Palmitoyl protein thioesterase-1 deficiency impairs synaptic vesicle recycling at nerve terminals, contributing to neuropathology in humans and mice

Abstract

Neuronal ceroid lipofuscinoses represent the most common childhood neurodegenerative storage disorders. Infantile neuronal ceroid lipofuscinosis (INCL) is caused by palmitoyl protein thioesterase-1 (PPT1) deficiency. Although INCL patients show signs of abnormal neurotransmission, manifested by myoclonus and seizures, the molecular mechanisms by which PPT1 deficiency causes this abnormality remain obscure. Neurotransmission relies on repeated cycles of exo-and endocytosis of the synaptic vesicles (SVs), in which several palmitoylated proteins play critical roles. These proteins facilitate membrane fusion, which is required for neurotransmitter exocytosis, recycling of the fused SV membrane components, and regeneration of fresh vesicles. However, palmitoylated proteins require depalmitoylation for recycling. Using postmortem brain tissues from an INCL patient and tissue from the PPT1-knockout (PPT1-KO) mice that mimic INCL, we report here that PPT1 deficiency caused persistent membrane anchorage of the palmitoylated SV proteins, which hindered the recycling of the vesicle components that normally fuse with the presynaptic plasma membrane during SV exocytosis. Thus, the regeneration of fresh SVs, essential for maintaining the SV pool size at the synapses, was impaired, leading to a progressive loss of readily releasable SVs and abnormal neurotransmission. This abnormality may contribute to INCL neuropathology.

Mutations in palmitoyl-protein thioesterase 1 alter exocytosis and endocytosis at synapses in Drosophila larvae

“…34 These results are also consistent with previous studies identifying potential presynaptic substrates of Ppt1 and its possible localization, which do not point to an interaction of Ppt1 with receptors or other post-synaptic targets. 26,41 Instead, most of the potential neuronal substrates, such as dynamin, SNAP25, CSP, VAMP2, syntaxin 1, SYTI, and GAD65, are pre-synaptic and control synaptic vesicle cycling. It is curious that the point mutation in the Ppt1 active site (Ppt1 A179T ) failed to modify mini EJP frequency.…”

“…Though we have not yet examined synaptic vesicle numbers in Drosophila Ppt1-mutant nerve terminals, several groups have shown that loss of PPT1 in mouse cortical neurons in vitro results in a decline in the size of the readily releasable synaptic vesicle pool as well as the total vesicle pool. 25,26 Such changes in vesicle number are likely due to reductions in endocytosis, perhaps due to alteration in the function of dynamin and other palmitoylated presynaptic proteins. Indeed, consistent with in vitro mouse results, we have observed a significantly reduced ability of Ppt1 loss-of-function nerve terminals to recover from repetitive stimulation, as well as reductions in in vivo endocytosis measured by FM1-43 uptake (Fig.…”

“…25,26 The biochemical basis for these phenotypes is less clear. Although we currently lack optimal reagents, such as an effective Drosophila anti-Ppt1 antibody that will allow us to confirm a presynaptic localization of Ppt1, we can speculate that a small pool of endogenous Ppt1 is likely to be at the synapse in the fly as has been observed by one group in mouse tissue.…”

“…24 In neural tissue, changes in endocytosis have been reported in cultured neurons of Ppt1-deficient mice and humans in the context of a reduction in the synaptic vesicle pool size, but the functional consequences of Ppt1 mutations on endocytosis remains to be characterized. 25,26 Many proteins involved in synaptic vesicle endo-and exocytosis such as dynamin, endophylin A, SNAP-25, synaptotagmin, the ) were placed in vials with 10 flies each. The flies were kept at 18 °C, heat shocked at 32 °C for 2 min, then moved to 20 °C for recovery.…”

“…Kim et al 26 have observed reduced levels of many of these presynaptic proteins in the soluble fraction of postmortem brains of INCL patients, as well as in Ppt1-knockout mice, suggesting that these proteins remain anchored to the synaptic membrane in the absence of depalmitoylation. Palmitoylation also seems to play a role in the correct localization of presynaptic proteins to the appropriate intracellular or plasma membrane domain, as shown in the Drosophila mutant of palmitoyl acyl transferase, hip14.…”