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Carmen C. Garcia
Pharmacology & Physiology

M.D. 1996, Universidad Central de Venezuela
M.Sc. 1999, University of Newcastle Upon Tyne

Thesis Advisor: Joseph McArdle, Ph.D.
Dept. of Pharmacology and Physiology

Wednesday, January 20, 2010
10:00 a.m., MSB H-609b


The vertebrate neuromuscular junction (NMJ) has been widely studied. The NMJ is a simple model of efficient neurotransmission. As for other synapses, the NMJ displays considerable plasticity, especially in response to pathology. Diabetes Mellitus affects central and peripheral synapses. In fact, peripheral diabetic neuropathy (DN), a frequent and debilitating complication of diabetes, is associated with functional and morphological changes of the NMJ. In order to understand the basis for these changes, we investigated NMJs of muscles from mice with experimentally-induced diabetes.
Swiss Webster mice were made diabetic with three inter-daily intraperitoneal injections of streptozotocin (STZ). Even in the presence of high glycemic levels, motor activity remained equivalent to control up to 6 weeks after the onset of hyperglycemia. Rotorod performance of diabetic mice began to decline significantly at 7 weeks. Compound muscle action potential (CMAP) amplitude varied in parallel with motor activity in our electromyography (EMG) tests. In contrast, the number of functional motor units (MUNE) significantly decreased as early as two weeks after onset of sustained hyperglycemia. The early MUNE abnormalities were accompanied by NMJ functional modifications. Specifically, there was a gradual decrement of evoked transmitter release.
Two electrode voltage clamp demonstrated that endplate current (EPC) amplitude and quantal content progressively declined. Reduction of quantal content occurred earlier in the fast twitch extensor digiturum longus (EDL) than in the slow twitch soleus muscle. Due to the increased susceptibility to hyperglycemia/hypo-insulinemia of the EDL, subsequent studies focused on this fast twitch muscle. We also identified defects in acetylcholine (ACh) release for motor nerves in the EDL muscle of db/db mice, a model of type 2 diabetes. Additional studies of the db/db model were not made.
The reduction of quantal content may result from altered soluble N-ethylmaleimide sensitive fusion protein attachment receptor (SNARE) proteins like the synaptosome-associated protein of 25000 Daltons (SNAP-25). Since SNAP-25 is the substrate of Botulinum neurotoxin A (BoNT/A), we examined the sensitivity of EDL muscles from STZ-diabetic mice to BoNT/A. Nerve evoked twitch force of diabetic muscle preparations was more sensitive to the paralytic effect of BoNT/A than control preparations. SNAP-25 is a target of reactive oxygen species (ROS). Furthermore, ROS production increases in diabetic conditions. Therefore, we measured SNAP-25 expression in cholinergic Neuro 2a (N2a) cells grown in high glucose media. After 6 days of culture in medium containing 10 or 15 mM glucose, N2a expressed lower levels of SNAP-25.
In addition to the alterations in motor nerve function, we also detected changes of the muscle endplate. Specifically, the amplitude and decay time of miniature endplate currents (mEPCs) significantly increased. These changes were postsynaptic in origin since the endplate response to iontophoretically applied ACh also increased in amplitude and decay time. The cause of these mEPC changes was attributed to a loss of endplate acetylcholinesterase (AChE) activity. RT-PCR showed down regulation of AChE transcripts. AChE activity was also impaired as demonstrated by histological and biochemical assays. Consistent with AChE deficiency, the decay time of mEPCs at NMJs of EDL muscles from diabetic mice were less sensitive than controls to prolongation by phenserine tatrate, a selective AChE inhibitor. Conversely, mEPC duration was more sensitive to prolongation by phenethylcymserine tartrate, a selective inhibitor of butyrylcholinesterase (BChE). Lack of sensitivity to the cone-snail toxin A-OIVA suggested that the appearance of slow mEPCs was not due to expression of the embryonic acetylcholine receptor.
When motor deficit became evident in the rotorod test (week 7-8), there was a significant decrease in muscle weight and a reduction of nerve evoked twitch force. After the initial increase of the amplitude of the endplate sensitivity to iontophoretically applied ACh, there was a decrease in ACh sensitivity at the 7 week time point. At 7 weeks, mEPC amplitude also declined. The decline of endplate response to endogenous or exogenous ACh was due to diabetes-induced disruption of ACh receptor (AChR) clusters, as observed with confocal microscopy. At the developing vertebrate NMJ, nerve-induced postsynaptic formation and maintenance of endplate AChR clusters is regulated by Agrin signaling via the muscle specific kinase (MuSK) and requires the intracellular scaffolding receptor-associated protein of the synapse (rapsyn). Therefore, we investigated the Agrin-MuSK signaling cascade. MuSK regulates endplate transcription of rapsyn and the AChR subunit. MuSK also regulates its own transcription. MuSK expression and function was unaltered for the EDL muscle of diabetic mice. mRNA expression of transcripts for rapsyn, subunit, and MuSK was equivalent to control at 8 weeks after the onset of STZ-induced hyperglycemia. These findings suggest that alterations of the Agrin-MuSK signaling pathway do not contribute to diabetes-induced fragmentation of AChR clusters. In contrast, protein levels of rapsyn initially decrease and then dramatically increase beginning at 7 weeks after the onset of STZ-induced diabetes. The upregulation of rapsyn may reflect a compensatory event in an attempt to maintain the integrity of AChRs.
This work clarifies the molecular mechanisms leading to NMJ dysfunction during diabetes. As diabetes progresses, the endplate apparatus deteriorates and neuromuscular transmission is significantly compromised. This NMJ pathology contributes to the characteristic muscle weakness seen in diabetes. Importantly, NMJ alteration begins prior to the detection of motor dysfunction. Thus, it is possible that early therapeutic intervention may prevent or delay the neuromuscular dysfunction during diabetic neuropathy.

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