A serine protease KLK8 emerges as a regulator of regulators in memory: Microtubule protein dependent neuronal morphology and PKA-CREB signaling

The multitude of molecular pathways underlying memory impairment in neurological disorders and aging-related disorders has been a major hurdle against therapeutic targeting. Over the years, neuronal growth promoting factors, intracellular kinases, and specific transcription factors, particularly cyclic AMP response element-binding protein (CREB), have emerged as crucial players of memory storage, and their disruption accompanies many cognitive disabilities. However, a molecular link that can influence these major players and can be a potential recovery target has been elusive. Recent reports suggest that extracellular cues at the synapses might evoke an intracellular signaling cascade and regulate memory function. Herein, we report novel function of an extracellular serine protease, kallikrein 8 (KLK8/Neuropsin) in regulating the expression of microtubule associated dendrite growth marker microtubule-associated protein (MAP2)c, dendrite architecture and protein kinase A (PKA)-CREB signaling. Both knockdown of KLK8 via siRNA transfection in mouse primary hippocampal neurons and via intra-hippocampal administration of KLK8 antisense oligonucleotides in vivo reduced expression of MAP2c, dendrite length, dendrite branching and spine density. The KLK8 mediated MAP2c deficiency in turn inactivated PKA and downstream transcription factor phosphorylated CREB (pCREB), leading to downregulation of memory-linked genes and consequent impaired memory consolidation. These findings revealed a protease associated novel pathway of memory impairment in which KLK8 may act as a "regulator of regulators", suggesting its exploration as an important therapeutic target of memory disorders

Protective Role of Ashwagandha Leaf Extract and Its Component Withanone on Scopolamine-Induced Changes in the Brain and Brain-Derived Cells

BACKGROUND:Scopolamine is a well-known cholinergic antagonist that causes amnesia in human and animal models. Scopolamine-induced amnesia in rodent models has been widely used to understand the molecular, biochemical, behavioral changes, and to delineate therapeutic targets of memory impairment. Although this has been linked to the decrease in central cholinergic neuronal activity following the blockade of muscarinic receptors, the underlying molecular and cellular mechanism(s) particularly the effect on neuroplasticity remains elusive. In the present study, we have investigated (i) the effects of scopolamine on the molecules involved in neuronal and glial plasticity both in vivo and in vitro and (ii) their recovery by alcoholic extract of Ashwagandha leaves (i-Extract). METHODOLOGY/PRINCIPAL FINDINGS:As a drug model, scopolamine hydrobromide was administered intraperitoneally to mice and its effect on the brain function was determined by molecular analyses. The results showed that the scopolamine caused downregulation of the expression of BDNF and GFAP in dose and time dependent manner, and these effects were markedly attenuated in response to i-Extract treatment. Similar to our observations in animal model system, we found that the scopolamine induced cytotoxicity in IMR32 neuronal and C6 glioma cells. It was associated with downregulation of neuronal cell markers NF-H, MAP2, PSD-95, GAP-43 and glial cell marker GFAP and with upregulation of DNA damage--γH2AX and oxidative stress--ROS markers. Furthermore, these molecules showed recovery when cells were treated with i-Extract or its purified component, withanone. CONCLUSION:Our study suggested that besides cholinergic blockade, scopolamine-induced memory loss may be associated with oxidative stress and Ashwagandha i-Extract, and withanone may serve as potential preventive and therapeutic agents for neurodegenerative disorders and hence warrant further molecular analyses

M1 muscarinic receptor is a key target of neuroprotection, neuroregeneration and memory recovery by i-Extract from Withania somnifera

