Subtle genomic DNA damage induces intraneuronal production of amyloid-ß (1-42) by increasing ß-secretase activity.

Aberrant accumulation of amyloid-ß (Aß) in brain is the major trigger for pathogenesis in Alzheimer's disease (AD). It is imperative to understand how Aß attains such toxic levels in the brain parenchyma. We detected that a subtle and tolerable amount of DNA damage, related to aging, increased intraneuronal Aß1-42 production both in cultured neuron and in cortex of rodent brain. Strikingly, we also observed elevated levels of mitochondrial fusion and of its major driver protein, MFN2. Hyperfusion of mitochondria may be seen as an adaptive stress response resulting from the induction of ER stress since we detected the activation of both PERK and IRE1α arms of unfolded protein response of ER stress. We found increased phosphorylation of PERK substrate eukaryotic initiation factor 2 α (eIF2α), and upregulation of the downstream effector proteins, ATF4 and CHOP. Concomitantly, increased XBP1 level, the direct effecter protein of IRE-1α, was observed. Reports suggest that eIF2α phosphorylation can increase BACE1 activity, the rate limiting enzyme in Aß production. Here, we show that inhibiting PERK, decreased Aß1-42 level while direct BACE1 inhibition, reduced the mitochondrial fusion. We found increased MFN2 expression in young 5xFAD mice when Aß plaques and neurodegeneration were absent. Thus, our study indicates that mild DNA damage leads to increased Aß1-42 production almost as a consequence of an initial ER stress-directed protective mitochondrial fusion in brain. We propose that an age-related subtle genomic DNA damage may trigger enhanced intraneuronal Aß1-42 production in an apparently healthy neuron way before the appearance of clinical symptoms in AD.

ICAM-1 protects neurons against Amyloid-ß and improves cognitive behaviors in 5xFAD mice by inhibiting NF-κB.

Alzheimer's disease (AD) is mainly characterized by amyloid beta (Aß) plaque deposition and neurofibrillary tangle formation due to tau hyperphosphorylation. It has been shown that astrocytes respond to these pathologies very early and exert either beneficial or deleterious effects towards neurons. Here, we identified soluble intercellular adhesion molecule-1 (ICAM-1) which is rapidly increased in astrocyte conditioned medium derived from Aß1-42 treated cultured astrocytes (Aß1-42-ACM). Aß1-42-ACM was found to be neuroprotective, however, Aß1-42-ACM deprived of ICAM-1 was unable to protect neurons against Aß1-42 mediated toxicity. Moreover, exogenous ICAM-1 renders protection to neurons from Aß1-42 induced death. It blocks Aß1-42-mediated PARP cleavage and increases the levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL, and decreases pro-apoptotic protein Bim. In an Aß-infused rat model of AD and in 5xFAD mouse, intra-peritoneal administration of ICAM-1 revealed a reduction in Aß load in hippocampal and cortical regions. Moreover, ICAM-1 treatment led to an increment in the expression of the Aß-degrading enzyme, neprilysin in 5xFAD mice. Finally, we found that ICAM-1 can ameliorate cognitive deficits in Aß-infused rat and 5xFAD mouse. Interestingly, ICAM-1 could block the NF-κB upregulation by Aß and inhibition of NF-κB recovers cognitive impairments in 5xFAD mice. Thus, our study finds a neuroprotective role of ICAM-1 and suggests that it can be a major candidate in cytokine-mediated therapy of AD.

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.

Forkhead Box O3a requires BAF57, a subunit of chromatin remodeler SWI/SNF complex for induction of p53 up-regulated modulator of apoptosis (Puma) in a model of Parkinson's disease.

