp recordings at the reversal potential for the inhibitory inputs (-79 mV) and at reversal potential for the excitatory inputs (+10 mV) to isolate spontaneous excitatory post-synaptic currents (sEPSC) and spontaneous inhibitory post-synaptic currents (sIPSCs) respectively. Voltage-clamp recordings of sEPSCs and sIPSCs revealed two types of activity. The first is the spontaneous postsynaptic currents (sPSCs) which arise from uncorrelated and random spontaneous activity of presynaptic excitatory and inhibitory neurons which make synapses onto the patched cell; the second is characterized by high amplitude bursts of sPSCs which arise from correlated activity of a large population of presynaptic connected neurons generated through network mechanisms. Integrals of the uncorrelated sPSCs in slices of WT and R192Q KI mice show that the excitatory synaptic charge of uncorrelated sEPSCs is larger in R192Q KI mice compared to WT mice, which is consistent with the enhanced glutamate release, and increased excitatory synaptic transmission found at PYR cells synapses of KI mice (Tottene et al, 2009). The inhibitory synaptic charge of uncorrelated sPSCs is not altered in KI mice which in accordance with unaltered inhibitory transmission at synapses of FS and other types of intemeurons contributing to the sIPSCs (Tottene et al, 2009). The analysis of the large bursts of correlated sPSCs showed that both excitatory and inhibitory burst charge are increased in R192Q KI mice but the ratio of excitatory to inhibitory burst charges is similar in WT and KI mice. These findings indicate that during the spontaneous network activity the excitation-inhibition balance is not altered in R192Q KI mice. The increase of both excitatory and inhibitory burst charges in KI mice is caused by an increase of burst frequency. As a consequence, the fraction of time spent by the cortical network in the spontaneous synchronous activity is larger in KI than WT mice.
L’emicrania emiplegica familiare di tipo 1 (FHM1), un raro sottotipo di emicrania con aura, è causata da mutazioni missenso nel gene che codifica per la subunità α1 dei canali del calcio (Ca2+) voltaggio dipendenti CaV2.1 (tipo P/Q). I topi omozigoti knock-in (KI) recanti la mutazione FHM1 R192Q presentano, in granuli di cervelletto e in cellule piramidali corticali, un aumento della densità di corrente Ca2+ di tipo P/Q (Tottene et al., 2009; van den Maagdenberg et al., 2004). Lo studio della trasmissione sinaptica in microcolture neuronali e in fettine acute di cervello di topi KI ha dimostrato: un aumento della forza della trasmissione sinaptica eccitatoria dovuto ad un aumento dell’influsso di Ca2+ attraverso i canali di tipo P/Q localizzati a livello presinaptico e un aumento della probabilità di rilascio di glutammato alle sinapsi corticali di cellule piramidali (PYR) dei topi mutanti. Al contrario, la trasmissione sinaptica inibitoria studiata tra interneuroni fast-spiking (FS) e cellule PYR è risultata essere inalterata nei topi KI nonostante i canali di tipo P/Q siano coinvolti nel rilascio anche a queste sinapsi inibitorie (Tottene et al., 2009). Queste evidenze suggeriscono che alterazioni episodiche del bilancio tra eccitazione ed inibizione in corteccia possano essere alla base dell’aumentata suscettibilità alla cortical spreading depression (CSD, il fenomeno che sottende l’aura emicranica). Per confermare questa ipotesi mi sono concentrato sugli effetti delle mutazioni FHM1 ad altre sinapsi corticali. La connessione sinaptica tra cellule PYR ed interneuroni positivi alla somatostatina (SOM+) corrispondenti alle cellule di Martinotti in ratto, è stata studiata in topo sfruttando la disponibilità di un ceppo transgenico (GFP-expressing interneurons, GIN) che esprimono la GFP (green fluorescent protein) in una sottopopolazione di interneuroni positivi alla somatostatina per lo più considerati cellule di Martinotti (Oliva et al., 2000; Fanselow et al., 2008). Ho eseguito esperimenti di doppio patch-clamp su fettine acute di cervello di corteccia somatosensoriale primaria per studiare la connessione eccitatoria tra cellule PYR e interneuroni