Arpita Konar

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.

Age-associated Cognitive Decline: Insights into Molecular Switches and Recovery Avenues

Age-associated cognitive decline is an inevitable phenomenon that predisposes individuals for neurological and psychiatric disorders eventually affecting the quality of life. Scientists have endeavored to identify the key molecular switches that drive cognitive decline with advancing age. These newly identified molecules are then targeted as recovery of cognitive aging and related disorders. Cognitive decline during aging is multi-factorial and amongst several factors influencing this trajectory, gene expression changes are pivotal. Identifying these genes would elucidate the neurobiological underpinnings as well as offer clues that make certain individuals resilient to withstand the inevitable age-related deteriorations. Our laboratory has focused on this aspect and investigated a wide spectrum of genes involved in crucial brain functions that attribute to senescence induced cognitive deficits. We have recently identified master switches in the epigenome regulating gene expression alteration during brain aging. Interestingly, these factors when manipulated by chemical or genetic strategies successfully reverse the age-related cognitive impairments. In the present article, we review findings from our laboratory and others combined with supporting literary evidences on molecular switches of brain aging and their potential as recovery targets.

Hippocampal chromatin-modifying enzymes are pivotal for scopolamine-induced synaptic plasticity gene expression changes and memory impairment.

The amnesic potential of scopolamine is well manifested through synaptic plasticity gene expression changes and behavioral paradigms of memory impairment. However, the underlying mechanism remains obscure and consequently ideal therapeutic target is lacking. In this context, chromatin‐modifying enzymes, which regulate memory gene expression changes, deserve major attention. Therefore, we analyzed the expression of chromatin‐modifying enzymes and recovery potential of enzyme modulators in scopolamine‐induced amnesia. Scopolamine administration drastically up‐regulated DNA methyltransferases (DNMT1) and HDAC2 expression while CREB‐binding protein (CBP), DNMT3a and DNMT3b remained unaffected. HDAC inhibitor sodium butyrate and DNMT inhibitor Aza‐2′deoxycytidine recovered scopolamine‐impaired hippocampal‐dependent memory consolidation with concomitant increase in the expression of synaptic plasticity genes Brain‐derived neurotrophic factor (BDNF) and Arc and level of histone H3K9 and H3K14 acetylation and decrease in DNA methylation level. Sodium butyrate showed more pronounced effect than Aza‐2′deoxycytidine and their co‐administration did not exhibit synergistic effect on gene expression. Taken together, we showed for the first time that scopolamine‐induced up‐regulation of chromatin‐modifying enzymes, HDAC2 and DNMT1, leads to gene expression changes and consequent decline in memory consolidation. Our findings on the action of scopolamine as an epigenetic modulator can pave a path for ideal therapeutic targets.

Neuropsin Expression Correlates with Dendritic Marker MAP2c Level in Different Brain Regions of Aging Mice

Neuropsin (NP) is a serine protease, implicated in synaptic plasticity and memory acquisition through cleavage of synaptic adhesion molecule, L1CAM. However, NP has not been explored during brain aging that entails drastic deterioration of plasticity and memory with selective regional vulnerability. Therefore, we have analysed the expression of NP and correlated with its function via analysis of endogenous cleavage of L1CAM and level of dendritic marker MAP2c in different regions of the aging mouse brain. While NP expression gradually decreased in the cerebral cortex during aging, it showed a sharp rise in both olfactory bulb and hippocampus in adult and thereafter declined in old age. NP expression was moderate in young medulla, but undetectable in midbrain and cerebellum. It was positively correlated with L1CAM cleavage and MAP2c level in different brain regions during aging. Taken together, our study shows age-dependent regional variation in NP expression and its positive correlation with MAP2c level, suggesting the involvement of NP in MAP2c mediated alterations in dendritic morphology during aging.

Bacopa monniera (CDRI-08) Upregulates the Expression of Neuronal and Glial Plasticity Markers in the Brain of Scopolamine Induced Amnesic Mice.

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.

