Altered neurotransmitter release machinery in mice deficient for the deubiquitinating enzyme Usp14

Homozygous ataxic mice (ax(J)) express reduced levels of the deubiquitinating enzyme Usp14. They develop severe tremors by 2-3 wk of age, followed by hindlimb paralysis, and death by 6-8 wk. While changes in the ubiquitin proteasome system often result in the accumulation of ubiquitin protein aggregates and neuronal loss, these pathological markers are not observed in the ax(J) mice. Instead, defects in neurotransmission were observed in both the central and peripheral nervous systems of ax(J) mice. We have now identified several new alterations in peripheral neurotransmission in the ax(J) mice. Using the two-microelectrode voltage clamp technique on diaphragm muscles of ax(J) mice, we observed that under normal neurotransmitter release conditions ax(J) mice lacked paired-pulse facilitation and exhibited a frequency-dependent increase in rundown of the end plate current at high-frequency stimulation (HFS). Combined electrophysiology and styryl dye staining revealed a significant reduction in quantal content during the initial and plateau portions of the HFS train. In addition, uptake of styryl dyes (FM dye) during HFS demonstrated that the size of the readily releasable vesicle pool was significantly reduced. Destaining rates for styryl dyes suggested that ax(J) neuromuscular junctions are unable to mobilize a sufficient number of vesicles during times of intense activity. These results imply that ax(J) nerve terminals are unable to recruit a sufficient number of vesicles to keep pace with physiological rates of transmitter release. Therefore, ubiquitination of synaptic proteins appears to play an important role in the normal operation of the neurotransmitter release machinery and in regulating the size of pools of synaptic vesicles.