SOM (da qui in poi chiamati interneuroni GIN) e la connessione inibitoria reciproca in topi GIN. Ho trovato che la connessione eccitatoria PYR-GIN presenta facilitazione a breve termine (short term facilitation) in risposta a treni di potenziali d’azione (action potentials, APs) a 25 Hz suggerendo che questi interneuroni possono essere reclutati da cellule PYR durante periodi di attività neuronale sostenuta; al contrario la sinapsi inibitoria GIN-PYR è caratterizzata da una debole depressione a breve termine (short term depression, STD) in risposta a treni di APs a 20 Hz. I canali del Ca2+ che controllano il rilascio di neurotrasmettitore ad entrambe le sinapsi eccitatorie ed inibitorie tra cellule PYR ed inteneuroni GIN non sono noti; pertanto ho utilizzato un approccio farmacologico perfondendo nella soluzione extracellulare inibitori di specifici canali del Ca2+, ω-Agatoxin IVA per inibire i canali di tipo P/Q e ω-Conotoxin GVIA per inibire i canali di tipo N, per determinare il loro coinvolgimento nel controllo del rilascio di neurotrasmettitore. Questi esperimenti hanno evidenziato che i canali di tipo P/Q con un ruolo predominante e i canali di tipo N sono coinvolti e cooperano nel rilascio di neurotrasmettitore sia alla sinapsi eccitatoria PYR-GIN che a quella inibitoria GIN-PYR. Dato il coinvolgimento dei canali di tipo P/Q sarà interessante studiare eventuali alterazioni della trasmissione sinaptica eccitatoria ed inibitoria tra cellule PYR ed inteneuroni GIN in topi KI per la mutazione R192Q incrociati con i topi GIN. Per verificare l’ipotesi generale che le mutazioni FHM1 possano portare ad uno sbilanciamento in corteccia tra inibizione ed eccitazione (a scapito di quest’ultima), ho collaborato con la Dott.ssa Alessandra Fabbro nello studio della carica sinaptica eccitatoria ed inibitoria totale che una cellula PYR dello strato 2/3 riceve durante l’attività di network in assenza di stimolazione. Abbiamo effettuato registrazioni di voltage clamp al potenziale di reversione degli input inibitori (-79 mV) e a quello degli input eccitatori (+ 10 mV) per poter isolare rispettivamente le correnti postsinaptiche eccitatorie (spontaneous excitatory postsynaptic currents, sEPSCs) e quelle inibitorie (spontaneous inhibitory postsynaptic currents, sIPSCs). Gli esperimenti di voltage clamp hanno messo in evidenza due tipi di attività. La prima è caratterizzata da correnti postsinaptiche spontanee (spontaneous postsynaptic currents, sPSCs) generate da input non correlati, eccitatori o inibitori, che arrivano in modo casuale da cellule eccitatorie PYR ed interneuroni inibitori connessi con la cellula da cui si esegue la registrazione; il secondo tipo di attività è caratterizzato da burst di PSCs di grandi ampiezze e che originano dall’attività correlata, forse attraverso meccanismi di network, di grandi popolazioni di cellule eccitatorie o inibitorie che formano sinapsi sulla cellula registrata. Il calcolo dell’integrale delle sPSCs non correlate in fette acute di corteccia somatosensoriale da topi WT e KI indicano che la carica eccitatoria delle sPESCs non correlate è aumentata nei topi KI in accordo con l’aumentato rilascio di glutammato e un aumentata trasmissione sinaptica eccitatoria alle sinapsi tra cellule PYR di topi KI (Tottene et al., 2009); al contrario la carica inibitoria mediata dalle sIPSCs non correlate risulta non essere alterata nei topi KI in accordo con l’inalterata trasmissione sinaptica inibitoria tra interneuroni FS e cellule PYR (Tottene et al., 2009). L’analisi delle aree dei grandi burst di input correlati ha evidenziato che sia la carica eccitatoria che quella inibitoria dei burst sono aumentate nei topi KI ma il rapporto tra carica eccitatoria ed inibitoria risulta essere inalterata nei topi KI. Questi risultati indicano che durante l’attività spontanea di network il bilancio tra eccitazione ed inibizione non è alterato. L’incremento della carica eccitatoria ed inibitoria nei topi KI è dovuto ad un aumento nella frequenza dei burst da cui consegue un aumento nel tempo trascorso dalla corteccia nell’attività spontanea correlata nei topi KI.