Parkinson's disease (PD) results from the selective loss of dopaminergic neurons of substantia nigra pars compacta region of the midbrain. It has been reported that the transcription factor forkhead Box O3a (FoxO3a) is activated and induces pro-apoptotic protein such as Bcl-2-interacting mediator of cell death (BIM) and p53 up-regulated modulator of apoptosis (PUMA) in variety of neuron death paradigms. Activity of FoxO3a is governed by its post-translational modifications which control its subcellular localization. Aim of this study was to determine whether FoxO3a is activated and up-regulates its pro-apoptotic genes to induce neuron death in PD. We exposed neuronal PC12 cells or primary cultures of dopaminergic neurons to 6-hydroxy dopamine (6-OHDA) and infused 6-OHDA in rat brain to develop PD models. We found that FoxO3a undergoes multiple post-translational modifications which render its nuclear localization in dopaminergic neuronal cells in response to 6-OHDA. The nuclear redistribution of FoxO3a is significantly increased in dopaminergic neurons of 6-OHDA infused rat brains as well. Moreover, FoxO3a is required for dopaminergic neurodegeneration in response to 6-OHDA as RNAi-mediated silencing of FoxO3a protects these cells from 6-OHDA toxicity. In a search of the downstream targets we identified PUMA as a direct target of FoxO3a. By knocking down FoxO3a we could successfully block the up-regulation of the pro-apoptotic protein PUMA in this model. Recently, it has been reported that chromatin remodeler SWItch/sucrose non-fermentable binds to FOXO and activates transcription. We found that Brg-associated factor 57 (BAF57), a subclass of SWItch/sucrose non-fermentable is up-regulated and play a necessary role in neuron death induced by 6-OHDA. Moreover, it is required for induction of PUMA by FoxO3a in this cellular model of PD. Taken together, our study suggest that FoxO3a is activated, translocates to nucleus, induces its pro-apoptotic target PUMA in the presence of chromatin remodeler BAF57 to execute neuron death in cellular models of PD.

TIMP-1: A key cytokine released from activated astrocytes protects neurons and ameliorates cognitive behaviours in a rodent model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by two pathologic species, extracellular amyloid-ß (Aß) plaques and intracellular neurofibrillary tangles. Astrocytes that maintain normal homeostasis in the brain undergo a set of molecular, cellular and functional changes called reactive astrogliosis in various neurological diseases including AD. It is hypothesized that reactive astrocytes initially tend to protect neurons by reducing Aß load and by secreting a plethora of cytokines, however, their functions have only been poorly investigated. Our studies on the kinetics of activation of cortical astrocytes following Aß-exposure revealed significant level of activation as early as in 6 h. The astrocyte conditioned medium (ACM) from 6 h Aß-treated astrocytes (Aß-ACM) provided significant neuroprotection of cultured cortical neurons against Aß insults. Analysis of the secreted proteins in Aß-ACM revealed a marked increase of Tissue inhibitor of Metalloproteinase-1 (TIMP-1) within 6 h. Interestingly, we found that neutralization of TIMP-1 with antibody or knockdown with siRNA in astrocytes abolished most of the neuroprotective ability of the 6 h Aß-ACM on Aß-treated cultured neurons. Furthermore addition of exogenous rat recombinant TIMP-1 protein protects primary neurons from Aß mediated toxicity. In a well characterized Aß-infused rodent model of AD, intra-cerebroventricular administration of TIMP-1 revealed a reduction in Aß load and apoptosis in hippocampal and cortical regions. Finally, we found that TIMP-1 can ameliorate Aß-induced cognitive dysfunctions through restoration of Akt and its downstream pathway and maintenance of synaptic integrity. Thus, our results not only provide a functional clarity for TIMP-1, secreted by activated astrocytes, but also support it as a major candidate in cytokine-mediated therapy of AD especially at the early phase of disease progression.

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.

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 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).

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.

A study of the expression and localization of toll-like receptors 2 and 9 in different grades of cervical intraepithelial neoplasia and squamous cell carcinoma.

TLRs are important molecules of innate immune response, those play central role in host pathogen interaction and recognition through pathogen associated molecular patterns (PAMPs). Previous studies have indicated the role of TLRs in many human malignancy and cervical cancer in terms of viral recognition and inflammatory changes in-vivo. The objective of this study was to evaluate the expression and localization of toll-like receptor (TLR) 2 and TLR9 in preinvasive and invasive cervical cancer patients and to investigate its use as a probable diagnostic tool for better management cervical cancer. This single institution study includes individuals with normal, precancerous lesions, cervical intraepithelial neoplastic (CIN) and invasive squamous cell carcinoma (SCC) of the cervix. Upon confirmation by histopathology, fluorescence based immunohistochemistry was performed in all patients for TLR2 and TLR9, followed by semi-quantitative estimation of the staining intensity and grade of expression. The expression pattern of TLR2 and TLR9 does not vary greatly from normal to precancerous lesions, but a significant variation was observed in advance stages, i.e. squamous cell carcinoma of the uterine cervix. Additionally the expression increased marginally in higher grades. In spite of their low difference in expression along different stages of cervical cancer, both TLR2 and TLR9 could detect the disease at an advance stages as depicted by the receiver operator characteristics curve analysis.

