␣-Synuclein (␣-syn) missense and multiplication mutations have been suggested to cause neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies. Before causing the progressive neuronal loss, ␣-syn mutations impair exocytosis, which may contribute to eventual neurodegeneration. To understand how ␣-syn mutations impair exocytosis, we developed a mouse model that selectively expressed PD-related human ␣-syn A53T (h-␣-syn A53T ) mutation at the calyx of Held terminals, where release mechanisms can be dissected with a patch-clamping technique. With capacitance measurement of endocytosis, we reported that h-␣-syn A53T , either expressed transgenically or dialyzed in the short term in calyces, inhibited two of the most common forms of endocytosis, the slow and rapid vesicle endocytosis at mammalian central synapses. The expression of h-␣-syn A53T in calyces also inhibited vesicle replenishment to the readily releasable pool. These findings may help to understand how ␣-syn mutations impair neurotransmission before neurodegeneration.

Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from nonspecific effects after cholesterol manipulation. Furthermore, it is unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase (COase) or methyl-β-cyclodextrin (MCD) impaired three different forms of endocytosis, i.e., slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of MCD reduced exocytosis, mainly by decreasing the readily releasable pool (RRP) and the vesicle replenishment after RRP depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses.

Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca 2ϩ and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca 2ϩ /calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca 2ϩ channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca 2ϩ /calmodulin.

Neurotransmission relies on membrane endocytosis to maintain vesicle supply and membrane stability. Endocytosis has been generally recognized as a major ATP-dependent function, which efficiently retrieves more membrane at elevated neuronal activity when ATP consumption within nerve terminals increases drastically. This paradox raises the interesting question of whether increased activity recruits ATP-independent mechanism(s) to accelerate endocytosis at the same time as preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at rat calyx of Held terminals by whole-cell membrane capacitance measurements. At room temperature, blocking ATP hydrolysis effectively abolished slow endocytosis and rapid endocytosis but only partially inhibited excess endocytosis following intense stimulation. The ATP-independent endocytosis occurred at calyces from postnatal days 8-15, suggesting its existence before and after hearing onset. This endocytosis was not affected by a reduction of exocytosis using the light chain of botulinum toxin C, nor by block of clathrin-coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre-existing at the surface, and was impaired by oxidation of cholesterol and inhibition of neutral sphingomyelinase. ATP-independent endocytosis became more significant at 34-35°C, and recovered membrane by an amount that, on average, was close to exocytosis. The results of the present study suggest that activity and temperature recruit ATP-independent endocytosis of pre-existing membrane (in addition to ATP-dependent endocytosis) to efficiently retrieve membrane at nerve terminals. This less understood endocytosis represents a non-canonical mechanism regulated by lipids such as cholesterol and sphingomyelinase.