P/Q-type voltage-gated calcium channels are essential for Ca2+ influx and neurotransmitter release in hippocampal synaptic transmission. Through alternative splicing, the exclusion or inclusion of the NP splice variant determines the classification of the P-type or Q-type channels, respectively, which differ in their sensitivity to the peptide toxin ω-Agatoxin IVA (Aga-IVA). Familial hemiplegic migraine type-1 (FHM-1) is an autosomal dominant subtype of migraine caused by gain-of-function missense mutations in the CaV2.1 subunit of P/Q-type channels. The S218L FHM-1 mutation is associated with a particularly severe clinical syndrome which includes ataxia, generalized seizures and fatal cerebral edema, and causes a hyperpolarizing shift in channel activation resulting in an increased proportion of P/Q-type channels being open at the resting membrane potential and is predicted to increase glutamate release. Increased sensitivity of Aga-IVA has been observed in synaptic signaling in CA1 hippocampal neurons of a S218L FHM-1 mouse model although the underlying mechanism is not known. Here, performing subunit and splice-variant specific quantitative real-time PCR on mouse hippocampal regions, I demonstrate that CaV channel subunits and P/Q-type splice variants are not differentially expressed between WT and S218L mice. Using whole-cell patch-clamp electrophysiology on CA1 neurons in mouse brain slices, I further show that the contribution of P/Q-, N- and R-type channels to excitatory miniature release in WT and S218L mice is highly variable. Examining the contribution of P/Q-type channels to evoked release, I show that P/Q-type channels are an important contributor towards the rate of excitatory spontaneous action potential evoked release in WT neurons. Further, that WT CA1 neurons exhibit a large unitary EPSC response evoked by paired-pulse stimulation and was reduced when P/Q-type channels were blocked. In contrast, EPSC amplitude in S218L neurons tended to be smaller compared to EPSC amplitudes from WT although this effect was not consistent. Together, these data suggest that in CA1 neurons P/Q-type channels are predominant in evoked synaptic transmission in WT neurons and that the S218L mutation appears to cause decreased action potential evoked Ca2+ influx. Further investigation is required to determine whether other VGCCs act to compensate evoked release in S218L neurons.
Medicine, Faculty of
Graduate

Familial Hemiplegic Migraine type 2 (FHM2) is a rare subtype of migraine with aura, caused by loss-of-function mutations in ATP1A2, the gene encoding for the alpha2 subunit of the Na+,K+-ATPase (NKA), which is expressed almost exclusively in astrocytes in the adult brain. In the brain of heterozygous knockin (KI) mice carrying the W887R FHM2 mutation, the alpha2 NKA protein is halved compared to WT. These mice show a lower threshold for induction and a higher velocity of propagation of experimental cortical spreading depression (CSD), the phenomenon underlying migraine aura. The aim of my PhD project was to investigate the unknown mechanisms leading to CSD facilitation in FHM2 mouse model. Given the colocalization of alpha2 NKA and the glial glutamate transporters in the astrocytic processes surrounding glutamatergic synapses, the working hypothesis is that glutamate clearance is impaired in FHM2-KI mice, thus leading to enhanced glutamatergic neurotransmission and hence CSD facilitation. By measuring the synaptically activated glutamate transporter current in cortical astrocytes, the lab demonstrated that the reduced expression of the alpha2 NKA in FHM2-KI mice leads to a reduced rate of glutamate clearance. We demonstrate that, in vitro, experimental CSD is facilitated in FHM2-KI compared to WT mice. I therefore investigated whether there is a causative link between the impaired glutamate clearance and CSD facilitation in FHM2-KI mice, using two different approaches: first, testing whether Ceftriaxone (Cef), a drug that increases the membrane expression of GLT-1 in neocortex rescues CSD facilitation. Cef-treatment increased slightly, but significantly, CSD threshold without affecting CSD propagation. In the