Endogenous Rho-Kinase Signaling Maintains Synaptic Strength by Stabilizing the Size of the Readily Releasable Pool of Synaptic Vesicles

Abstract

Rho-associated kinase (ROCK) regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. There is, however, little information about whether ROCK modulates the electrical activity and information processing of neuronal circuits. At neonatal stage, ROCK␣ is expressed in hypoglossal motoneurons (HMNs) and in their afferent inputs, whereas ROCK␤ is found in synaptic terminals on HMNs, but not in their somata. Inhibition of endogenous ROCK activity in neonatal rat brainstem slices failed to modulate intrinsic excitability of HMNs, but strongly attenuated the strength of their glutamatergic and GABAergic synaptic inputs. The mechanism acts presynaptically to reduce evoked neurotransmitter release. ROCK inhibition increased myosin light chain (MLC) phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. Functional and ultrastructural changes induced by ROCK inhibition were fully prevented/reverted by MLC kinase (MLCK) inhibition. Furthermore, ROCK inhibition drastically reduced the phosphorylated form of p21-associated kinase (PAK), which directly inhibits MLCK. We conclude that endogenous ROCK activity is necessary for the normal performance of motor output commands, because it maintains afferent synaptic strength, by stabilizing the size of the readily releasable pool of synaptic vesicles. The mechanism of action involves a tonic inhibition of MLCK, presumably through PAK phosphorylation. This mechanism might be present in adults since unilateral microinjection of ROCK or MLCK inhibitors into the hypoglossal nucleus reduced or increased, respectively, whole XIIth nerve activity.

Myosin Light Chain Kinase Accelerates Vesicle Endocytosis at the Calyx of Held Synapse

“…Since the global deletion of MLCK, myosin IIA, or myosin IIB is lethal at birth, future studies using cultured embryonic neurons and conditional knock-out animals will be necessary to study the role of MLCK/myosin in endocytosis on the basis of specifically controlled expression of MLCK and myosin. Indeed, a new study reports that knock-out of myosin IIB decreases activitydependent uptake of FM1-43 and horseradish peroxidase in cultured hippocampal neurons (Chandrasekar et al, 2013), suggesting that MLCK/myosin positively modulates endocytosis across different synapses.…”

“…MLCK is typically activated by Ca 2ϩ /calmodulin and can phosphorylate the regulatory light chain of myosin, which triggers interaction between myosin and actin. Although MLCK may also be directly or indirectly activated by other molecules such as the neural cell adhesion molecule (Polo-Parada et al, 2005), protein kinase C (Maeno-Hikichi et al, 2011), Rho/Rho-kinase (Garcia et al, 1999), and p21-activated kinases (Sanders et al, 1999), its modulation of synaptic endocytosis most likely involves Ca 2ϩ / calmodulin that has been indicated to regulate endocytosis in neurons Sun et al, 2010;Yamashita et al, 2010;Yao and Sakaba, 2012) and neuroendocrine cells (Artalejo et al, 1996). Calmodulin inhibition at the calyx impairs distinct ) evoked by depol 20ms in a control calyx.…”

“…Blebbistatin and deletion of myosin IIB also inhibit endocytosis in hippocampal synapses (Chandrasekar et al, 2013). Although a role of actin in endocytosis is not yet established at mammalian central synapses (Sankaranarayanan et al, 2003), it has been demonstrated in other synapses and nonneuronal cells (Brodin et al, 2000;Merrifield et al, 2005;Girao et al, 2008;Mooren et al, 2012). In the lamprey synapses, actindepolymerizing agents can inhibit clathrin-mediated endocytosis and arrest clathrin-coated pits on the plasma membrane (Shupliakov et al, 2002;Bourne et al, 2006).…”

“…In hippocampal neurons, knockdown of calmodulin expression decreases endocytosis rate (Sun et al, 2010). These observations further highlight calmodulin as a versatile Ca 2ϩ sensor to regulate distinct synaptic functions including endocytosis, ion channel modulation (Xu and Wu, 2005;Dick et al, 2008;Catterall et al, 2013), vesicle replenishment , and synaptic plasticity (Lee et al, 2010). One downstream target of Ca 2ϩ /calmodulin is myosin light chain kinase (MLCK), which can phosphorylate the regulatory light chain of myosin (Nairn and Picciotto, 1994).…”

The less things change, the more they are different: contributions of long-term synaptic plasticity and homeostasis to memory

“…The levels of spontaneous release of conventional neurotransmitters (glutamate) or other factors (neurotrophins or cytokines) expressed at each synapse as a consequence of the activity-induced plasticity might maintain appropriate levels of local synthesis required for local forms of L-LTP and L-LTD (Sutton et al 2004(Sutton et al , 2006. The molecules affecting synaptic strength at any moment in time could represent presynaptic proteins and signaling pathways critical for transmitter release (Gonzalez-Forero et al 2012), postsynaptic transmitter receptors, and proteins participating in their cycling into and out of the plasma membrane (Malinow and Malenka 2002;Fortin et al 2012), neurotrophins and other secreted factors and their receptors (English et al 2012;Hruska and Dalva 2012), factors or regulators of protein translation and degradation (Cajigas et al 2010;Kandel 2012), and kinases that are constitutively active (Chain et al 1999;Hernandez et al 2003). These local molecules and their downstream signaling coupled with proteins and mRNA transported from the cell body would maintain the new level of synaptic transmission that encodes the behavioral modification or memory.…”