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Background and aim The aging-dependent activation of glycogen synthase kinase-3β (GSK-3β) has been suggested to be important in the onset of dementia. To discover novel therapeutic Kampo medicines for dementia, we examined the effects of orengedokuto (OGT; 黃連解毒湯 huáng lián jiědú tāng) and san'oshashinto (SST; 三黃瀉心湯 sān huáng xiè xīn tāng) on memory deficits and GSK-3β activity in senescence-accelerated prone mice (SAMP8). Experimental procedure The object recognition test (ORT) and conditioned fear memory test (CFT) were employed to elucidate short-term working memory and long-term fear memory. The activity of GSK-3β and the phosphorylation of related molecules were measured using a kinase assay and Western blotting. Results and conclusion OGT and SST attenuated memory deficits in SAMP8 in ORT, but not in CFT. In ex vivo experiments, cortical GSK-3β activity was significantly stronger in SAMP8 than in SAMR1. The enhanced cortical GSK-3β activity in SAMP8 was accompanied by a significant increase in the level of phosphorylated collapsin response mediator protein-2 (CRMP2), an important factor that is involved in the regulation of microtubule stability. OGT and SST attenuated not only increases in cortical GSK-3β activity, but also the levels of phosphorylated CRMP2 in SAMP8. In vitro experiments, flavonoids contained in these kampo medicines, inhibited GSK-3β activity in concentration-dependent manners. These results suggest that OGT and SST prevent aging-induced short-term working memory deficits by inhibiting aging-dependent elevations in the cortical GSK-3β activity and subsequent CRMP2 phosphorylation., Graphical abstract Image 1, Highlights • OGT and SST attenuated short-term working memory deficits in SAMP8. • Age-dependent cortical GSK-3β activation was suppressed by OGT and SST. • OGT and SST also attenuated the levels of phosphorylated CRMP2 in SAMP8.

Dietary sphingolipids such as glucosylceramide (GlcCer) are potential nutritional factors associated with prevention of metabolic syndrome. Our current understanding is that dietary GlcCer is degraded to ceramide and further metabolized to sphingoid bases in the intestine. However, ceramide is only found in trace amounts in food plants and thus is frequently taken as GlcCer in a health supplement. In the present study, we successfully prepared konjac ceramide (kCer) using endoglycoceramidase I (EGCase I). Konjac, a plant tuber, is an enriched source of GlcCer (kGlcCer), and has been commercialized as a dietary supplement to improve dry skin and itching that are caused by a deficiency of epidermal ceramide. Nerve growth factor (NGF) produced by skin cells is one of the itch factors in the stratum corneum of the skin. Semaphorin 3A (Sema 3A) has been known to inhibit NGF-induced neurite outgrowth of epidermal nerve fibers. It is well known that the itch sensation is regulated by the balance between NGF and Sema 3A. In the present study, while kGlcCer did not show an in vitro inhibitory effect on NGF-induced neurite outgrowth of PC12 cells, kCer was demonstrated to inhibit a remarkable neurite outgrowth. In addition, the effect of kCer was similar to that of Sema 3A in cell morphological changes and neurite retractions, but different from C2-Ceramide. kCer showed a Sema 3A-like action, causing CRMP2 phosphorylation, which results in a collapse of neurite growth cones. Thus, it is expected that kCer is an advanced konjac ceramide material that may have neurite outgrowth-specific action to relieve uncontrolled and serious itching, in particular, from atopic eczema., Highlights • We prepared konjac ceramide (kCer) from konjac GlcCer using endoglycoceramidase I. • kCer inhibits NGF-induced neurite outgrowth by semaphorin 3A-like action. • Konjac GlcCer does not show NGF-induced neurite outgrowth such as kCer.

Nogo-A is a myelin associated protein and one of the most potent neurite growth inhibitors in the central nervous system. Interference with Nogo-A signaling has thus been investigated as therapeutic target to promote functional recovery in CNS injuries. Still, the finding that Nogo-A presents a fairly ubiquitous expression in many types of neurons in different brain regions, in the eye and even in the inner ear suggests for further functions besides the neurite growth repression. Indeed, a growing number of studies identified a variety of functions including regulation of neuronal stem cells, modulation of microglial activity, inhibition of angiogenesis and interference with memory formation. Aim of the present commentary is to draw attention on these less well-known and sometimes controversial roles of Nogo-A. Furthermore, we are addressing the role of Nogo-A in neuropathological conditions such as ischemic stroke, schizophrenia and neurodegenerative diseases.