second approach I investigated whether pharmacological reduction of the glutamate clearance in WT mice to a value similar to that of FHM2-KI mice lowered the threshold for CSD induction and increased the velocity of CSD propagation. I found that the concentration of a glutamate transporters inhibitor able to slow the glutamate clearance in WT mice similarly than in FHM2-KI, also lowered the threshold for CSD induction similarly than in FHM2-KI and increased the velocity of CSD propagation, but less than in FHM2-KI, suggesting that the reduced rate of glutamate clearance can account for most of the facilitation of CSD induction and for a fraction of the facilitation of CSD propagation in the FHM2 mutants. Given the key role of N-methyl-D-aspartate receptors (NMDARs) in CSD initiation, I investigated whether in FHM2-KI mice there is an excessive NMDARs activation due to increased glutamate spillover. Measurements of post-synaptic NMDAR currents (NMDARs-EPSC) elicited in cortical layer 2/3 pyramidal neurons (Pyrs) by extracellular stimulation in layer 1 in acute cortical slices of the barrel cortex revealed an increased amplitude and a slowing of the decay of the NMDARs-EPSC in FHM2-KI mice. Quantitatively, the changes in FHM2-KI were larger after trains of pulses at high frequency than after a single stimulus. These data support the conclusion that defective glutamate clearance in FHM2 leads to increased activation of Pyrs NMDARs consequent to increased glutamate spillover, and suggest that this may largely account for CSD facilitation in FHM2. I investigated the pharmacological profile of the NMDARs activated by increased glutamate spillover. I found that in WT mice the large majority of the NMDARs-EPSC is due to activation of triheteromeric GluN2A-2BRs and only a small fraction is due to activation of diheteromeric GluN2BRs, while in FHM2-KI a larger fraction of the NMDARs-EPSC is due to activation of GluN2BRs compared to WT mice. This is consistent with preferential recruitment of GluN2BRs by increased glutamate spillover in FHM2 KI mice. The comparison of the pharmacological data suggests that the increased activation of GluN2BRs might quantitatively account for most, if not all, the increased activation of NMDARs in FHM2-KI compared to WT mice. These findings in FHM2 KI, together with previous findings in FHM1 mouse model, support the idea that excessive glutamatergic transmission and excessive activation of NMDARs play a crucial role in CSD facilitation in FHM mouse models. Previous findings obtained in the lab revealed unaltered inhibitory transmission at several cortical inhibitory synapses in contrast with increased excitatory transmission at cortical Pyrs synapses, together with the α2NKA expression at excitatory but not inhibitory cortical synapses, point to a dysfunctional regulation of E/I balance in FHM. As a first test of this hypothesis, we decided to investigate whether FHM mutations alter the dynamic regulation of the E/I balance in L2/3 during recurrent network activity induced by optogenetic activation of L2/3 Pyrs with different types of light stimuli, that presumably mimic different types of physiological activity. I performed in utero electroporation at day 15.5 of gestation allows to selectively express channelrhodopsin-2 (ChR2), a non-specific cation channel that depolarize upon blue light illumination (443 nm), in L2/3 Pyrs; this allows to selectively activate L2/3 Pyrs in thalamocortical slices and to record light-evoke excitatory and inhibitory currents in voltage-clamped neurons not expressing ChR2. My work in the last period was dedicated to implement this technique in particular, I optimized I) the concentrations of cDNA and Fast Green FCF to be injected into the embryo; II) the electroporation and injection parameters; III) the mating strategy in order to obtain a sufficient number of pregnant mice and surviving pups after electroporation. If we succeed in resolving the remaining technical problems regarding the electroporation method, we are now ready to investigate whether FHM mutations alter the dynamic regulation of the E/I balance in L2/3 during recurrent network activity.
