Medha Plus - A novel polyherbal formulation ameliorates cognitive behaviors and disease pathology in models of Alzheimer's disease.

Alzheimer's disease (AD) is a multi-faceted neurodegenerative disorder that leads to drastic cognitive impairments culminating in death. Pathologically, it is characterized by amyloid-ß (Aß) plaques, neurofibrillary tangles and neurodegeneration in brain. Complete cure of AD remains elusive to date. Available synthetic drugs only provide symptomatic reliefs targeting single molecule, hence, are unable to address the multi-factorial aspects in AD pathogenesis. It is imperative to develop combinatorial drugs that address the multiple molecular targets in AD. We show a unique polyherbal formulation of Brahmi, Mandukaparni, Shankhpushpi, Yastimadhu, Kokilaksha and Shunthi called 'Medha Plus' (MP), conventionally used for improving memory and reducing anxiety, was able to ameliorate cognitive deficits and associated pathological hallmarks of AD. Viability assays revealed that MP prevented Aß-induced loss of neurites as well as neuronal apoptosis in cellular models. An array of behavioral studies showed that MP was able to recover AD-associated memory deficits in both Aß-injected rats and 5XFAD mice. Immunohistochemical studies further revealed that MP treatment reduced Aß depositshpi and decreased apoptotic cell death in the hippocampus. Enzymatic assays demonstrated anti-oxidative and anti-acetyl cholinesterase properties of MP especially in hippocampus of Aß-injected rats. An underlying improvement in synaptic plasticity was observed with MP treatment in 5XFAD mice along with an increased expression of phospho-Akt at serine 473 indicating a role of PI3K/Akt signaling in correcting these synaptic deficits. Thus, our strong experiment-driven approach shows that MP is an incredible combinatorial drug that targets multiple molecular targets with exemplary neuroprotective properties and is proposed for clinical trial.

Modulation of α-Synuclein Fibrillation by Ultrasmall and Biocompatible Gold Nanoclusters.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, the pathogenesis of which is closely linked to the misfolding and aggregation of the neuronal protein α-Synuclein (A-Syn). Numerous molecules that inhibit/modulate the pathogenic aggregation of A-Syn in an effort to tackle PD pathogenesis have been reported, but none so far have been successful in treating the disease at the clinic. One major reason for this is the poor blood-brain barrier (BBB) permeability of most of the molecules being used. Therefore, using BBB-permeable (and biocompatible) nanomaterials as fibrillation modulators is gaining importance. In the present work, we show how nontoxic and ultrasmall gold nanoclusters (AuNCs) can systematically modulate the pathogenic fibrillation of A-Syn in vitro, based on the chemical nature of their capping agents, using two reported easily synthesizable AuNCs as models. In addition, we detect the BBB permeability in mice of one of these AuNCs solely by making use of its intrinsic fluorescence. Thus, our work exemplifies how AuNCs can be potential therapeutics against PD; while also acting as fluorescent probes for their own BBB permeability.

P38K and JNK pathways are induced by amyloid-ß in astrocyte: Implication of MAPK pathways in astrogliosis in Alzheimer's disease.

Astrocyte activation is one of the crucial hallmarks of Alzheimer's disease (AD) along with amyloid-ß (Aß) plaques, neurofibrillary tangles and neuron death. Glial scar and factors secreted from activated astrocytes have important contribution on neuronal health in AD. In this study, we investigated the mechanisms of astrocyte activation both in in vitro and in vivo models of AD. In this regard, mitogen activated protein kinase (MAPK) signalling cascades that control several fundamental and stress related cellular events, has been implicated in astrocyte activation in various neurological diseases. We checked activation of different MAPKs by western blot and immunocytochemistry and found that both JNK and p38K, but not ERK pathways are activated in Aß-treated astrocytes in culture and in Aß-infused rat brain cortex. Next, to investigate the downstream consequences of these two MAPKs (JNK and p38K) in Aß-induced astrocyte activation, we individually blocked these pathways by specific inhibitors in presence and absence of Aß and checked Aß-induced cellular proliferation, morphological changes and glial fibrillary acidic protein (GFAP) upregulation. We found that activation of both JNK and p38K signalling cascades are involved in astrocyte proliferation evoked by Aß, whereas only p38K pathway is implicated in morphological changes and GFAP upregulation in astrocytes exposed to Aß. To further validate the implication of p38K pathway in Aß-induced astrocyte activation, we also observed that transcription factor ATF2, a downstream phosphorylation substrate of p38, is phosphorylated upon Aß treatment. Taken together, our study indicates that p38K and JNK pathways mediate astrocyte activation and both the pathways are involved in cellular proliferation but only p38K pathway contributes in morphological changes triggered by Aß.

