Abstract
Heterozygous de novo mutations in the neuronal protein Munc18-1 are linked to epilepsies, intellectual disability, movement disorders, and neurodegeneration. These devastating diseases have a poor prognosis and no known cure, due to lack of understanding of the underlying disease mechanism. To determine how mutations in Munc18-1 cause disease, we use newly generated S. cerevisiae strains, C. elegans models, and conditional Munc18-1 knockout mouse neurons expressing wild-type or mutant Munc18-1, as well as in vitro studies. We find that at least five disease-linked missense mutations of Munc18-1 result in destabilization and aggregation of the mutant protein. Aggregates of mutant Munc18-1 incorporate wild-type Munc18-1, depleting functional Munc18-1 levels beyond hemizygous levels. We demonstrate that the three chemical chaperones 4-phenylbutyrate, sorbitol, and trehalose reverse the deficits caused by mutations in Munc18-1 in vitro and in vivo in multiple models, offering a novel strategy for the treatment of varied encephalopathies.
Highlights
Heterozygous de novo mutations in the neuronal protein Munc[] are linked to epilepsies, intellectual disability, movement disorders, and neurodegeneration
When we analyzed the distribution of disease-linked missense mutations in Munc[] in its primary and secondary sequence, we found no specific area or domain of Munc[] to be affected (Supplementary Fig. 1), suggesting loss of function of Munc[] mutants as the underlying disease mechanism
We chose these five mutants because: (1) multiple mutations at these residues are associated with disease (Supplementary Fig. S1 and ref. 10), (2) diseaselinked residues are conserved in the C. elegans homolog UNC-18 (Supplementary Fig. 2a), permitting the study of motor phenotypes in our newly generated C. elegans disease models in vivo, (3) homologous residues are mutated in Munc[] in the immune disease familial hemophagocytic lymphohistiocytosis type 530, enabling extension of our findings to another organ system and disease, (4) residue P335 has been proposed to function as a flexible hinge point in Munc[], modulating the binding of Munc[] to syntaxin-1, VAMP2, and the SNARE complex, and regulating neurotransmitter release[31,32,33], and (5) we maintained expression of full-length protein while replicating disease-linked mutations to enable antibody-based detection
Summary
Heterozygous de novo mutations in the neuronal protein Munc[] are linked to epilepsies, intellectual disability, movement disorders, and neurodegeneration. Heterozygous mice are viable and display normal synaptic vesicle fusion, but reveal a reduction in the readily releasable pool of synaptic vesicles[25] Together, these data define a critical regulatory function of Munc[] in neurotransmitter release, in particular in determining the number of readily releasable vesicles, and raise the possibility that Munc[] mutations in humans cause severe disease by a loss-of-function mechanism, i.e., haploinsufficiency, but by asserting an additional dominantnegative effect on the wild-type allele. Recent studies have suggested that mutations in Munc[] could result in a thermo-labile protein[28], and temperature-sensitive structural changes associated with the C180Y mutation have been reported for a GFP-tagged C180Y variant in PC12 cells[29] It remains unclear how mutations in Munc[] cause varied autosomaldominant disorders, and a systematic and detailed understanding of their etiology is needed in order to develop effective strategies to counteract their deleterious effects. We demonstrate that the three chemical chaperones 4-phenylbutyrate, sorbitol, and trehalose are able to stabilize Munc[] protein levels, reversing the insolubility and aggregation of mutant Munc[], and to rescue neuronal deficits in vitro and in vivo, providing a novel therapeutic approach for Munc18-1-associated encephalopathies
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