Academic journal article Genetics

Disruption of Endocytosis with the Dynamin Mutant Shibirets1 Suppresses Seizures in Drosophila

Academic journal article Genetics

Disruption of Endocytosis with the Dynamin Mutant Shibirets1 Suppresses Seizures in Drosophila

Article excerpt

ONE of the complexities in understanding and treating epilepsy disorders is that even though understanding of themolecular lesions underlying the conditions has improved, 20-30% patients display resistance to currently available antiepileptic drugs (AEDs) (Schuele and Lüders 2008). Many AEDs aim to decrease excitability by globally restoring ionic balances via binding to Na+, K+, or Ca2+ channels (Meldrum 1996). However, this global approach may not be sufficient to prevent the action potentials and seizures caused by all epilepsy mutations given that a mutation may affect a single population of neurons differently than themutation in the context of the interconnected neural circuits. For example, haploinsufficiency of the sodium channel Na(V)1.1 in mice actually caused hyperexcitability and spontaneous seizures as a result of preferential loss of sodium currents in inhibitory interneurons compared to the sodium currents in pyramidal cells (Yu et al. 2006). In humans, approximately 70% of the cases of Dravet syndrome (DS) or severe myoclonic epilepsy of infancy (SMEI) are caused by SCN1A mutations, a large portion of them resulting in truncations that likely would eliminate the function of the channel, canceling out the effects of any Na+ channel-targeted drugs (Marini et al. 2011).

Seizures are thought to initiate within a localized area and then spread to downstream neurons within the circuit, involving a large number of chemical synapses. For action potentials associatedwith the seizure to cross a chemical synapse, docked synaptic vesicles must fuse with the plasma membrane and release neurotransmitter into the synaptic cleftvia exocytosis. Equally important for synaptic transmission is endocytosis, in which the plasma membrane is recycled to form new synaptic vesicles (Saheki and De Camilli 2012). Given the limited number of vesicles that can fit within the active zone of a synaptic terminal, replenishment of the vesicles locally by endocytosis is essential for sustained or high-frequency synaptic transmission. Because synaptic vesicles are critical for the generation and spread of seizures throughout the nervous system, regardless of the nature of the epilepsy mutation, we hypothesized that synaptic transmission mutations that interact or disrupt synaptic vesicles could be candidates to suppress seizures in Drosophila models of seizure disorders and epilepsy (Parker et al. 2011a).

Behavioral screens in Drosophila have found genes essential for synaptic transmission by isolating temperature-sensitive mutations, which often cause paralysis when shifted to a restrictive temperature because of their important roles in neural transmission (Grigliatti et al. 1973; Siddiqi and Benzer 1976). One such mutant, shibire, encodes dynamin, a GTPase required late in the process of endocytosis and synaptic vesicle recycling that is responsible for fission of the vesicle from the membrane (van der Bliek and Meyerowrtz 1991). The shibire mutant shits1 is a temperature-sensitive missense mutation in the GTPase domain of the protein, andwhen flies are heated to the restrictive temperature (29) and stimulated, nerve terminals become completely depleted of synaptic vesicles, paralyzing the flies (Koenig et al. 1983; van der Bliek and Meyerowrtz 1991). Ultrastructural analysis of the depleted nerve terminals shows the presynaptic membrane filled with clathrin-coated pits corresponding to vesicles stuck at the late stage of endocytosis, waiting to be pinched offby dynamin (Koenig and Ikeda 1989). When flies are returned to permissive temperatures, dynamin resumes normal function, and vesicular recycling resumes, refilling the depleted releasable pool of vesicles and allowing the shits1 mutation to act as a conditional block of endocytosis. In addition to impairments with vesicle endocytosis, ionic currents recorded from the dorsal longitudinal muscle (DLM) neuromuscular synapse in shits1 flies showed rapid synaptic fatigue within 20 msec to highfrequency stimulation (HFS), suggesting an additional role for dynamin in maintenance of the releasable pool of vesicles because the time to recycle a newly formed vesicle is between 15 and 30 sec (Kawasaki et al. …

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