L’emicrania emiplegica familiare di tipo 2 (FHM2) è un sottotipo di emicrania con aura, causata da una mutazione con perdita di funzione nel gene ATP1A2, codificante per la subunità alpha2 della Na+,K+ATPase (alpha2NKA). La alpha2 NKA è espressa quasi esclusivamente negli astrociti nel cervello adulto. Nel cervello dei topi eterozigoti in cui è stata inserita la mutazione umana W887R che causa l’FHM2 (FHM2-KI), i livelli di espressione della proteina alpha2 NKA sono dimezzati rispetto ai topi wild-type (WT). I topi FHM2-KI hanno una ridotta soglia di innesco e un’aumentata velocità di propagazione della cortical spreading depression (CSD), il fenomeno alla base dell’aura emicranica. Lo scopo del mio dottorato è stato studiare i meccanismi alla base della facilitazione della CSD in un modello murino di FHM2. Data la colocalizzazione della alpha2 NKA con i trasportatori gliali del glutammato (GluTs) nei processi astrocitari delle sinapsi eccitatorie, l’ipotesi è che la rimozione del glutammato negli FHM2-KI sia compromessa e che questo comporti un’aumentata neurotrasmissione glutammatergica, e quindi la facilitazione della CSD. Attraverso la misura della corrente attivata sinapticamente mediata dai trasportatori del glutammato (GluTs), è stato dimostrato che la ridotta espressione della alpha2 NKA comporta una ridotta ricaptazione del glutammato negli FHM2-KI. E’ stato dimostrato che, in vitro, la CSD sperimentale è facilitata negli FHM2-KI rispetto ai WT. Ho studiato se vi fosse una relazione causa-effetto tra la ridotta rimozione del glutammato e la facilitazione della CSD negli FHM2-KI, mediante due diversi approcci: nel primo ho testato l’effetto del Ceftrixone (Cef), un farmaco che aumenta l’espressione in membrana dei GLT-1 nella neocorteccia, sul ripristino di soglia e velocità di propagazione della CSD degli FHM2-KI ai valori dei WT. Il trattamento farmacologico degli FHM2-KI con Cef aumenta significativamente la soglia per l’innesco, senza influenzare la velocità di propagazione della CSD. Con il secondo approccio ho testato se la riduzione farmacologica della rimozione del glutammato nei WT, in misura simile agli FHM2-KI, abbassasse la soglia per l’innesco della CSD e ne aumentasse la velocità di propagazione come nella mutazione. Ho identificato la concentrazione di un inibitore dei GluTs capace di mimare l’effetto della mutazione ed ho trovato che questa concentrazione abbassa la soglia per l’innesco della CSD in modo simile agli FHM2-KI e aumenta la velocità di propagazione anche se in maniera inferiore rispetto agli FHM2-KI, suggerendo che la ridotta rimozione del glutammato rende conto della maggior parte della facilitazione dell’induzione e di parte della velocità di propagazione. Dato il ruolo dei recettori N-methyl-D-aspartate (NMDARs) nell’innesco della CSD, ho studiato se negli FHM2-KI vi fosse un aumentata attivazione degli NMDARs dovuta allo spillover del glutammato. La misura della corrente postsinaptica eccitatoria mediata dagli NMDARs (NMDARs-EPSC) evocata nei neuroni piramidali dello strato 2/3 della corteccia somatosensoriale mediante stimolazione extracellulare nello strato 1 in fettina di cervello, ha dimostrato che l’ampiezza è aumentata ed il tempo di decadimento rallentato negli FHM2-KI rispetto ai WT. Ho studiato il profilo farmacologico degli NMDARs attivati dallo spillover del glutammato. Ho trovato che nei topi WT, la maggior parte dell’NMDARs-EPSC è dovuto all’attivazione dei recettori trieteromerici GluN2A-2B (GluN2A-2BRs) e solo una piccola parte ai dieteromerici GluN2B (GluN2BRs), mentre negli FHM2-KI una frazione maggiore dell’NMDARs-EPSC è dovuta ai GluN2BRs. Questo è consistente con un reclutamento preferenziale dei GluN2BRs dall’aumentato spillover del glutammato. Il confronto dei dati farmacologici suggerisce che l’aumentata attivazione dei GluN2BRs potrebbe quantitativamente rendere conto della maggior parte, se non completamente, dell’aumentata attivazione degli NMDARs negli FHM2-KI rispetto ai WT. Questi dati negli FHM2 e dati precedenti ottenuti nei topi FHM1 KI, supportano l’idea che un’eccessiva neurotrasmissione glutammatergica ed un’eccessiva attivazione dei recettori NMDA abbiano un ruolo fondamentale nella facilitazione