1 We investigated whether withanolide A (WL-A), isolated from the Indian herbal drug Ashwagandha (root of Withania somnifera), could regenerate neurites and reconstruct synapses in severely damaged neurons. We also investigated the effect of WL-A on memory-deficient mice showing neuronal atrophy and synaptic loss in the brain. Axons, dendrites, presynapses, and postsynapses were visualized by immunostaining for phosphorylated neurofilament-H (NF-H), microtubule-associated protein 2 (MAP2), synaptophysin, and postsynaptic density-95 (PSD-95), respectively. 2 Treatment with Ab(25-35) (10 mM) induced axonal and dendritic atrophy, and pre-and postsynaptic loss in cultured rat cortical neurons. Subsequent treatment with WL-A (1 mM) induced significant regeneration of both axons and dendrites, in addition to the reconstruction of pre-and postsynapses in the neurons. 3 WL-A (10 mmol kg À1 day À1 , for 13 days, p.o.) recovered Ab(25-35)-induced memory deficit in mice. At that time, the decline of axons, dendrites, and synapses in the cerebral cortex and hippocampus was almost recovered. 4 WL-A is therefore an important candidate for the therapeutic treatment of neurodegenerative diseases, as it is able to reconstruct neuronal networks.

The aim of this study was to investigate the effects and the mechanism of diosgenin, a famous plant-derived steroidal sapogenin, on memory deficits in Alzheimer's disease (AD) model mice. Diosgenin-treated 5XFAD mice exhibited significantly improved performance of object recognition memory. Diosgenin treatment significantly reduced amyloid plaques and neurofibrillary tangles in the cerebral cortex and hippocampus. Degenerated axons and presynaptic terminals that were only observed in regions closely associated with amyloid plaques were significantly reduced by diosgenin treatment. The 1,25D3-membrane-associated, rapid response steroid-binding protein (1,25D3-MARRS) was shown to be a target of diosgenin. 1,25D3-MARRS knockdown completely inhibited diosgenin-induced axonal growth in cortical neurons. Treatment with a neutralizing antibody against 1,25D3-MARRS diminished the axonal regeneration effect of diosgenin in Aβ(1–42)-induced axonal atrophy. This is the first study to demonstrate that the exogenous stimulator diosgenin activates the 1,25D3-MARRS pathway, which may be a very critical signaling target for anti-AD therapy.

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We previously screened neurite outgrowth activities of several Ginseng drugs in human neuroblastoma, and demonstrated that protopanaxadiol (ppd)-type saponins were active constituents. Since ppd-type saponins are known to be completely metabolized to 20-O-b-D-glucopyranosyl-20(S)-protopanaxadiol (M1) by intestinal bacteria when taken orally, M1 and ginsenoside Rb 1 , as a representative of ppd-type saponins, were examined for cognitive disorder. In a mouse model of Alzheimer's disease (AD) by Ab(25-35) i.c.v. injection, impaired spatial memory was recovered by p.o. administration of ginsenoside Rb 1 or M1. Although the expression levels of phosphorylated NF-H and synaptophysin were reduced in the cerebral cortex and the hippocampus of Ab(25-35)-injected mice, their levels in ginsenoside Rb 1 -and M1-treated mice were almost completely recovered up to control levels. Potencies of the effects were not different between ginsenoside Rb 1 and M1 when given orally, suggesting that most of the ginsenoside Rb 1 may be metabolized to M1, and M1 is an active principal of ppd-type saponins for the memory improvement. In cultured rat cortical neurons, M1 showed extension activity of axons, but not dendrites. The axon-specific outgrowth was seen even when neuritic atrophy had already progressed in response to administration of Ab(25-35) as well as in the normal condition. These results suggest that M1 has axonal extension activity in degenerated neurons, and improve memory disorder and synaptic loss induced by . M1 was shown to be effective in vitro and in vivo, indicating that Ginseng drugs containing ppd-type saponins may reactivate neuronal function in AD by p.o. administration.

The reconstruction of neuronal networks in the damaged brain is necessary for the therapeutic treatment of neurodegenerative diseases. We have screened the neurite outgrowth activity of herbal drugs, and identified several active constituents. In each compound, neurite outgrowth activity was investigated under amyloid-β-induced neuritic atrophy. Most of the compounds with neurite regenerative activity also demonstrated memory improvement activity in Alzheimer’s disease-model mice. Protopanaxadiol-type saponins in Ginseng drugs and their metabolite, M1 (20-O-β-D-glucopyranosyl-(20S)-protopanaxadiol), showed potent regeneration activity for axons and synapses, and amelioration of memory impairment. Withanolide derivatives (withanolide A, withanoside IV, and withanoside VI) isolated from the Indian herbal drug Ashwagandha, also showed neurite extension in normal and damaged cortical neurons. Trigonelline, a constituent of coffee beans, demonstrated the regeneration of dendrites and axons, in addition to memory improvement.