Small-Molecule Cdc25A Inhibitors Protect Neuronal Cells from Death Evoked by NGF Deprivation and 6-Hydroxydopamine.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that are presently incurable. There have been reports of aberrant activation of cell cycle pathways in neurodegenerative diseases. Previously, we have found that Cdc25A is activated in models of neurodegenerative diseases, including AD and PD. In the present study, we have synthesized a small library of molecules targeting Cdc25A and tested their neuroprotective potential in cellular models of neurodegeneration. The Buchwald reaction and amide coupling were crucial steps in synthesizing the Cdc25A-targeting molecules. Several of these small-molecule inhibitors significantly prevented neuronal cell death induced by nerve growth factor (NGF) deprivation as well as 6-hydroxydopamine (6-OHDA) treatment. Lack of NGF signaling leads to neuron death during development and has been associated with AD pathogenesis. The NGF receptor TrkA has been reported to be downregulated at the early stages of AD, and its reduction is linked to cognitive failure. 6-OHDA, a PD mimic, is a highly oxidizable dopamine analogue that can be taken up by the dopamine transporters in catecholaminergic neurons and can induce cell death by reactive oxygen species (ROS) generation. Some of our newly synthesized molecules inhibit Cdc25A phosphatase activity, block loss of mitochondrial activity, and inhibit caspase-3 activation caused by NGF deprivation and 6-OHDA. Hence, it may be proposed that Cdc25A inhibition could be a therapeutic possibility for neurodegenerative diseases and these Cdc25A inhibitors could be effective treatments for AD and PD.

Single point mutations at the S129 residue of α-synuclein and their effect on structure, aggregation, and neurotoxicity.

Parkinson's disease is an age-related neurological disorder, and the pathology of the disease is linked to different types of aggregates of α-synuclein or alpha-synuclein (aS), which is an intrinsically disordered protein. The C-terminal domain (residues 96-140) of the protein is highly fluctuating and possesses random/disordered coil conformation. Thus, the region plays a significant role in the protein's solubility and stability by an interaction with other parts of the protein. In the current investigation, we examined the structure and aggregation behavior of two artificial single point mutations at a C-terminal residue at position 129 that represent a serine residue in the wild-type human aS (wt aS). Circular Dichroism (CD) and Raman spectroscopy were performed to analyse the secondary structure of the mutated proteins and compare it to the wt aS. Thioflavin T assay and atomic force microscopy imaging helped in understanding the aggregation kinetics and type of aggregates formed. Finally, the cytotoxicity assay gave an idea about the toxicity of the aggregates formed at different stages of incubation due to mutations. Compared to wt aS, the mutants S129A and S129W imparted structural stability and showed enhanced propensity toward the α-helical secondary structure. CD analysis showed proclivity of the mutant proteins toward α-helical conformation. The enhancement of α-helical propensity lengthened the lag phase of fibril formation. The growth rate of ß-sheet-rich fibrillation was also reduced. Cytotoxicity tests on SH-SY5Y neuronal cell lines established that the S129A and S129W mutants and their aggregates were potentially less toxic than wt aS. The average survivability rate was ∼40% for cells treated with oligomers (presumably formed after 24 h of incubation of the freshly prepared monomeric protein solution) produced from wt aS and ∼80% for cells treated with oligomers obtained from mutant proteins. The relative structural stability with α-helical propensity of the mutants could be a plausible reason for their slow rate of oligomerization and fibrillation, and this was also the possible reason for reduced toxicity to neuronal cells.

Brain-enriched miR-128: Reduced in exosomes from Parkinson's patient plasma, improves synaptic integrity, and prevents 6-OHDA mediated neuronal apoptosis.

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with the death of mid-brain dopaminergic neurons. Unfortunately, no effective cure or diagnostic biomarkers for PD are available yet. To address this, the present study focuses on brain-enriched small non-coding regulatory RNAs called microRNAs (miRNAs) that are released into the circulation packaged inside small extracellular vesicles called exosomes. We collected blood samples from PD patients and isolated exosomes from the plasma. qPCR-based detection revealed a particular neuron-enriched miR-128 to be significantly decreased in the patient-derived exosomes. Interestingly, a concomitant decreased expression of miR-128 was observed in the cellular models of PD. Fluorescent live cell imaging and flow-cytometry revealed that over-expression of miR-128 can prevent 6-OHDA-mediated mitochondrial superoxide production and induction of neuronal death respectively. This neuroprotective effect was found to be induced by miR-128-mediated inhibition of FoxO3a activation, a transcription factor involved in apoptosis. miR-128 over-expression also resulted in down-regulation of pro-apoptotic FoxO3a targets- FasL and PUMA, at both transcript and protein levels. Further downstream, miR-128 over-expression inhibited activation of caspases-8, -9 and -3, preventing both the intrinsic and extrinsic pathways of apoptosis. Additionally, over expression of miR-128 prevented down-regulation of synaptic proteins- Synaptophysin and PSD-95 and attenuated neurite shortening, thereby maintaining overall neuronal integrity. Thus, our study depicts the intracellular role of miR-128 in neuronal apoptosis and neurodegeneration and its implications as a biomarker being detectable in the circulating exosomes of PD patient blood. Thus, characterization of such exosomal brain-enriched miRNAs hold promise for effective detection and diagnosis of PD.

