Huntingtin‐associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells

Abstract

Key pointsr Huntingtin-associated protein 1 (HAP1) is expressed in neurons and endocrine cells, in which it is thought to regulate vesicle trafficking.r HAP1 is a binding partner of the Huntington's disease (HD)-causing protein huntingtin, and binding is stronger in HD. r These results establish HAP1 as a significant player in exocytosis control with potential relevance for HD and for a number of neuronal and homeostatic pathways.Abstract Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1 −/− and HAP1 +/+ littermate mice. Numbers of Ca 2+ -dependent and Ca 2+ -independent full fusion events in HAP1 −/− cells are significantly decreased compared with those in HAP1 +/+ cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca 2+ entry. Whereas release per full fusion event is unchanged in HAP1 −/− cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1 −/− cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation.

Depressed excitability and ion currents linked to slow exocytotic fusion pore in chromaffin cells of the SOD1G93A mouse model of amyotrophic lateral sclerosis

“…The drastic different morphology of both spikes suggests they can serve as markers for ALS and AD. A recent study also indicates an alteration of preexocytotic and exocytotic steps in chromaffin cells of a mouse model of HD; this study concludes that the HAP1 regulates exocytosis via two potentially interlinked mechanisms: control of vesicle docking and the size of the RRP, and the regulation of fusion pore stabilization (41).…”

“…Sympathetic neuronal-like chromaffin cells are increasingly being used to explore these questions. For instance, altered exocytosis in chromaffin cells has been shown in the APP/PS1 mouse model of Alzheimer's disease (AD) (14); in the knockout mouse model of the huntingtin-associated protein 1 (HAP1), a model of Huntington's disease (HD) (41); and in mice carrying specific mutations in proteins of the exocytotic machine (63,67). We have studied here the cell excitability, ion currents, Ca 2ϩ signals, and quantal release of catecholamine in chromaffin cells from wild-type and mSOD1 mice.…”

Loss of Huntingtin stimulates capture of retrograde dense-core vesicles to increase synaptic neuropeptide stores

“…Recent studies demonstrate involvement of Htt adaptor protein HAP1 in insulin release from pancreatic beta cells (Pan et al, 2016). HAP1 is also important in several steps of exocytosis in adrenal chromaffin cells (Mackenzie et al, 2014). Therefore, to test the role of Htt on DCV-mediated release, we examined neuropeptide secretion by synaptic boutons in response to 70 Hz stimulation.…”

Extracellular and intracellular sphingosine‐1‐phosphate distinctly regulates exocytosis in chromaffin cells

“…; Mackenzie et al . ). Briefly, the adrenal glands were dissected out of adult Sphk1 KO (IMSR Cat# JAX:019095, ), kindly provided by Dr. Richard Proia of the NIH (Bethesda, MD, USA) (Allende et al .…”