Capsaicin has been suggested to act not only on thin primary afferents but also on the hypothalamus, but the neurotransmitter(s) of central capsaicin-sensitive neurons are unknown. The present study was conducted to determine whether any central, especially hypothalamic, glutamatergic terminals were sensitive to capsaicin. Capsaicin evoked glutamate release from slices of hypothalamus and lumbar dorsal horn, but not cerebellum. Such capsaicin action was Ca2+ dependent and inhibited by the capsaicin antagonist capsazepine. Vanilloid receptor subtype 1 mRNA was widely distributed in the brain, with a marked level in the hypothalamus and cerebellum, but not in the spinal cord. The results suggest that there are glutamatergic terminals sensitive to capsaicin in the hypothalamus.

Natural medicines are attractive sources of leading compounds that can be used as interventions for neurodegenerative disorders. The complexity of their chemical components and undetermined bio-metabolism have greatly hindered both the use of natural medicines and the identification of their active constituents. Here, we report a systematic strategy for evaluating the bioactive candidates in natural medicines used for Alzheimer’s disease (AD). We found that Drynaria Rhizome could enhance memory function and ameliorate AD pathologies in 5XFAD mice. Biochemical analysis led to the identification of the bio-effective metabolites that are transferred to the brain, namely, naringenin and its glucuronides. To explore the mechanism of action, we combined the drug affinity responsive target stability with immunoprecipitation-liquid chromatography/mass spectrometry analysis, identifying the collapsin response mediator protein 2 protein as a target of naringenin. Our study indicates that biochemical analysis coupled with pharmacological methods can be used in the search for new targets for AD intervention.

Capsaicin receptors are expressed in primary sensory neurons and excited by heat and protons. We examined the in¯ammation-induced changes of the level of VR1 capsaicin receptor mRNA in sensory neurons and the sensitivity of primary afferents to capsaicin. Carrageenan treatment induced axonal transport of VR1 mRNA, but not that of preprotachykinin mRNA, from the dorsal root ganglia to central and peripheral axon terminals. The sensitivity of central terminals to capsaicin, which was estimated by measuring the capsaicin-evoked release of glutamate from the dorsal horn, was increased by peripheral in¯ammation, and such an increase was suppressed by inhibiting the RNA translation in the dorsal horn with cycloheximide and an intrathecal injection of VR1 antisense oligonucleotides. Thus, peripheral in¯ammation induces the axonal transport of VR1 mRNA, which may be involved in the hypersensitivity of primary afferents to capsaicin and the production of in¯ammatory hyperalgesia. Keywords: axonal transport, capsaicin sensitivity, carrageenan in¯ammation, glutamate release, primary afferent, VR1 capsaicin receptor mRNA.Subcutaneous injection of carrageenan into the hind paw induces in¯ammation, a decrease in nociceptive threshold (Kayser et al. 1991) and hyperexcitability of primary afferents (Coggeshall et al. 1983). Nociceptive primary afferents are sensitive to capsaicin. Capsaicin induces the excitation of nociceptors (Such and Jancso 1986; Holzer 1991) and the release of pain transmitters such as glutamate (Ueda et al. 1993; and neuropeptides (Yaksh et al. 1980;Saria et al. 1986). Capsaicin receptor is called a`proton sensor' and`hot sensor' because of its sensitivity to protons and heat stimulation, respectively (Caterina et al. 1997). When in¯ammation occurs in the periphery, the concentration of protons is increased in injured tissues. Therefore, the activation of capsaicin receptors on the peripheral terminals of primary afferents may be partly involved in in¯ammatory hyperalgesia (Caterina et al. 2000). With regard to the spinal cord, glutamate (Okano et al. 1998) and neuropeptides (Satoh et al. 1992;Okano et al. 1998) are also involved in in¯ammatory hyperalgesia. Peripheral in¯ammation increases capsaicin-evoked release of glutamate and neuropeptides ) from the dorsal horn. Although an increase in the biosynthesis of neurotransmitters in primary sensory neurons may be partly responsible for an increase in capsaicin-evoked release of the neurotransmitters Ohno et al. 1990), it is unclear whether peripheral in¯ammation alters, especially increases, the sensitivity of primary afferent