Smokeless tobacco induces toxicity and apoptosis in neuronal cells: a mechanistic evaluation.

Smokeless tobacco (SLT) or chewing tobacco has been a highly addictive practice in India across ages, posing major threat to the systemic health and possibly neurodegeneration. Earlier studies showed components of SLT could be harmful to neuronal health. However, mechanism of SLT in neurodegeneration remained unexplored. This study investigated the detrimental role of SLT on differentiated neuronal cell lines, PC12 and SH-SY5Y by using graded doses of water soluble lyophilised SLT. Reduced cell viability, compromised mitochondrial structure and functions were observed when neuronal cell lines were treated with SLT (6 mg/mL) for 24 h. There was reduction of oxidative phosphorylation and aerobic glycolysis as determined by diminution of ATP production (2.5X) and basal respiration (1.9X). Mitochondrial membrane potential was dropped by 3.5 times. Bid, a pro-apoptotic Bcl-2 family protein, has imperative role in regulating mitochondrial outer membrane permeabilization and subsequent cytochrome c release leading to apoptosis. This article for the first time indicated the involvement of Bid in SLT mediated neurotoxicity and possibly neurodegeneration. SLT treatment enhanced expression of cleaved-Bid in time dependent manner. The involvement of Bid was further confirmed by using Bid specific shRNA which reversed the effects of SLT and conferred significant protection from apoptosis up to 72 h. Thus, our results clearly indicated that SLT induced neuronal cell death occurred via production of ROS, alteration of mitochondrial morphology, membrane potential and oxidative phosphorylation, inactivation of survival pathway and activation of apoptotic markers mediated by Bid. Therefore, Bid could be a potential future therapeutic target for SLT induced neurodegeneration.

Human placental laminin: Role in neuronal differentiation, cell adhesion and proliferation.

Human placental extract has wound healing potential. Immuno-blots revealed presence of laminin in placental extract (70 +/- 0.257 µg/ml; n=3). It was purified using immuno-affinity chromatography. SDS-PAGE and SEHPLC indicated a188 kDa protein with some small peptides. Since placental laminin existed in its truncated form, its roles in cellular migration, differentiation and wound healing were verified. Induction of cellular migration and motility in rat fibroblasts were enhanced by placental laminin as observed from scratch wound assay. Promotion of neuronal differentiation of PC12 cells by placental laminin was observed by phase contrast microscopy. Confocal images showed presence of laminin on the cell surface and along the axonal processes. Significant interaction between integrin receptors and laminin responsible for cellular differentiation was demonstrated from co-localization experiments. Union between integrin receptor and its synthetic antagonist revealed retarded pattern of neurite outgrowth in laminin treated cells. Animal model studies revealed faster wound healing in the presence of placental laminin. Induction of re-epithelialization and angiogenesis in wound area by cellular proliferation and adhesion were observed. The cytokine levels showed an initial rise and gradual fall over the duration of wound healing on application of the fragmented laminin. Thus, roles of placental laminin in neuronal differentiation and wound healing were indicated.

Destabilization of ß-amyloid aggregates by thrombin derived peptide: plausible role of thrombin in neuroprotection.

ß-amyloid (Aß) aggregates involved in Alzheimer's disease (AD) are resistant to proteases but could be destabilized by small peptides designed to target specific hydrophobic regions of Aß that take part in aggregate assembly. Since thrombin and AD are intricately connected, and elastase modulates thrombin activity, elastase-digested thrombin peptides were verified for intervention in the Aß-aggregation pathway. Intact or elastase-digested thrombin destabilized Aß fibril, as demonstrated by thioflavin T assay. Peptides were synthesized employing thrombin as a template, of which, a hexapeptide (T3) showed maximum destabilization at 1 µm. ExPASy peptide cutter software coupled with mass spectrometric analysis confirmed the generation of T3 peptide from elastase-digested thrombin. TEM micrographs revealed that 30-day incubation of preformed Aß fibrils or monomers with T3 resulted in destabilization or inhibition, respectively, leading mostly to particles of 1.74 ± 0.17 nm, which roughly corresponded to Aß monomer. Surface plasmon resonance employing CM5 chip coupled with Aß40 mouse monoclonal antibody showed a drop in response when T3 was incubated with Aß fibrils between 2 and 8 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and confocal microscopy demonstrated the ability of T3 to rescue neuroblastoma cells from Aß oligomer-induced cytotoxic damage. Although no [Aß-T3] adduct could be detected by mass spectrometry, an initial interaction appeared to facilitate the process of destabilization/inhibition of aggregation. T3 was comparable to standard ß-sheet breaker peptides, LPFFD and KLVFF in terms of Aß aggregate destabilization. High hydrophobicity values coupled with recognition and breaking elements make T3 a potential candidate for future therapeutic applications.

