Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity.

Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes including KCNQ3 which encodes Kv7.3. Neuronal Kv7 channels are mostly heterotetramers of Kv7.2 and Kv7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in Kv7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reduces KCNQ3 transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decrease KCNQ3 and BDNF transcripts as well as the density of Kv7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and Kv7.3 density at the AIS during homeostatic scaling of AP firing rate.

Prolonged seizure activity causes caspase dependent cleavage and dysfunction of G-protein activated inwardly rectifying potassium channels.

Recurrent high-frequency epileptic seizures cause progressive hippocampal sclerosis, which is associated with caspase-3 activation and NMDA receptor-dependent excitotoxicity. However, the identity of caspase-3 substrates that contribute to seizure-induced hippocampal atrophy remains largely unknown. Here, we show that prolonged high-frequency epileptiform discharges in cultured hippocampal neurons leads to caspase-dependent cleavage of GIRK1 and GIRK2, the major subunits of neuronal G protein-activated inwardly rectifying potassium (GIRK) channels that mediate membrane hyperpolarization and synaptic inhibition in the brain. We have identified caspase-3 cleavage sites in GIRK1 (387ECLD390) and GIRK2 (349YEVD352). The YEVD motif is highly conserved in GIRK2-4, and located within their C-terminal binding sites for Gßγ proteins that mediate membrane-delimited GIRK activation. Indeed, the cleaved GIRK2 displays reduced binding to Gßγ and cannot coassemble with GIRK1. Loss of an ER export motif upon cleavage of GIRK2 abolishes surface and current expression of GIRK2 homotetramic channels. Lastly, kainate-induced status epilepticus causes GIRK1 and GIRK2 cleavage in the hippocampus in vivo. Our findings are the first to show direct cleavage of GIRK1 and GIRK2 subunits by caspase-3, and suggest the possible role of caspase-3 mediated down-regulation of GIRK channel function and expression in hippocampal neuronal injury during prolonged epileptic seizures.