Activity-dependent regulation of synaptic strength and cellular mechanisms of migraine

Lead Research Organisation: University College London
Department Name: Institute of Neurology

Abstract

Migraine is a chronic neurological disorder where affected patients experience recurrent attacks of moderate to severe headaches that are often accompanied by other debilitating symptoms such as nausea, vomiting and sensitivity to light, smell, or sound. Migraine affects over 10% of the population and represents a major disease burden for society. The neuronal mechanisms of this syndrome remain however poorly understood.
Some inherited cases of migraine, as well as other episodic neurological disorders such as ataxia (incoordination due to abnormal cerebellar function) and epilepsy, are caused by mutations of ion channels that gate calcium, sodium, or potassium fluxes across presynaptic membranes during action potentials. These "presynaptic neurological channelopathies" are thought to destabilise neuronal networks by affecting the release of neurotransmitters. The effects of the disease mutations on the channel functions in channelopathies can be precisely determined by electrophysiological methods. Therefore, understanding the mechanisms of channelopathies provides invaluable insights into the pathogenesis of more common forms of migraine, ataxia and epilepsy.
The conventional way to study channelopathies is to determine the precise effects of a mutation at the single channel level and then to relate these effects to changes in synaptic transmission in neuronal models of disease. However, this straightforward approach often leads to paradoxical results. Using our pilot data we hypothesise that the missing key to understanding these diseases is homeostatic compensation of synaptic transmission, which, although abundantly documented in experimental studies, has been largely overlooked in studies of pathogenic mechanisms. Homeostatic synaptic plasticity is a negative feedback mechanism, which compensates for increases or decreases in neuronal activity by adjusting the strengths of innervating synapses. Our preliminary data argue that channelopathies do invoke homeostatic changes. Furthermore, an understanding of homeostatic compensation could go a long way to resolve the long-standing puzzle why most neurological channelopathies are episodic disorders, which generally do not interfere with brain function between manifestations of the disease.
In this project we propose for the first time to systematically study the role of homeostatic mechanisms in channelopathies using mouse models of Familial Hemiplegic Migraine Type 1 (FHM1). FHM1 is caused by mutations in the CACNA1A gene that encodes the pore forming subunit of P/Q-type presynaptic calcium channels that are the major triggers of neurotransmitter release in the brain. We have recently developed a set of new imaging methods, which allow us to study the relationship between calcium entry and vesicular exocytosis, and to probe presynaptic ion channel function in individual small presynaptic terminals. Using these techniques we will determine to what extent the gain of function effects of two different FHM1 CACNA1A mutations (S218L and R192Q) are homeostatically compensated in different types of brain neuronal networks. This should provide first insights into the role and the limitations of homeostatic compensation for inherited ion channel dysfunction, which can be used as a novel framework to understanding the abnormal behaviour of neuronal circuits in paroxysmal neurological disorders. In a long term our results may identify new target mechanisms to prevent or mitigate the clinical manifestations of intermittent disturbances of synaptic transmission.

Technical Summary

Although compensatory homeostatic synaptic mechanisms caused by changes in the activity of neuronal networks are abundantly documented in experimental studies, they have been largely overlooked in studies of episodic neurological disorders (such as migraine, epilepsy, and ataxia) which are all thought to be due to de-stabilised neuronal network function triggered by disease precipitating stimuli.
The main goal of this project is to investigate for the first time the role of activity-dependent homeostatic compensatory mechanisms for inherited ion channel dysfunction using two mouse knock-in models of Familial Hemiplegic Migraine Type 1 (FHM1). FHM1 manifests as severe migraine with aura and is caused by dominant missense mutations in the CACNA1A gene, which encodes the pore-forming Cav2.1 subunit of P/Q-type calcium channels, a major trigger of action potential (AP)-evoked neurotransmitter release in the brain. Using a combination of FM-dye and Ca2+ fluorescence imaging, presynaptic AP recordings, whole-cell patch-clamp recordings, and optogenetic photostimulation we will test how chronic manipulations of network activity affect the major presynaptic parameters in different types of small neocortical synapses expressing S218L and R192Q Cav2.1 mutant channels. Specifically we will address the following questions:
Is homeostatic compensation of AP-evoked release in S218L and R192Q neurons mediated by (i) changes in vesicular release probability at the active zone or (ii) changes in the number of release-ready vesicles?
How do S218L and R192Q affect presynaptic AP waveform?
How do S218L and R192Q mutations affect excitatory transmission in different types of neuronal connections in ex vivo acute cortical slices?
This should provide the first insights into the role and the limitations of homeostatic compensation for FHM1, which can be used as a novel framework to understanding the abnormal neuronal circuit behaviour in paroxysmal neurological disorders.

