Laurie Daniell

Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.

Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice.

The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.

PIP Kinase Iγ Is the Major PI(4,5)P2 Synthesizing Enzyme at the Synapse

Abstract Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P 2 in neurons. We report that phosphatidylinositol phosphate kinase type Iγ (PIPKIγ), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIγ antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIγ undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIγ in the synthesis of a PI(4,5)P 2 pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.

Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C

rab3A, a low molecular weight GTP-binding protein of synaptic vesicles with a putative function in synaptic vesicle docking, interacts in a GTP-dependent manner with rabphilin-3A, a peripheral membrane protein that binds Ca2+ and phospholipids. We now show that rabphilin-3A is an evolutionarily conserved synaptic vesicle protein that is attached to synaptic vesicle membranes via its N terminus and exhibits a heterogeneous distribution among synapses. In rab3A-deficient mice, rabphilin-3A is decreased in synapses belonging to neurons that primarily express rab3A and accumulates in the perikarya of these neurons. In contrast, neurons expressing significant levels of rab3C still contain normal levels of rabphilin-3A in a synaptic pattern, and rabphilin-3A binds rab3C in vitro. These results suggest that analogous to the membrane recruitment of raf by ras, rab3A and rab3C may function in recruiting rabphilin-3A to the synaptic vesicle membrane in a GTP-dependent manner.

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

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. Exo/endocytosis 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.

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein–protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

A putative role for intramolecular regulatory mechanisms in the adaptor function of amphiphysin in endocytosis.

Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.