Memory loss is one of the most tragic symptoms of Alzheimer's disease. Our laboratory has recently demonstrated that 'i-Extract' of Ashwagandha (Withania somnifera) restores memory loss in scopolamine (SC)-induced mice. The prime target of i-Extract is obscure. We hypothesize that i-Extract may primarily target muscarinic subtype acetylcholine receptors that regulate memory processes. The present study elucidates key target(s) of i-Extract via cellular, biochemical, and molecular techniques in a relevant amnesia mouse model and primary hippocampal neuronal cultures. Wild type Swiss albino mice were fed i-Extract, and hippocampal cells from naïve mice were treated with i-Extract, followed by muscarinic antagonist (dicyclomine) and agonist (pilocarpine) treatments. We measured dendritic formation and growth by immunocytochemistry, kallikrein 8 (KLK8) mRNA by reverse transcription polymerase chain reaction (RT-PCR), and levels of KLK8 and microtubule-associated protein 2, c isoform (MAP2c) proteins by western blotting. We performed muscarinic receptor radioligand binding. i-Extract stimulated an increase in dendrite growth markers, KLK8 and MAP2. Scopolamine-mediated reduction was significantly reversed by i-Extract in mouse cerebral cortex and hippocampus. Our study identified muscarinic receptor as a key target of i-Extract, providing mechanistic evidence for its clinical application in neurodegenerative cognitive disorders

Bacopa monniera

Preclinical studies on animal models have discerned the antiamnesic and memory-enhancing potential of Bacopa monniera (Brahmi) crude extract and standardized extracts. These studies primarily focus on behavioral consequences. However, lack of information on molecular underpinnings has limited the clinical trials of the potent herb in human subjects. In recent years, researchers highlight plasticity markers as molecular correlates of amnesia and being crucial to design therapeutic targets. In the present report, we have investigated the effect of a special extract of B. monniera (CDRI-08) on the expression of key neuronal (BDNF and Arc) and glial (GFAP) plasticity markers in the cerebrum of scopolamine induced amnesic mice. Pre- and postadministration of CDRI-08 ameliorated amnesic effect of scopolamine by decreasing acetyl cholinesterase activity and drastically upregulating the mRNA and protein expression of BDNF, Arc, and GFAP in mouse cerebrum. Interestingly, the plant extract per se elevated BDNF and Arc expression as compared to control but GFAP was unaltered. In conclusion, our findings provide the first molecular evidence for antiamnesic potential of CDRI-08 via enhancement of both neuronal and glial plasticity markers. Further investigations on detailed molecular pathways would encourage therapeutic application of the extract in memory disorders

Median Facial Cleft in Amniotic Band Syndrome

Amniotic band syndrome manifests at birth with a variety of malformations ranging from constriction ring to defects incompatible to life, in various parts of the body. Although some theories have been proposed for the development of this syndrome, the exact cause remains unknown. The median facial cleft is an extremely rare manifestation of amniotic band syndrome with a relative paucity of reports available in the literature. Here, we report one such case

Ashwagandha i-Extract and withanone treatment and recovery of glial cell differentiation marker GFAP in scopolamine-treated C6 cells.

<p>Level of GFAP expression in C6 glioma cells treated with either scopolamine or scopolamine and i-Extract/withaferin A/withanone as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027265#s4" target="_blank">material and methods</a>. Levels detected by western blotting (A) and immunocytostaining (B) are shown. Quantitation from two independent experiments was performed using SigmaStat (Aspire Software International). Actin was used as an internal control.</p

Recovery of neuronal cell differentiation markers in scopolamine-treated cells in response to Ashwagandha i-Extract and withanone treatment.

<p>Level of MAP2, NF-H, GAP43 and PSD-95 expression in IMR32 neuroblastoma cells treated with either scopolamine or scopolamine and i-Extract/withaferin A/withanone as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027265#s4" target="_blank">material and methods</a>. Levels of expression as detected by western blotting (A) and immunocytostaining (B) are shown. Quantitation was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027265#pone-0027265-g005" target="_blank">Figure 5</a>.</p

Treatment with Ashwagandha i-Extract/withanone and protection against scopolamine-induced DNA damage and oxidative stress.

<p>Level of expression of DNA damage marker (γH2AX) (A) and oxidative stress marker (reactive oxygen species, ROS) (B) in C6 cells treated with either scopolamine or scopolamine and i-Extract/withaferin A/withanone as detected by immunocytochemistry with specific antibodies and as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027265#s4" target="_blank">material and methods</a> are shown. Quantitation of γH2AX (C) and ROS (D) are shown, in which 200–300 cells were counted and scored with high intensity of signal.</p