Multifunctional transcriptional coactivator PC4 is a global co-regulator of p53-dependent stress response and gene regulation.

Human positive coactivator 4 (PC4), a multifunctional chromatin-associated protein, is known to directly interact with p53 and modulate expressions of a few p53-dependent genes. However, the role of PC4 in p53's myriad of other regulatory functions is not known. The p53-PC4 interaction was selectively perturbed by a small peptide which led to abrogation of genotoxic stress-induced up-regulation of many p53-dependent genes and reduction of apoptosis in A549 cells. Over-expression of a PC4 point mutant, incapable of binding p53, recapitulated many of the effects of the peptide. Global gene expression profiling in A549 cells, upon peptide treatment, revealed PC4's involvement in the regulation of many p53-dependent pathways, including the Hippo pathway. Introduction of the peptide in neuronal cells significantly reduced its amyloid-ß-induced death. Thus, PC4 emerges as a global co-regulator of p53 and a therapeutic target against pathogeneses where the p53-dependent cell death process plays a crucial role.

RNAi-Mediated Silencing of FOXO Factors.

RNAi-mediated silencing of a particular gene is an important tool in the field of biology. Knocking down experiments has revealed the role of different proteins and their downstream targets. Here, we describe the procedure of silencing transcription factor of Forkhead box class "O" (FOXO) using shRNA.

The pro-apoptotic protein Bmf co-operates with Bim and Puma in neuron death induced by ß-amyloid or NGF deprivation.

The pro-apoptotic Bcl-2 homology 3 domain only (BH3-only) proteins are central regulators of cell death in various physiological and pathological conditions, including Alzheimer's disease (AD). Bcl-2 modifying factor (Bmf) is one such BH3-only protein that is implicated in various death paradigms such as anoikis, seizures, cancer and autoimmunity. It also co-operates with other BH3-only proteins such as Bim in various death paradigms. However, its role in neurodegeneration is under-investigated. Here, we report for the first time the essential role of Bmf and its co-operativity with direct activator BH3-only proteins Bim and Puma in neuron death induced by beta-amyloid (Aß) toxicity or NGF deprivation. Oligomeric Aß is main pathologic species in AD and NGF deprivation is relevant for both developmental as well as pathologic neuron death. We find that Bmf over-expression causes cell death and Bmf knockdown protects neurons against death evoked by Aß or NGF deprivation. We also find that Bmf co-operates with other important BH3-only proteins such as Bim and Puma in neuron death induced by Aß or NGF deprivation. Simultaneous knocking down of these molecules by their respective shRNAs provide enhanced protection against Aß. Taken together, our results elucidate the essential role of Bmf and its co-operative effects with already known neuron death inducers, Bim and Puma, in neuron death evoked by Aß treatment or NGF deprivation.

Primary pelvic hydatid cyst in a postmenopausal female: a surgical challenge.

Primary pelvic hydatid cysts are a rare entity and are often overlooked as a differential diagnosis of a pelvic-space-occupying lesion particularly in non-endemic regions. Unpreparedness and a hasty decision on the surgical approach may end in life-threatening complications and systemic dissemination of the disease. We report the case of a 55-year-old postmenopausal woman with a history of two previous unsuccessful surgeries to remove pelvic cystic lesions due to dense adhesions between the surrounding gut wall, bladder, and the cyst wall. Clinical and imaging findings failed to diagnose the nature of the cysts, and a laparotomy was contemplated. On the third surgical attempt, the clinical suspicion was considered and by meticulous dissection the cysts were removed thoroughly without undue complications. In the postoperative follow-up period there was no sign of disease recurrence or dissemination.

Primary pelvic hydatid cyst in a postmenopausal female: a surgical challenge

Primary pelvic hydatid cysts are a rare entity and are often overlooked as a differential diagnosis of a pelvic-space-occupying lesion particularly in non-endemic regions. Unpreparedness and a hasty decision on the surgical approach may end in life-threatening complications and systemic dissemination of the disease. We report the case of a 55-year-old postmenopausal woman with a history of two previous unsuccessful surgeries to remove pelvic cystic lesions due to dense adhesions between the surrounding gut wall, bladder, and the cyst wall. Clinical and imaging findings failed to diagnose the nature of the cysts, and a laparotomy was contemplated. On the third surgical attempt, the clinical suspicion was considered and by meticulous dissection the cysts were removed thoroughly without undue complications. In the postoperative follow-up period there was no sign of disease recurrence or dissemination.

Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-ß-induced Neuronal Death.

Amyloid-ß (Aß)-induced neuron death is considered central to the pathogenesis of Alzheimer's disease (AD). Among several death modalities, autophagy and apoptosis play important roles in Aß-induced neuron death suggesting that there may be regulatory mechanisms that initiate both cell death pathways. However, molecules that govern both pathways have not been identified. Here, we report that, upon Aß treatment, tribbles pseudokinase 3 (Trib3, an ortholog of Drosophila Tribbles) is up-regulated in neurons both in vivo and in vitro Increased Trib3 levels inhibited the activity of the kinase Akt by interacting with it. As a result, forkhead box O1 (FoxO1), a transcription factor that is negatively regulated by Akt, was activated, translocated to the nucleus, and induced the pro-apoptotic gene BCL2-like 11 (Bim). Conversely, FoxO1 responded to Aß insult by binding to the Trib3 gene promoter, enhancing its expression. Our investigations further revealed that Trib3 also induces autophagy. We found that Trib3 indirectly activates unc-51-like autophagy-activating kinase1 (Ulk1) by impeding phosphorylation of, and thus inactivating, a negative regulator of Ulk1, mechanistic target of rapamycin. Ulk1 activation augmented autophagosome formation and reduced autophagy flux. Thus, Trib3 was required for formation of autophagosomes, which accumulated in neurons as autophagic flux was thwarted. Most importantly, silencing endogenous Trib3 strongly protected neurons from Aß insult. Our results suggest that a self-amplifying feed-forward loop among Trib3, Akt, and FoxO1 in Aß-treated neurons induces both apoptosis and autophagy, culminating in neuron death. Thus, Trib3 may serve as a potential therapeutic target for AD.

Amyloid-ß induced astrocytosis and astrocyte death: Implication of FoxO3a-Bim-caspase3 death signaling.

Astrocytes, the main element of the homeostatic system in the brain, are affected in various neurological conditions including Alzheimer's disease (AD). A common astrocytic reaction in pathological state is known as astrocytosis which is characterized by a specific change in astrocyte shape due to cytoskeletal remodeling, cytokine secretion and cellular proliferation. Astrocytes also undergo apoptosis in various neurological conditions or in response to toxic insults. AD is pathologically characterized by progressive deposition of amyloid-ß (Aß) in senile plaques, intraneuronal neurofibrillary tangles, synaptic dysfunction and neuron death. Astrocytosis and astrocyte death have been reported in AD brain as well as in response to Aß in vitro. However, how astrocytes undergo both proliferation and death in response to Aß remains elusive. In this study, we used primary cultures of cortical astrocytes and exposed them to various doses of oligomeric Aß. We found that cultured astrocytes proliferate and manifest all signs of astrocytosis at a low dose of Aß. However, at high dose of Aß the activated astrocytes undergo apoptosis. Astrocytosis was also noticed in vivo in response to Aß in the rat brain. Next, we investigated the mechanism of astrocyte apoptosis in response to a high dose of Aß. We found that death of astrocyte induced by Aß requires a set of molecules that are instrumental for neuron death in response to Aß. It involves activation of Forkhead transcription factor Foxo3a, induction of its pro-apoptotic target Bim and activation of its downstream molecule, caspase3. Hence, this study demonstrates that the concentration of Aß decides whether astrocytes do proliferate or undergo apoptosis via a mechanism that is required for neuron death.

The regulation of p53 up-regulated modulator of apoptosis by JNK/c-Jun pathway in ß-amyloid-induced neuron death.