Planned Impact

(1) Potential clinical and patient impact.
In addition to the academic beneficiaries we anticipate that this project has the potential to have a significant impact on the development of new treatments for episodic neurological disorders such as migraine, ataxia, and epilepsy.
Ion channels underlie neuronal excitability and all brain activity. Although they are implicated in the manifestations of almost all neurological and neuropsychiatric diseases, the most direct evidence for their pathophysiological importance comes from genetic mutations that disrupt their normal function. The channelopathies are known to cause a range of neurological disorders including migraine, cerebellar ataxia and epilepsy. This project seeks to understand the mechanisms of an important channelopathy that affects presynaptic signalling. Familial Hemiplegic Migraine Type 1 (FHM1) is caused by mutations of the pore-forming subunit of the calcium channel Cav2.1, which contributes to triggering neurotransmitter release. Little is known of the synaptic consequence of these mutations for neurotransmitter release and action potential propagation in the axon, let alone how neuronal and circuit excitability are altered, so the disease mechanisms of FHM1 remain unresolved even though the molecular lesion is known.
Thus the detailed investigation of the effects of FHM1 mutations on transmitter release and homeostatic plasticity in mouse FHM1 models should provide unique insights into pathophysiological mechanisms of this disease. The short-term beneficiaries from this project will be sufferers of FHM1, because the fundamental understanding of presynaptic pathology may pave the way for directed treatments.
The information that channelopathies can provide on synaptic signalling and the function of neuronal circuits is also relevant to common forms of paroxysmal neurological disorders, most notably epilepsy and migraine. Epilepsy has a prevalence of 4-10 in 1000 people and accounts for 20% of all neurological consultations, and it is increasingly believed that idiopathic forms of this group of diseases are caused in large part by polygenic ion channel disturbances. Migraine affects 10% of the population, and represents a major burden to society and to the individual, and understanding the role of synaptic homeostatic compensation in FHM1 may provide clues to potential drug therapy for idiopathic forms of the disease.

(2) Potential industry impact.
We anticipate that during the project we will continue to collaborate with leading manufacturers of scientific equipment in order to translate new methods developed by our group to production of new scientific instruments. For example we have already contributed (i) to the development of a new field stimulation perfusion chamber for imaging in neuronal cultures which is now being used in more than 50 labs (RC-49MFS chamber, Warner Instruments, USA), and (ii) to the development of a new software package, which allows on-line enhancement of images during visualised patch-clamp experiments (PatchVision, Scientifica, UK).

(3) Skills for staff on project.
The staff will gain advanced skills in fluorescence microscopy, patch-clamp electrophysiology and scanning ion conductance microscopy. Because skilled electrophysiologists are in high demand (this is evident both when we try to recruit, and when students graduate from the group), we anticipate that the skills honed on this project will make the involved researchers especially competitive for future positions.