della CSD nei modelli di FHM. Precedenti studi in laboratorio hanno rivelato un’inalterata neurotrasmissione inibitoria a diverse sinapsi inibitorie in contrasto con un’aumentata trasmissione eccitatoria alle sinapsi corticali ed, assieme con l’espressione della alpha2 NKA alle sinapsi corticali eccitatorie ma non inibitorie, suggeriscono una regolazione disfunzionale del bilanciamento eccitazione/inibizione nei topi FHM. Per testare questa ipotesi abbiamo deciso di studiare se le mutazioni che causano l’FHM alterino la regolazione eccitazione/inibizione nello strato 2/3 della corteccia somatosensoriale durante l’attività di network ricorrente indotta da attivazione optogenetica delle cellule piramidali dello strato 2/3, con tipi differenti stimoli luminosi, che mimano differenti tipologie di attività fisiologiche. Attraverso la tecnica dell’elettroporazione in utero abbiamo cercato di esprimere selettivamente la channelrhodopsin-2, un canale cationico non-specifico che si depolarizza in seguito ad illuminazione con luce blu (443 nm), nelle cellule piramidali dello strato 2/3. Questo permette di attivare selettivamente cellule piramidali dello strato 2/3 in fettine di cervello talamo-corticali e registrare correnti eccitatorie ed inibitorie evocate dagli stimoli luminosi in neuroni piramidali che non esprimono channelrhodopsin-2. Nell’ultimo periodo mi sono occupata di implementare questa tecnica in particolare migliorando I) la concentrazione di cDNA e Fast Green FCF da iniettare negli embrioni; II) i parametri di elettroporazione ed iniezione; III) le strategie di accoppiamento per ottenere un maggior numero di topi incinta e di cuccioli che sopravvivono all’intera procedura. Se riusciremo a risolvere gli ultimi problemi tecnici relativi alla tecnica dell’elettroporazione in utero siamo pronti a studiare se le mutazioni FHM alterino la regolazione dinamica del bilanciamento eccitazione/inibizione nello strato 2/3 durante l’attività ricorrente di network.

This project was an effort to improve the speed and affordability of genetic testing for neurological disorders. The research involved the development of a genetic diagnostic testing technique using Next Generation Sequencing technologies to investigate Familial Hemiplegic Migraine and related neurological conditions. The method successfully expanded the scope of testing, resulting in the ability to detect genetic changes that would have gone unnoticed in previous tests and significantly reduced the cost of testing for patients and the healthcare system.

This dissertation focused on expanding the knowledge and understanding of common and complex migraine genetics. The central objectives of this research was to 1) identify new migraine causative genes using modern sequencing techniques and; 2) investigate potential genetic associations between common and complex migraine. This study has successfully identified 1) a susceptibility association between a potential familial hemiplegic migraine (FHM) gene and common migraine and; 2) implicated 4 new genes as potentially FHM causative. As a result of this study we foresee the development of a more comprehensive genetic test with improved diagnostic success rates.

Hemiplegic migraine (HM) is a rare monogenic subtype of migraine with aura that includes reversible motor weakness and can be either familial or sporadic. Three genes have been associated with HM. Migraine can also be part of more elaborate monogenic syndromes. When the family history includes early-onset cerebrovascular diseases and dementia, the monogenic small-vessel diseases CADASIL, RVCL-S, and COL4A1-associated syndromes should be considered. The mitochondrial disease MELAS is also associated with migraine-like and stroke-like episodes, often with a progressive nature, hearing loss and short stature. Like familial HM type 1, EA2 and SCA-6 are associated with CACNA1A mutations. All three disorders can include episodic and progressive ataxia and imaging may reveal cerebellar atrophy. Episodic hemiplegia or quadriplegia also occurs in the rare disorder alternating hemiplegia of childhood, with an age at onset before 18 months and often in association with tonic/dystonic attacks and developmental delay.