Chihiro Tohda

Neuritic regeneration and synaptic reconstruction induced by withanolide A

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 Aβ(25–35) (10 μM) induced axonal and dendritic atrophy, and pre‐ and postsynaptic loss in cultured rat cortical neurons. Subsequent treatment with WL‐A (1 μM) induced significant regeneration of both axons and dendrites, in addition to the reconstruction of pre‐ and postsynapses in the neurons. 3 WL‐A (10 μmol kg−1 day−1, for 13 days, p.o.) recovered Aβ(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.

Search for Natural Products Related to Regeneration of the Neuronal Network

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.

Existence of capsaicin-sensitive glutamatergic terminals in rat hypothalamus.

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.

Axonal transport of VR1 capsaicin receptor mRNA in primary afferents and its participation in inflammation-induced increase in capsaicin sensitivity

Capsaicin receptors are expressed in primary sensory neurons and excited by heat and protons. We examined the inflammation‐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 inflammation, 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 inflammation induces the axonal transport of VR1 mRNA, which may be involved in the hypersensitivity of primary afferents to capsaicin and the production of inflammatory hyperalgesia.

Aβ(25–35)-Induced Memory Impairment, Axonal Atrophy, and Synaptic Loss are Ameliorated by M1, A Metabolite of Protopanaxadiol-Type Saponins

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-β-D-glucopyranosyl-20(S)-protopanaxadiol (M1) by intestinal bacteria when taken orally, M1 and ginsenoside Rb1, as a representative of ppd-type saponins, were examined for cognitive disorder. In a mouse model of Alzheimers disease (AD) by Aβ(25–35) i.c.v. injection, impaired spatial memory was recovered by p.o. administration of ginsenoside Rb1 or M1. Although the expression levels of phosphorylated NF-H and synaptophysin were reduced in the cerebral cortex and the hippocampus of Aβ(25–35)-injected mice, their levels in ginsenoside Rb1- and M1-treated mice were almost completely recovered up to control levels. Potencies of the effects were not different between ginsenoside Rb1 and M1 when given orally, suggesting that most of the ginsenoside Rb1 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 Aβ(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 Aβ(25–35). 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.

Axon- or dendrite-predominant outgrowth induced by constituents from Ashwagandha.

We previously reported that the methanol extract of Ashwagandha (roots of Withania somnifera Dunal) induced dendrite extension in a human neuroblastoma cell line. In this study, we found that six of the 18 compounds isolated from the methanol extract enhanced neurite outgrowth in human neuroblastoma SH-SY5Y cells. Double immunostaining was performed in rat cortical neurons using antibodies to phosphorylated NF-H as an axonal marker, and to MAP2 as a dendritic marker. In withanolide A-treated cells, the length of NF-H-positive processes was significantly increased compared with vehicle-treated cells, whereas, the length of MAP2-positive processes was increased by withanosides IV and VI. These results suggest that axons are predominantly extended by withanolide A, and dendrites by withanosides IV and VI.

Promotion of axonal maturation and prevention of memory loss in mice by extracts of Astragalus mongholicus.

Neurons with atrophic neurites may remain alive and therefore may have the potential to regenerate even when neuronal death has occurred in some parts of the brain. This study aimed to explore effects of drugs that can facilitate the regeneration of neurites and the reconstruction of synapses even in severely damaged neurons.

Withanoside IV and its active metabolite, sominone, attenuate Aβ(25–35)-induced neurodegeneration

At the present, medication of dementia is limited to symptomatic treatments such as the use of cholinesterase inhibitors. To cure dementia completely, that is regaining neuronal function, reconstruction of neuronal networks is necessary. Therefore, we have been exploring antidementia drugs based on reconstructing neuronal networks in the damaged brain and found that withanoside IV (a constituent of Ashwagandha; the root of Withania somnifera) induced neurite outgrowth in cultured rat cortical neurons. Oral administration of withanoside IV (10 µmol/kg/day) significantly improved memory deficits in Aβ(25–35)‐injected (25 nmol, i.c.v.) mice and prevented loss of axons, dendrites, and synapses. Sominone, an aglycone of withanoside IV, was identified as the main metabolite after oral administration of withanoside IV. Sominone (1 µm) induced axonal and dendritic regeneration and synaptic reconstruction significantly in cultured rat cortical neurons damaged by 10 µm Aβ(25–35). These data suggest that orally administrated withanoside IV may ameliorate neuronal dysfunction in Alzheimers disease and that the active principle after metabolism is sominone.

Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-N-SH cells.

Extension of dendrites and axons in neurons may compensate for and repair damaged neuronal circuits in the dementia brain. Our aim in the present study was to explore drugs activating neurite outgrowth and regenerating the neuronal network. We found that the methanol extract of Ashwagandha (roots of Withania somnifera; 5 μg/ml) significantly increased the percentage of cells with neurites in human neuroblastoma SK-N-SH cells. The effect of the extract was dose- and time-dependent. mRNA levels of the dendritic markers MAP2 and PSD-95 by RT-PCR were found to be markedly increased by treatment with the extract, whereas those of the axonal marker Tau were not. Immunocytochemistry demonstrated the specific expression of MAP2 in neurites extended by the extract. These results suggest that the methanol extract of Ashwagandha promotes the formation of dendrites.

Current and future therapeutic strategies for functional repair of spinal cord injury

Spinal cord injury (SCI) causes serious, chronic dysfunction which is difficult to treat. Disability, including long-lasting motor and sensory dysfunction, typically results from damage to the descending and ascending spinal tracts and interneurons and, secondarily, to the neuronal degeneration that occurs proximal and distal to the spinal insult. Numerous strategies are being implemented to protect neurons from damage, to enhance axon growth and to foster cell proliferation. Described in this report are recent clinical trials aimed at testing strategies to restore locomotion after SCI. While laboratory animal studies have indicated that it may be possible to minimize neuronal damage resulting from spinal cord injury, little progress has been made in reducing or reversing the events associated with the chronic phase of this condition. The strategy aiming to inhibit single molecule sometimes shows controversial results. In SCI, a lot of players participate in motor and sensory dysfunctions. Therefore, sufficient functional recovery may be achieved by regulating multiple targets. Regrowth of tracts connecting the brain and spinal cord, and axonal sprouting of propriospinal interneurons are fundamentally important for neuronal network working. In addition, remyelination, protection of neuronal death, inhibition of inflammation, and upregulation of beneficial influence of astrocytes are also quite crucial to supporting the axonal refining. Combination of several strategies might be useful as a practical therapy. Several compounds such as a Sema3A inhibitor, estrogen, withanoside IV and their relating compounds or other neurotrophic factor-mimicking agents may be candidates for useful SCI therapeutic drugs since those have multi-effects on damaged spinal cord. [corrected].