Michael W. Swank

Regulation of Dendritic Morphogenesis by Ras–PI3K–Akt–mTOR and Ras–MAPK Signaling Pathways

Dendritic arborization and spine formation are critical for the functioning of neurons. Although many proteins have been identified recently as regulators of dendritic morphogenesis, the intracellular signaling pathways that control these processes are not well understood. Here we report that the Ras–phosphatidylinositol 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling pathway plays pivotal roles in the regulation of many aspects of dendrite formation. Whereas the PI3K–Akt–mTOR pathway alone controlled soma and dendrite size, a coordinated activation together with the Ras-mitogen-activated protein kinase signaling pathway was required for increasing dendritic complexity. Chronic inhibition of PI3K or mTOR reduced soma and dendrite size and dendritic complexity, as well as density of dendritic filopodia and spines, whereas a short-term inhibition promoted the formation of mushroom-shaped spines on cells expressing constitutively active mutants of Ras, PI3K, or Akt, or treated with the upstream activator BDNF. Together, our data underscore the central role of a spatiotemporally regulated key cell survival and growth pathway on trophic regulation of the coordinated development of dendrite size and shape.

Book Review: Protein Kinase Signal Transduction Cascades in Mammalian Associative Conditioning

One of the most intriguing and intensely studied questions facing contemporary neuroscientists involves the elucidation of the physiological mechanisms that underlie learning and memory. Recent advances have given us a much more detailed understanding of the signal transduction mechanisms subserving learning in the intact animal. One fact that has become clear is that activation of protein kinases and phosphorylation of their downstream effectors play a critical role. Four protein kinase cascades have garnered considerable attention in the study of information storage at both the synaptic and behavioral levels: Ca++/phospholipid-dependent protein kinase (PKC), Ca++/calmodulin-dependent protein kinase II (CaMKII), cAMP-dependent protein kinase (PKA), and extracellular signal-regulated kinase (ERK). This review will concentrate on studies of two behavioral tasks, conditioned fear and conditioned taste aversion, that provide evidence for the involvement of these kinase systems in associative learning. The authors will also examine a number of potential kinase substrates and how each could participate in the formation of long-term memories.

Phosphorylation of ERK/MAP kinase is required for long-term potentiation in anatomically restricted regions of the lateral amygdala in vivo

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) is transiently activated in anatomically restricted regions of the lateral amygdala (LA) following Pavlovian fear conditioning and that blockade of ERK/MAPK activation in the LA impairs both fear memory consolidation and long-term potentiation (LTP) in the amygdala, in vitro. The present experiments evaluated the role of the ERK/MAPK signaling cascade in LTP at thalamo-LA input synapses, in vivo. We first show that ERK/MAPK is transiently activated/phosphorylated in the LA at 5 min, but not 15 or 60 min, after high-frequency, but not low-frequency, stimulation of the auditory thalamus. ERK activation induced by LTP-inducing stimulation was anatomically restricted to the same regions of the LA previously shown to exhibit ERK regulation following fear conditioning. We next show that intra-LA infusion of U0126, an inhibitor of ERK/MAPK activation, impairs LTP at thalamo-LA input synapses. Collectively, results demonstrate that ERK/MAPK activation is necessary for synaptic plasticity in anatomically defined regions of the LA, in vivo.

Ca2+ permeation and/or binding to CaV1.1 fine-tunes skeletal muscle Ca2+ signaling to sustain muscle function

BackgroundCa2+ influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca2+ permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed.MethodsWe generated a mouse with a Ca2+ binding and/or permeation defect in the voltage-dependent Ca2+ channel, CaV1.1, and used Ca2+ imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca2+ imaging techniques to define pathways modulated by Ca2+ binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles.ResultsUsing mice with a pore mutation in CaV1.1 required for Ca2+ binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca2+ stores during sustained activity. Decreases in these Ca2+-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca2+ binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers.ConclusionsWhile not essential for excitation-contraction coupling, Ca2+ binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.

A novel method of loading samples onto mini-gels for SDS-PAGE: increased sensitivity and Western blots using sub-microgram quantities of protein.

Commercially available mini-gels for sodium dodecyl sulphate (SDS)-PAGE and Western blotting are limited both by the number of lanes that can be loaded per gel and the minimum amount of protein per lane that must be loaded. Here we describe a method for loading protein samples onto existing commercially available mini-gels that allows loading of 50 or more lanes per gel. The enhanced sensitivity of the method allows Western blotting with sub-microgram quantities of protein. Samples are loaded onto filter paper strips mounted on a plastic backing sheet, and film-wrapped strips on a separate dummy loader interdigitate with the sample strips, creating a physical barrier to lateral diffusion. The sample loader sandwich is placed on top of the stacking gel, and is compatible with all commercially available SDS-PAGE systems. Comparison of 15-lane mini-gels with 30-lane micro-loader strips reveals up to a 10-fold increase in sensitivity with the new method. Using 50- and 66-lane micro-loaders, sub-microgram quantities of protein produce reliable and quantifiable signal by Western blotting. Manipulation of the ionic conditions within dummy loader strips provides a mechanism for enhancing lateral resolution, allowing for the possibility of further miniaturization.

Regulation of neuronal structure and function by mTOR-dependent and -independent signaling pathways

Introduction: Melatonin the main product secreted by the pineal gland has been shown to increase neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) stimulation. Besides microtubule and intermediate filament reorganization, neurite formation involves microfilament rearrangements in microspikes, lamellipodia and filopodia to form growth cones in the neurite growing tips. In this work, we studied the effect of melatonin on growth cone formation and the possible participation of Rho associated kinase (ROCK) a downstream kinase in the PKC signaling pathway. Methods: N1E-115 cells were incubated for 6 h with either the vehicle, 1 nM melatonin, 2 nM of the PKC activator phorbol12-myristate-13acetate (PMA), or with melatonin or PMA in the presence of the PKC Inhibitor, bisindolylmaleimide (5 M), the Rho inhibitor C3 (100 ng/ml) or the ROCK inhibitor, Y27632 (10 M) and melatonin or PMA. Microfilaments organized in growth cones were stained with rhodamine-phalloidin and observed by fluorescence microscopy. ROCK activity was determined in cells cultured and in whole cell extracts with the mentioned compounds by using the ROCK substrate long S6 peptide. Results: The results showed that melatonin caused an increase in the number of cells with growth cones. Similar results were obtained with the PKC activator PMA. While, the PKC inhibitor bisindolylmaleimide, Rho inhibitor C3, or the ROCK inhibitorY27632, abolished the microfilament reorganizations elicited by either melatonin or PMA. Both ROCK and PKC activity measured in whole cell extracts and in N1E-115 cells were increased in the presence of melatonin and PMA. Discussion: The results indicate that ROCK activity is stimulated by melatonin downstream the PKC pathway in N1E-115 cells and in whole cell extracts. Also, data suggest that melatonin induces activation of PKC which in turn activates RHO/ROCK pathway increasing growth cone formation that precludes neurite enlargement elicited by melatonin.