A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of β-amyloid peptides (Aβ) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma Aβ and a decrease in brain Aβ monomer after 7 d, indicating increased transport of Aβ from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the Aβ-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain Aβ levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain Aβ. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain Aβ, indicating that increase in liver LRP and sLRP occurring independent of Aβ concentration could result in clearance of Aβ. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for Aβ clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.herbal extract | dementia | neurodegenerative disease A lzheimer's disease (AD) is characterized by progressive dysfunction of memory and higher cognitive functions. Pathological hallmarks include senile plaques, neurofibrillary tangles, dystrophic neurites, gliosis, and neuroinflammation. Cholinesterase inhibitors and the NMDA antagonist memantine, the commonly used drugs for AD, provide symptomatic relief but do not alter the course of disease. No curative treatment is available, and research focuses on drugs for slowing disease progression or providing prophylaxis.The majority of AD cases are sporadic in nature. The small fraction of familial cases are caused primarily by mutations in three genes: amyloid precursor protein (APP), presenilin1 (PS1), and presenilin 2 (PS2). These mutations result in abnormal processing of APP and increased generation of β amyloid peptide 1-42 (Aβ42), which aggregates as β sheets (1). Treatment strategies have focused on reducing β-amyloid load through (i) inhibition of γ-or β-secretases or activation of α-secretase; (ii) inhibition of Aβ aggregation; (iii) activation of proteases, such as neprilysin (NEP); and (iv) active and passive immunotherapy (2, 3).Among other mechanisms, influx and efflux of brain Aβ are regulated by receptor for advanced glycation end products (RAGE) and low-density lipoprotein receptor-related protein (LRP), respectively (4). The soluble form of LRP in plasma (sLRP) is a peripheral sink for Aβ that aids i...

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Activation of the mixed lineage kinase and c-Jun N-terminal kinase (JNK) has been reported in models of PD. Our focus was to discern whether distinct pathways were activated in cell-specific manner within the SNpc. We now demonstrate the selective phosphorylation of p38 MAP kinase within the dopaminergic neurons, whereas JNK activation occurs predominantly in the microglia. p38 activation results in downstream phosphorylation of p53 and increased p53 mediated transcription of Bax and Puma in the ventral midbrain. Treatment with p38 inhibitor, SB239063 protected primary dopaminergic neurons derived from human progenitor cells from MPP ϩ mediated cell death and prevented the downstream phosphorylation of p53 and its translocation to the nucleus in vivo, in the ventral midbrain. The increased staining of phosphorylated p38 in the surviving neurons of SNpc in human brain sections from patients with PD and in MPTP treated mice but not in the ventral tegmental area provides further evidence suggesting a role for p38 in the degeneration of dopaminergic neurons of SNpc. We thus demonstrate the cell specific activation of MAP kinase pathways within the SNpc after MPTP treatment emphasizing the role of multiple signaling cascades in the pathogenesis and progression of the disease. Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression.

Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, α-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of β-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.

Environmental and genetic causes are implicated in the etiopathogenesis of Parkinson's disease (PD), a neurodegenerative movement disorder. DJ-1, a putative gene recessively linked to early onset PD, functions as an antioxidant, transcriptional co-activator, and molecular chaperone. We examined DJ-1 status following global perturbation of protein thiol homeostasis by depleting cellular antioxidant glutathione or downregulating glutaredoxin 1, a thiol disulfide oxidoreductase, wherein both paradigms generate oxidative stress. While these perturbations did not affect expression of DJ-1 mRNA, downregulation of glutaredoxin 1 but not glutathione depletion caused loss of DJ-1 protein, translocation of Daxx (a death-associated protein) from nucleus, and cell death. Overexpression of wild-type DJ-1, but not the cysteine mutants, prevented Daxx translocation and cytotoxicity. Protease inhibitors prevented constitutive DJ-1 loss. Residual DJ-1 was present in reduced state, indicating that DJ-1 when oxidized was degraded through proteolysis. Thus, loss of DJ-1 occurring through its oxidative modification and subsequent proteolysis mediated through dysregulation of thiol disulfide oxidoreductase may contribute to pathogenesis of sporadic PD, thus providing a link between environmental challenges and constitutive levels of this vital protein.

Impairment of Akt phosphorylation, a critical survival signal, has been implicated in the degeneration of dopaminergic neurons in Parkinson's disease. However, the mechanism underlying pAkt loss is unclear. In the current study, we demonstrate pAkt loss in ventral midbrain of mice treated with dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), when compared to ventral midbrain of control mice treated with vehicle alone. Thiol residues of the critical cysteines in Akt are oxidized to a greater degree in mice treated with MPTP, which is reflected as a 40% loss of reduced Akt. Association of oxidatively modified Akt with the phosphatase PP2A, which can lead to enhanced dephosphorylation of pAkt, was significantly stronger after MPTP treatment. Maintaining the protein thiol homeostasis by thiol antioxidants prevented loss of reduced Akt, decreased association with PP2A, and maintained pAkt levels. Overexpression of glutaredoxin, a protein disulfide oxidoreductase, in human primary neurons helped sustain reduced state of Akt and abolished MPP(+)-mediated pAkt loss. We demonstrate for the first time the selective loss of Akt activity, in vivo, due to oxidative modification of Akt and provide mechanistic insight into oxidative stress-induced down-regulation of cell survival pathway in mouse midbrain following exposure to MPTP.