Mutations in Munc18-1 (also called STXBP1) lead to devastating infantile epileptic encephalopathies with profound intellectual disability, intractable seizures, ataxia, and numerous other symptoms. Munc18-1 controls neurotransmitter release at the synapse, but recent evidence demonstrates that a non-synaptic Munc18-1- dependent process, a role in general secretory trafficking, is also perturbed with Munc18-1 loss. Yet, the causative molecular mechanism for the mistrafficking seen in Munc18-1-/- neurons remains unclear. Preliminary data reveal that mutations in Munc18-1 cause reduced levels of the Golgi SNARE protein syntaxin-5 (stx5) and stx5-containing SNARE complexes, as well as an abnormal Golgi morphology. The central hypothesis of this proposal, based on strong preliminary data, is Munc18-1 mutations cause syntaxin-1 mislocalization and induce loss of function of stx5, leading to trafficking defects in the Golgi and subsequent non-synaptic neuronal dysfunction. This impairment in secretory trafficking may partly trigger the developmental dysfunction seen in Munc18-1 related syndromes. The objective of this proposal to demonstrate how loss of stx5 leads to neuronal, non-synaptic dysfunction. The rationale for these studies is that revealing the role of stx5 in maintaining neuronal function will have translational importance in the development of rational treatments for Munc18-1 linked syndromes. Guided by strong preliminary data, this hypothesis will be tested in two specific aims:
Aim 1) Determine how mutant Munc18-1 causes an abnormal Golgi phenotype, and Aim 2) Determine how the stx5 reduction seen in Munc18-1-/- and mutant Munc18-1 neurons affects intracellular protein trafficking and neuron activity. In the first aim, the nature and cause of this abnormal Golgi morphology will be determined using primary neurons. First, changes to resident Golgi, ER, and cytoskeletal proteins will be characterized in Munc18-1-/- and mutant Munc18-1 neurons. Additionally, the effect of stx5 reduction on the Golgi phenotype will be determined, as well as the effect of syntaxin-1 mislocalization. In the second aim, changes to secretory cargo mistrafficking due to reduction in stx5 will be determined. Furthermore, the effect of stx5 reduction on neuronal function and neurite and synapse morphology will be examined. This research is significant, because it will determine the mechanisms and importance of disturbed secretory trafficking in neurons and the role of stx5 in particular, and will have translational importance in the development of new treatment strategies. This research is innovative, because of (1) its novel hypothesis that non-synaptic dysfunction of Munc18-1 contributes to these syndromes, and (2) its multidisciplinary approach combining biochemical and cell biological approaches to gain insight into how mutations in Munc18-1 lead to non-synaptic dysfunction.
Despite a known genetic cause, no effective treatments exist for Munc18-1 related disorders, which cause intractable epilepsy, severe intellectual disability, and other severe neurologic symptoms in children. The aim of this proposal is to investigate the non-synaptic neuronal deficits that are caused by these genetic mutations. An understanding of these deficits will be essential to testing future corrective strategies for these devastating diseases and others caused by similar mechanisms.
