Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice

Abstract

Synaptojanin 1 is a polyphosphoinositide phosphatase implicated in synaptic vesicle recycling. We used FM1-43 imaging and electron microscopy in cultured cortical neurons from control and synaptojanin 1 knockout mice to study how the absence of this protein affects specific steps of the synaptic vesicle cycle. Exo͞endocytosis after a moderate stimulus was unchanged. However, during prolonged stimulation, the regeneration of fusion-competent synaptic vesicles was severely impaired. In stimulated nerve terminals, there was a persistent accumulation of clathrin-coated vesicles and a backup of newly reformed vesicles in the cytomatrix-rich area around the synaptic vesicle cluster. These findings demonstrate that synaptojanin 1 function is needed for the progression of recycling vesicles to the functional synaptic vesicle pool. N eurotransmission and its reliability depend on the efficient and precise recycling of synaptic vesicles in the presynaptic nerve terminal (1, 2). Vesicle fusion and neurotransmitter release are followed by rapid retrieval and repackaging of synaptic vesicle membrane components into new functional vesicles. Morphologic, biochemical, genetic, and functional evidence points to an important role of the clathrin-mediated recycling pathway in this process (1,(3)(4)(5). Alternative and parallel pathways may also participate in recycling (1, 2, 6-9).Growing evidence suggests that phosphoinositides play an important role in the synaptic vesicle cycle via their interaction with endocytic and actin regulatory proteins (10-13). Phosphoinositides, PI(4,5)P 2 in particular, bind clathrin adaptors such as AP-2 (14-16), AP180 (17,18), and epsin (19,20), as well as the GTPase dynamin (21, 22), which is critically required for the fission of endocytic vesicles (23, 24). They also regulate a variety of proteins that control actin nucleation (13,25,26). Actin, in turn, is thought to play an important role in clathrin-mediated recycling by generating and organizing cytoskeletal scaffolds at endocytic zones (27-29) and, possibly, by contributing to the translocation of newly formed endocytic vesicles to the synaptic vesicle cluster (30-34).Synaptojanin 1 is a polyphosphoinositide phosphatase highly enriched in presynaptic nerve terminals and localized to recycling intermediates in synaptosomes (35,36). Its COOHterminal proline-rich domain interacts with several accessory factors implicated in synaptic vesicle recycling, including amphiphysin (35), endophilin (37), (38), intersectin (34, 39), and syndapin͞pacsin (40, 41). Its NH 2 -terminal Sac1-like module hydrolyzes the phosphates of PI(3)P, PI(4)P, and PI(3,5)P 2 (42), whereas its central phosphatase module hydrolyzes the 5Ј phosphate of PI(4,5)P 2 and PI(3,4,5)P 3 (43,44). By means of these enzymatic activities, synaptojanin can dephosphorylate polyphosphoinositides to phosphatidylinositol and thereby function as a negative regulator of the interactions between membranes, coat proteins, and regulatory factors for actin.In mice knockout studies, the absence of ...

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

“…The SJ1-mediated change in phosphoinositide composition of the newly formed endocytic vesicle may favor uncoating by decreasing the affinity of clathrin adaptors with the membrane while simultaneously promoting the recruitment of auxilin, a cofactor for the ''uncoating ATPase'' HSC70 (39,40). The importance of SJ1 in clathrin uncoating is strongly supported by the accumulation of clathrin-coated vesicles in nerve terminals of SJ1 knockout mice (3,28).…”

“…This biochemical link to dynamin, together with the accumulation of coated vesicles at synapses of SJ1 knockout mice, has led to the hypothesis that the main function of SJ1-mediated hydrolysis of PI(4,5)P 2 is to facilitate clathrin uncoating after fission (3,5,28).…”

Phosphoinositides in Constitutive Membrane Traffic

“…Synaptojanin 1 is found in nerve terminals associated with membranes coated with clathrin (127,227). Mice deficient in synaptojanin 1 (67,181) and mutants in Unc26, the C. elegans synaptojanin ortholog (134), have neurological defects and nerve endings that accumulate coated vesicles, suggesting that there is a defect in vesicle uncoating. Synaptojanin 2 has a broader tissue distribution and differs from synaptojanin 1 at the COOH terminus (254).…”

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

“…In yeast, Sac1p is an important regulator for maintaining the secretory function of ER by controlling the supply of ATP inside the ER lumen [27]. In synaptojanin 1 mutant mice, the clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings, which suggests that synaptojanin 1 facilitates the progression of recycling vesicles back to the synaptic vesicle pool [6,19]. Here, we show that rSac3 can affect the neurite outgrowth of NGF-stimulated PC12 cells, and that this function is dependent on the enzymatic activity of PIPPase ( Figures 5 and 6).…”

“…The C-terminal region of synaptojanin 1 may interact with the SH3 domain of different proteins [14][15][16][17][18]. Mice that are deficient in synaptojanin 1 have elevated levels of PI(4,5)P 2 and PI(3,4,5)P 3 in the brain cytosol and accumulate coated vesicles at nerve terminals, suggesting that synaptojanin 1 functions in recycling vesicles back to the synaptic vesicle pool [19]. Synaptojanin 2 is expressed as multiple alternative splice forms in various tissues [12], and may be involved in clathrin-coated pit formation and lipid hydrolysis, which are required during early clathrin-mediated endocytosis [20].…”