Brain Aging: A Critical Reappraisal

Despite remarkable scientific advancements in recent years, much about the human brain still remains a mystery, particularly in the context of brain aging and associated disorders. Understanding of the factors that influence brain integrity late in life will help to maintain healthy brain functions. This is indeed a difficult task because in several cases, normal brain aging switches to pathological aging associated with drastic deterioration in cognitive abilities, motor skills and mood resulting in neurological diseases, ranging from late onset neurodegenerative diseases such as Alzheimer’s and Parkinson’s to early onset neuropsychiatric disorders such as schizophrenia and bipolar disorder. Brain aging is accompanied by several anatomical, cellular and molecular alterations including reduction in brain volume, protein turnover, increase in protein aggregation, impairment in neural plasticity, perturbed calcium homeostasis, neuronal survival and neuroinflammation, eventually affecting brain functions with increased incidence of neurological disorders. Factors like stress, depression, hypertension as well as obesity accelerate the aging of brain contributing to neurodegeneration and associated cognitive deficits. During the past decades, technical advances including microarray, neuroimaging and behavioral paradigms have helped to get a holistic picture of age-associated alterations in the brain. Here we review the current understanding of age-related structural and functional changes in the brain and how these changes might contribute to vulnerability for developing age-related neurological diseases and designing potential therapeutic avenues. Taken together, the findings suggest that adoption of certain neuroprotective strategies like dietary restriction, antioxidant supplementation, low alcohol intake, less exposure to stressors, environmental enrichment and lifestyle modulations involving exercise and intellectual brain training programs can be beneficial to delay the loss of brain integrity during aging.

Recovery of Age-Related Memory Loss: Hopes and Challenges

Advancing age is associated with drastic decline in memory and is a predisposing factor for neurodegenerative and neuropsychiatric disorders. Such decline results from aging of the brain involving loss of morphological integrity, alterations at the level of genes, enzymes and hormones, metabolism, oxidative stress, protein processing and synaptic function. Multiple biological scales ranging from genes to neural network and behavior and the individual variability that span age associated memory loss have added complexity to the recovery strategies. However, recent advancement in neuroscience research has not only removed the myth of unrecoverable memory loss during aging but also proposed a multitude of recovery approaches. These approaches include herbal interventions, dietary restrictions, antioxidant supplementation, environmental enrichment, lifestyle modulation and molecular targeting. Our laboratory is particularly interested in unraveling the molecular mechanism of age related memory loss and delineate therapeutic targets. Studies on animal models and humans reveal drastic changes in the expression and function of a wide array of molecules including chromatin modifying enzymes, immediate early genes, neurotrophins, presynaptic and postsynaptic proteins and neurite growth markers in vulnerable brain regions of cerebral cortex and hippocampus during aging. Such molecular changes are well translated into behavioral paradigms of memory impairment. In this chapter, we review age associated changes in brain, mechanisms of memory loss and recovery strategies. Essentially, we highlight the molecular correlates of brain aging and their potential as therapeutic targets for age associated memory loss.

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.

A cardinal sin when researching neuropsin/KLK8: Thou shalt validate antibodies

We read with great interest the recent article by Herring et al. [1], who presented a highly detailed explication of potential roles for kallikrein 8 (KLK8) in Alzheimer’s disease (AD); their work highlights how KLK8 inhibition attenuates AD pathology in mice [1]. They illustrated increased KLK8 protein in AD brain, progressing by Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) stage. But increase in KLK8 in TgCRND8 (APPSWE/IND) mice suggests a role for increased amyloidbeta (Ab) protein precursor (APP) levels, increased Ab levels, and/or disrupted Ab42/40 ratios in altering KLK8 levels. What is not clear is the sequence of the biochemical cascade. Did excess APP levels cause increase in KLK8 or vice versa? They also demonstrated behavioral and neuropathologic benefits for blockading KLK8 in situ in the transgenic mice, including reduction in Ab plaque load, t hyperphosphorylation, and changes in APP processing. Likewise, KLK8 blockade produced memory deficits in wild-type mice. The mechanism they proposed included both KLK8 activity on ephrin receptor B2 (EPHB2) and several other KLK8 substrates. KLK8 increase has long been known in the study of AD [2]. However, they did not show effects of KLK8 on other early memoryrelevant gene products, such as cyclic adenine monophosphate response element binding protein (CREB) activity. How KLK8 relates to cholinergic pathways is also not discussed. Cholinergic dysfunction is well known to associate with AD, and in particular, muscarinic acetylcholine receptor (mAChR) agonists are potential drug candidates for treatment of AD [3]. Unfortunately, the matters were not as nicely tied up as one might wish. The report fails to sufficiently indicate the nature of immunoreactive bands detected by Western immunoblotting techniques, and their Western results form the basis of interpretation of their whole work. This is related to a problem that is pandemic in molecular biology (including protein) research validation. Notably, Herring et al. did not