Impaired synaptic vesicle reformation in synaptojanin 1 knockout mice: Insights into the role of phosphoinositides in synaptic vesicle recycling

Neurotransmission depends upon the efficient and precise recycling of the synaptic vesicle pool in the presynaptic nerve terminal. Several indirect lines of evidence suggest that phosphoinositides play a role in this process. Synaptojanin 1, a presynaptic polyphosphoinositide phosphatase is implicated in synaptic vesicle recycling as a major regulator of phosphoinositides. To directly address how the regulation of phosphoinositides underlies critical events in the synaptic vesicle cycle, a knockout mouse was generated that lacked the expression of synaptojanin 1. Mice deficient in synaptojanin 1 exhibit 100% perinatal mortality, failure to thrive, and neurologic defects. Biochemical analysis of knockout brain cytosol reveal a decrease in phosphoinositide phosphatase activity and a correlated increase in PI(4,5)P₂ and PI(3,4,5)P₃ levels. Extraction of lipids from primary cultured neurons reveal a 1.6-fold steady-state increase in PI(4,5)P₂ levels. These biochemical changes correlate morphologically with an increase in clathrin-coated membranes in both in vitro and in vivo systems. Electrophysiologic studies demonstrate an increase in synaptic depression and a delay in recovery, changes in synaptic function consistent with defects in vesicle recycling. Measurements of FM1-43 turnover in electrically stimulated knockout neurons demonstrate kinetic changes in specific steps of the synaptic vesicle cycle. Overall synaptic vesicle turnover is intact, and exo/endocytosis after a short stimulus is unchanged. However, the total size of the recycling synaptic vesicle pool is ∼40% smaller in knockout neurons. Moreover, during prolonged stimulation, the regeneration of fusion-competent synaptic vesicles is severely impaired. Pulse-chase electron microscopy using electron-dense tracers correlates these functional changes with a persistent increase in clathrin-coated vesicles in stimulated knockout synapses that is only transient in control synapses. Furthermore, there is a backup of newly reformed vesicles and endosome-like cisternae in the cytomatrix-rich area around the synaptic vesicle cluster. Taken together, these findings in a mammalian genetic system provide direct evidence that synaptojanin 1 function is important for the regeneration of the functional synaptic vesicle pool. They provide support for a model in which the regulation of phosphoinositides plays a key role in interactions between membranes, coat proteins, and the actin cytoskeleton that are critical for synaptic vesicle recycling.