Publications

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Begum R (2016) Action potential broadening in a presynaptic channelopathy. in Nature communications

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Bello OD (2018) Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis. in Proceedings of the National Academy of Sciences of the United States of America

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Chamberland S (2018) Action potential counting at giant mossy fiber terminals gates information transfer in the hippocampus. in Proceedings of the National Academy of Sciences of the United States of America

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Vivekananda U (2017) Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization. in Proceedings of the National Academy of Sciences of the United States of America

 
Description BBSRC LIDO Industrial Case PhD studentship for a project "Molecular mechanisms of Synaptotagmin 1 mediated synaptic vesicle fusion"
Amount £106,212 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description Strategic Award in Biomedical Science, Synaptopathies: genetics, biophysics and circuit mechanisms of paroxysmal neurological disorders
Amount £4,200,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2015 
End 04/2020
 
Description Application of scanning ion conductance microscopy (SICM) to study small central synapses 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have participated in SICM patch-clamp experiments to study distribution of voltage gated calcium channels in small hippocampal synapses in culture.
Collaborator Contribution This collaboration allowed us to obtain data on sub-synaptic distribution of voltage gated calcium channels. This complements our data on presynaptic calcium imaging in small central synapses. Currently we are investigating using SCIM modulation of presynaptic action potential waveforms in mouse models of migraine and ataxia.
Impact Publications: Novak,P., Gorelik,J., Vivekananda,U., Shevchuk,A.I., Ermolyuk,Y.S., Bailey,R.J., Bushby,A.J., Moss,G.W., Rusakov,D.A., Klenerman,D., Kullmann,D.M., Volynski,K.E., and Korchev,Y.E. (2013). Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels. Neuron 79, 1067-1077. Vivekananda,U., Novak,P., Bello,O.D., Korchev,Y.E., Krishnakumar,S.S., Volynski,K.E., and Kullmann,D.M. (2017). Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization. Proc. Natl. Acad. Sci. U. S. A. 114, 2395-2400.
Start Year 2009
 
Description Computational modelling of presynaptic calcium dynamics and vesicular fusion 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided the data and the theoretical models for presynaptic calcium dynamics and vesicular release
Collaborator Contribution Dr. Yulia Timofeeva developed computational models of three-dimensional calcium diffusion and calcium triggered vesicle fusion.
Impact This is a multi-disciplinary collaboration including synaptic physiology on one side (K. Volynski) and computational modelling on the other (Y. Timofeeva). We have demonstrated that stochastic opening of individual Ca2+ channels contributes to both evoked and spontaneous glutamate release. Our results argue that stochastic Ca2+ channel opening can engage the same signalling cascade that underlies fast evoked neurotransmitter release, thereby challenging the common view that action potential-evoked and action potential-independent release are mediated by non-overlapping sources of Ca2+. These results have been published in our recent manuscript: Ermolyuk YS, Alder FG, Surges R, Pavlov I, Timofeeva Y, Kullmann DM, Volynski KE*. Differential triggering of spontaneous glutamate release by P/Q-, N-, and R-type Ca2+ channels. Nat. Neurosci. 16, 1754-1763 (2013). Using computational modelling we have also shown that at physiologically relevant concentrations, Ca2+ buffering by calmodulin plays a dominant role in inhibiting vesicular release and in modulating short-term synaptic plasticity. Timofeeva,Y., and Volynski,K.E. (2015). Calmodulin as a major calcium buffer shaping vesicular release and short-term synaptic plasticity: facilitation through buffer dislocation. Front Cell Neurosci. 9:239. doi: 10.3389/fncel.2015.00239. eCollection@2015., 239.
Start Year 2012
 
Description Molecular mechanisms of synaptotagmin 1 mediated fast synaptic vesicle fusion. 
Organisation Yale University
Country United States 
Sector Academic/University 
PI Contribution Our group performs functional testing in live neurons of synaptotagmin 1 mutants that have specific biochemical properties and that have been created in Prof. James Rothman lab. In particular, we test how each given mutation affects vesicular release using electrophysiology and fluorescence microscopy.
Collaborator Contribution Prof. James Rothman lab designs and tests synaptotagmin 1 mutations with specific molecular properties using biochemical and structural methods.
Impact This is a multidisciplinary collaboration, which includes biochemistry, structural biology, electrophysiology, and fluorescence imaging. Until now, we have established a set of methods for screening of synaptotagmin 1 mutations that affect its oligomerisation. We anticipate that this should allow us to obtain novel important insights into the basic mechanisms of fast neurotransmitter release in brain synapses.
Start Year 2015