VAMP2 associated SNAREopathies: from mechanism to therapeutic approaches

Recent years have seen the discovery of genetic mutations underlying many neurodevelopmental diseases, which, although individually rare, collectively account for a substantial burden to individuals, their families and society. How these mutations result in learning disability, autism, speech problems, and epilepsy is gradually emerging, prompting the search for new treatments to mitigate the symptoms. However, there is an important group of disorders for which the consequences of the mutations at the microscopic level remain very poorly understood: these disorders are caused by mutations that affect how neurons signal to other neurons or muscle cells. Such signalling takes place at synapses and is fundamental for all brain functions, as well as the control of movements and the function of many other organs. At the core of synaptic communication is the precise discharge of packets of chemical messengers (neurotransmitters). We now have a highly detailed understanding of how the so-called SNARE proteins assemble to bring these packets, known as synaptic vesicles, to the membrane and then discharge the neurotransmitter into the synaptic cleft upon arrival of an electrical signal. We refer to the neurodevelopmental disorders caused by defects of SNAREs and functionally associated proteins collectively as 'SNAREopathies'.
The goal of our proposal is to understand how these defects alter the discharge of neurotransmitters from synaptic vesicles and the consequences for brain circuit function and also investigate whether these effects could potentially be reversed by harnessing powerful new tools for genetic therapy.
An especially difficult challenge for many SNAREopathies is that they are suspected of acting in a genetically dominant manner. That is, if only one of the two copies of the gene encoding a SNARE protein carries a mutation, it may suppress the function of the entire SNARE protein complex, including the normal, unaffected copy of the gene. We have chosen to focus exclusively on mutations of a gene that encodes the essential SNARE protein known as vesicle-associated membrane protein 2, or VAMP2.
Our proposal brings together complementary expertise. We are able to characterise the molecular machinery of SNARE proteins by stripping neurotransmitter vesicle trafficking down to the essential steps of assembly of VAMP2 with its partners and the detailed rearrangements that occur upon the arrival of a signal to discharge the vesicle contents. We will combine this approach with highly sensitive measurements of how precisely neurotransmitter is released at individual synapses and how this process is affected by different VAMP2 mutations. We will also address the consequences for the function of small neuronal circuits in a mouse model carrying a human mutation. Finally, guided by a quantitative understanding of how many copies of each of the key molecules interact to support normal vesicle trafficking, we will design a therapy aimed at rescuing the effects of the mutation, and ask which aspects of the disease are potentially amenable to rescue in the mouse model: if the therapy is delivered once symptoms have manifested, can we only suppress seizures, or can we also improve learning and other defects which might have arisen during early development before symptom onset?
The proposed research will provide much-needed clarity on the mechanisms of VAMP2 mutations, build towards a viable treatment option which can be translated to the clinic in due course, and define a research pipeline that can be applied to other SNAREopathies.

De novo mutations of presynaptic SNARE proteins (VAMP2, Syntaxin, SNAP25) and associated chaperone and calcium-sensing proteins are increasingly recognised as an important cause of neurodevelopmental disorders characterised by learning disability, motor abnormalities, autistic features and epilepsy. The mechanisms of such 'SNAREopathies' are very poorly understood, explained in large part by the relative inaccessibility of the presynaptic compartment of neuronal networks. We propose to address this knowledge gap by combining our complementary expertise in: (i) SNARE protein biochemistry, including reconstituted single-vesicle fusion assays, (ii) electrophysiological and fluorescence microscopy applied to glutamate release from individual synaptic boutons in neuronal cultures, and (iii) behavioural and electrophysiological investigations in a conditional knock-in mouse model. We will focus exclusively on mutations of the vesicular protein VAMP2, including those that appear to exert a dominant negative disease mechanism, presenting an additional challenge to design advanced therapies. Nevertheless, based on evidence that the number of VAMP2 molecules in individual vesicles is tightly regulated and that VAMP2 overexpression is well tolerated, we hypothesise that AAV-mediated overexpression is a viable therapeutic strategy. We will test this prediction in a conditional VAMP2 knock-in mouse disease model and ask which aspects of the phenotype are amenable to rescue if the therapy is delivered after symptom onset. The work will test the feasibility of a systematic mechanistic investigation of the entire spectrum of SNAREopathies, and provide proof of principle that they can be mitigated by genetic therapy.