Neuronal loss in selective areas of brain underlies the pathology of Alzheimer's disease (AD). Recent evidences place oligomeric ß-amyloid (Aß) central to the disease. However, mechanism of neuron death in response to Aß remains elusive. Activation of the c-Jun N-terminal kinase (JNK) pathway and induction of the AP-1 transcription factor c-Jun are reported in AD. However, targets of JNK/c-Jun in Aß-induced neuron death are mostly unknown. Our study shows that pro-apoptotic proteins, Bim (Bcl-2 interacting mediator of cell death) and Puma (p53 up-regulated modulator of apoptosis) are targets of c-Jun in Aß-treated neurons. We demonstrate that the JNK/c-Jun pathway is activated, in cultures of cortical neurons following treatment with oligomeric Aß and in AD transgenic mice, and that inhibition of this pathway by selective inhibitor blocks induction of Puma by Aß. We also find that both JNK and p53 pathways co-operatively regulate Puma expression in Aß-treated neurons. Moreover, we identified a novel AP1-binding site on rat puma gene which is necessary for direct binding of c-Jun with Puma promoter. Finally, we find that knocking down of c-Jun by siRNA provides significant protection from Aß toxicity and that induction of Bim and Puma by Aß in neurons requires c-Jun. Taken together, our results suggest that both Bim and Puma are target of c-Jun and elucidate the intricate regulation of Puma expression by JNK/c-Jun and p53 pathways in neurons upon Aß toxicity. JNK/c-Jun pathway is shown to be activated in neurons of the Alzheimer's disease (AD) brain and plays a vital role in neuron death in AD models. However, downstream targets of c-Jun in this disease have not been thoroughly elucidated. Our study shows that two important pro-apoptotic proteins, Bim (Bcl-2 interacting mediator of cell death) and Puma (p53 up-regulated modulator of apoptosis) are targets of c-Jun in Aß-treated neurons. We demonstrate that the JNK/c-jun pathway is activated, in cultures of cortical neurons following treatment with oligomeric Aß and in AD transgenic mice, and that inhibition of this pathway by selective inhibitor blocks induction of Puma by Aß. We have also observed functional co-operation of both JNK and p53 pathway in regulation of Puma under Aß toxicity. Most importantly, we identified a novel AP1-binding site on rat puma gene which is necessary for direct binding of c-Jun with Puma promoter. Thus, our results suggest that both Bim and Puma are target of c-Jun and elucidate the intricate regulation of Puma expression by JNK/c-Jun and p53 pathways in neurons upon Aß toxicity.

The essential role of p53-up-regulated modulator of apoptosis (Puma) and its regulation by FoxO3a transcription factor in ß-amyloid-induced neuron death.

Neurodegeneration underlies the pathology of Alzheimer disease (AD). The molecules responsible for such neurodegeneration in AD brain are mostly unknown. Recent findings indicate that the BH3-only proteins of the Bcl-2 family play an essential role in various cell death paradigms, including neurodegeneration. Here we report that Puma (p53-up-regulated modulator of apoptosis), an important member of the BH3-only protein family, is up-regulated in neurons upon toxic ß-amyloid 1-42 (Aß(1-42)) exposure both in vitro and in vivo. Down-regulation of Puma by specific siRNA provides significant protection against neuron death induced by Aß(1-42). We further demonstrate that the activation of p53 and inhibition of PI3K/Akt pathways induce Puma. The transcription factor FoxO3a, which is activated when PI3K/Akt signaling is inhibited, directly binds with the Puma gene and induces its expression upon exposure of neurons to oligomeric Aß(1-42). Moreover, Puma cooperates with another BH3-only protein, Bim, which is already implicated in AD. Our results thus suggest that Puma is activated by both p53 and PI3K/Akt/FoxO3a pathways and cooperates with Bim to induce neuron death in response to Aß(1-42).

Efficacy of cyclin dependent kinase 4 inhibitors as potent neuroprotective agents against insults relevant to Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with no cure till today. Aberrant activation of cell cycle regulatory proteins is implicated in neurodegenerative diseases including AD. We and others have shown that Cyclin dependent kinase 4 (Cdk4) is activated in AD brain and is required for neuron death. In this study, we tested the efficiency of commercially available Cdk4 specific inhibitors as well as a small library of synthetic molecule inhibitors targeting Cdk4 as neuroprotective agents in cellular models of neuron death. We found that several of these inhibitors significantly protected neuronal cells against death induced by nerve growth factor (NGF) deprivation and oligomeric beta amyloid (Aß) that are implicated in AD. These neuroprotective agents inhibit specifically Cdk4 kinase activity, loss of mitochondrial integrity, induction of pro-apoptotic protein Bim and caspase3 activation in response to NGF deprivation. The efficacies of commercial and synthesized inhibitors are comparable. The synthesized molecules are either phenanthrene based or naphthalene based and they are synthesized by using Pschorr reaction and Buchwald coupling respectively as one of the key steps. A number of molecules of both kinds block neurodegeneration effectively. Therefore, we propose that Cdk4 inhibition would be a therapeutic choice for ameliorating neurodegeneration in AD and these synthetic Cdk4 inhibitors could lead to development of effective drugs for AD.