Publisher Summary This chapter discusses the inactivation of intracellular Rho to stimulate axon growth and regeneration. In tissue culture, inactivation of the Rho signaling pathway is effective in promoting neurite growth on growth inhibitory central nervous system (CNS) substrates by two different methods: (1) inactivation of Rho with C3 transferase and (2) inactivation by dominant negative mutation of Rho. The Rho family of GTPases regulates actin-mediated cell motility in all cells. In neurons, intracellular Rho GTPases regulate the response of growth cones to both chemorepulsive guidance cues and growth inhibitory proteins. Growth inhibitory proteins that induce growth cone collapse activate Rho, and molecules that promote neurite growth inactivate Rho. The chapter discusses whether targeting Rho GTPase activity in neurons can allow them to ignore growth inhibitory signaling and grow directly on inhibitory substrates. The inactivation of Rho not only allows axon growth on myelin and chondroitin sulfate proteoglycan (CSPG) substrates, but also allows axon regeneration after injury in the CNS. Inactivation of Rho with C3 to promote regeneration and functional recovery after SCI is simple, and the chapter reveals the potential for a new, straightforward technique to promote axon regeneration.

Publisher Summary This chapter describes Rho guanosine 5'-triphosphates (GTPases) and axonal growth cone collapse. The axonal growth cone is the specialized distal tip of the extending axon and consists of both lamellipodia and filopodia structures. Control of growth cone dynamics is critical for neurite outgrowth and guidance. Modulating the activity state of Rho, Rac, and Cdc42 in neurons can modulate the growth cone response to collapsing agents. The chapter discusses two methods of introducing Rho family GTPase into cultured neurons. These methods are designed to facilitate an analysis of the role of such GTPase in growth cone collapse by axonal guidance factors. The chapter presents a protocol to measure levels of activated Rac and Cdc42 in cultured neurons following the introduction of growth cone collapsing agents.

The structural mechanisms responsible for the gradual elaboration of the cytoplasmic elongation of neurons are reviewed. In addition to discussing recent work, important older work is included to inform newcomers to the field how the current perspective arose. The highly specialized axon and the less exaggerated dendrite both result from the advance of the motile growth cone. In the area of physiology, studies in the last decade have directly confirmed the classic model of the growth cone pulling forward and the axon elongating from this tension. Particularly in the case of the axon, cytoplasmic elongation is closely linked to the formation of an axial microtubule bundle from behind the advancing growth cone. Substantial progress has been made in understanding the expression of microtubule-associated proteins during neuronal differentiation to stiffen and stabilize axonal microtubules, providing specialized structural support. Studies of membrane organelle transport along the axonal microtubules produced an explosion of knowledge about ATPase molecules serving as motors driving material along microtubule rails. However, most aspects of the cytoplasmic mechanisms responsible for neurogenesis remain poorly understood. There is little agreement on mechanisms for the addition of new plasma membrane or the addition of new cytoskeletal filaments in the growing axon. Also poorly understood are the mechanisms that couple the promiscuous motility of the growth cone to the addition of cytoplasmic elements.

Publisher Summary This chapter presents some investigations of the navigational hypothesis for growth cone behavior. It analyses growth cone behaviors at boundaries between combinations of permissive and inhibitory substratum-bound molecules. In addition, the chapter investigates the involvement of one cytoplasmic regulatory factor—namely, cytoplasmic [Ca 2+ ], in controlling growth cone behaviors. The results suggest that: (1) growth cone behavior changes at boundaries between substratum adsorbed molecules, (2) interactions with surface bound molecules can trigger changes in growth cone [Ca 2+ ], (3) recurrent spikes of cytoplasmic [Ca 2+ ] can be generated in growth cones, and (4) growth cones contain Ca 2+ -regulated proteins that may be important in controlling the organization and functions of actin filaments and microtubules. These issues are relevant to nerve regeneration, as axons that are separated from their synaptic targets must re-express the activities necessary for growth cone migration and axonal elongation. Often, neurons with injured axons show a rapidly increased expression of the major cytoskeletal components of growing neurites, actin, and tubulin. Perhaps increased expression of cell surface receptors for environmental cues and the cytoplasmic signaling systems that mediate regulation of growth cone motility are also required for successful axonal regeneration.

Publisher Summary The nerve growth cone is the irregularly shaped terminal enlargement of a growing neurite. It is its leading edge that is endowed with the capacity of finding the path to the appropriate target area and forming a synapse with an appropriate target cell. Experimental findings indicate that growth cones contain protein kinase activities that resemble those of synaptosomes qualitatively. However, major quantitative differences exist. Particularly striking is the simple pattern of major substrates of calcium-dependent kinases. Three of these substrates, pp40, pp46, and pp80, and/or their phosphorylation are developmentally regulated. Thus, they are candidates for the molecules involved in growth regulation. The prominent role of protein kinase C in the phosphorylation of growth cone proteins is of further significance. Protein kinase C has been implicated in a number of growth-regulatory processes, as well as leukocyte chemotaxis. These functions can be expected to have equivalents in the nerve growth cone because the primary growth factor effects appear to occur at this site, at least in the case of nerve growth factor-sensitive peripheral neurons.