Neuropharmacology

British Journal of Pharmacology (2005) 144, 961–971. doi:10.1038/sj.bjp.0706122

Neuritic regeneration and synaptic reconstruction induced by withanolide A
Tomoharu Kuboyama1,2, Chihiro Tohda1 and Katsuko Komatsu1,2

1Research Center for Ethnomedicines, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
221st Century COE Program, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan
Correspondence: Katsuko Komatsu, E-mail: katsukok@ms.toyama-mpu.ac.jp

Received 14 October 2004; Revised 22 November 2004; Accepted 29 November 2004; Published online 14 February 2005.

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.
Treatment with A(25–35) (10 M) induced axonal and dendritic atrophy, and pre- and postsynaptic loss in cultured rat cortical neurons. Subsequent treatment with WL-A (1 M) induced significant regeneration of both axons and dendrites, in addition to the reconstruction of pre- and postsynapses in the neurons.
WL-A (10 mol kg-1 day-1, for 13 days, p.o.) recovered A(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.
WL-A is therefore an important candidate for the therapeutic treatment of neurodegenerative diseases, as it is able to reconstruct neuronal networks.
Keywords: Axon, dendrite, presynapse, postsynapse, Morris water maze, A(25–35), Withania somnifera, Ashwagandha, withanolide

Abbreviations: MAP2, microtubule-associated protein 2; NF-H, neurofilament-H; NGF, nerve growth factor; PSD-95, post-synaptic density-95

Top of pageIntroduction
Despite a number of ongoing investigations, neurodegenerative diseases remain incurable. Although patients suffering from neurodegenerative diseases can benefit from medication with numerous drugs, the progression of these diseases cannot yet be halted. In patients with Alzheimer's disease, neuritic atrophy and synaptic loss are considered the major causes of cognitive impairment, as based on the results of neuropathological post-mortem studies of the brain (De Kosky & Scheff, 1990; Terry et al., 1991; Dickson & Vickers, 2001). In the brains of patients suffering from other neurodegenerative diseases such as Parkinson's disease, Huntington's disease, and Creutzfeldt–Jakob disease, the atrophy of neurites has also been observed (Jackson et al., 1995; Liberski & Budka, 1999; Mattila et al., 1999). Such atrophy leads to the destruction of neuronal networks, and subsequently to fatal dysfunction of the brain systems in these patients. The prevention of, or at least a decrease in the magnitude of the cause of each disease, may prevent the progression of symptoms, but such inhibition is not associated with the repair of already severely damaged brain function. We hypothesized that reconstructing neuronal networks in the injured brain would be the most necessary step in the fundamental recovery of brain function. In order to reconstruct neuronal networks, neuritic regeneration and synaptic reconstruction must take place in the damaged brain. Therefore, we aimed to explore compounds that would facilitate the regeneration of neurites and the reconstruction of synapses, even in severely damaged neurons, and to show evidence of the effects in vivo as well as in vitro.

Ashwagandha (root of Withania somnifera Dunal) is the most popular herbal drug in Ayurvedic medicine, and has been used traditionally and commonly as a tonic and nootropic agent. It has also been reported to be associated with improvements in scopolamine-induced memory deficits in mice (Dhuley, 2001). We previously demonstrated that a methanol extract of Ashwagandha was associated with neurite extension and, in particular, that of dendrites (Tohda et al., 2000). In addition, we identified that six constituents isolated from the methanol extract induced neurite outgrowth in human neuroblastoma SH-SY5Y cells (Zhao et al., 2002). In normal cortical neurons, predominant axonal outgrowth was observed in the treatment with withanolide A (WL-A), which was one of the major active constituents isolated from Ashwagandha (Kuboyama et al., 2002).

Amyloid is a major pathological cause of Alzheimer's disease due to the formation of a -sheet structure (Simmons et al., 1994); amyloid forms deposits in the brain, and subsequently induces neuronal cell death (Bobinski et al., 1997), neuritic atrophy (Canning et al., 1993; Knowles et al., 1999), and synaptic loss (Terry et al., 1991). A(25–35) is an active partial fragment of amyloid . This fragment also forms a -sheet structure (Pike et al., 1995) and induces neuronal cell death (Yankner et al., 1990; Pike et al., 1995), neuritic atrophy (Grace et al., 2002; Tohda et al., 2004), synaptic loss (Grace et al., 2002; Tohda et al., 2003; 2004), and memory impairment (Maurice et al., 1996; Tohda et al., 2003; 2004).


In this study, we investigated the effects of WL-A on neuritic regeneration and synaptic reconstruction in cultured neurons damaged by A(25–35) and cognitive-deficient mice by A(25–35)-injection. In particular, synaptic formations at both the presynaptic region (axon and presynapse) and the postsynaptic region (dendrite and postsynapse) were observed discriminatively.

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