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.

Predictors and Pathway of Maternal Near Miss: A Case-Control Study in a Tertiary Care Facility in Kolkata.

Background: Use of maternal near-miss (MNM) cases as an adjunct has been advocated to understand the processes of obstetric care because they share similar pathways as maternal deaths. Identifying the predictors and care pathway is crucial to improve the quality of care and end preventable maternal deaths. Materials and Methods: This case-control study was conducted at a tertiary care facility in Kolkata from May 2019 to March 2020. Women admitted with complications during pregnancy, childbirth, or within 42 days of postpartum, who met the World Health Organization (WHO) near-miss criteria, were identified as cases, and equivalent age-group matched controls were recruited. Sample size of 60 cases and 60 controls was estimated, assuming a power of 80%, level of significance 0.05, and case-control ratio of 1. After obtaining approval from the institutional ethics committee and informed written consent from the participants, data was collected through face-to-face interview and review of records. Statistical analysis including care pathway analysis (using three-delay model) was performed using Statistical Package for Social Sciences version 16. Results: Joint family type (adjusted odds ratio [AOR] [CI] = 5.06 [1.48, 7.28]), lack of antenatal checkups (AOR [CI] = 7.85 [1.47, 12.09]), previous history of cesarean section (AOR [CI] = 3.94 [1.09, 14.33]), first delay in seeking care (AOR [CI] = 13.84 [3.62, 32.83]), and preexisting medical disorders (AOR [CI] = 11.03 [4.62, 22.80]) were identified as significant predictors of MNM in the adjusted model. Significant difference in the proportion of first and second delays in the care pathway was observed between cases and controls. Conclusions: Identification of risk factors of MNM and pattern of delays in the care pathway will help improving quality of obstetric care.

Sertad1 plays an essential role in developmental and pathological neuron death.

Developmental and pathological death of neurons requires activation of a defined pathway of cell cycle proteins. However, it is unclear how this pathway is regulated and whether it is relevant in vivo. A screen for transcripts robustly induced in cultured neurons by DNA damage identified Sertad1, a Cdk4 (cyclin-dependent kinase 4) activator. Sertad1 is also induced in neurons by nerve growth factor (NGF) deprivation and Abeta (beta-amyloid). RNA interference-mediated downregulation of Sertad1 protects neurons in all three death models. Studies of NGF withdrawal indicate that Sertad1 is required to initiate the apoptotic cell cycle pathway since its knockdown blocks subsequent pathway events. Finally, we find that Sertad1 expression is required for developmental neuronal death in the cerebral cortex. Sertad1 thus appears to be essential for neuron death in trophic support deprivation in vitro and in vivo and in models of DNA damage and Alzheimer's disease. It may therefore be a suitable target for therapeutic intervention.

Astrocyte subtype-specific approach to Alzheimer's disease treatment.

Astrocytes respond to any pathological condition in the central nervous system (CNS) including Alzheimer's disease (AD), and this response is called astrocyte reactivity. Astrocyte reaction to a CNS insult is a highly heterogeneous phenomenon in which the astrocytes undergo a set of morphological, molecular and functional changes with a characteristic secretome profile. Such astrocytes are termed as 'reactive astrocytes'. Controversies regarding the reactive astrocytes abound. Recently, a continuum of reactive astrocyte profiles with distinct transcriptional states has been identified. Among them, disease-associated astrocytes (DAA) were uniquely present in AD mice and expressed a signature set of genes implicated in complement cascade, endocytosis and aging. Earlier, two stimulus-specific reactive astrocyte subtypes with their unique transcriptomic signatures were identified using mouse models of neuroinflammation and ischemia and termed as A1 astrocytes (detrimental) and A2 astrocytes (beneficial) respectively. Interestingly, although most of the A1 signature genes were also detected in DAA, as opposed to A2 astrocyte signatures, some of the A1 specific genes were expressed in other astrocyte subtypes, indicating that these nomenclature-based signatures are not very specific. In this review, we elaborate the disparate functions and cytokine profiles of reactive astrocyte subtypes in AD and tried to distinguish them by designating neurotoxic astrocytes as A1-like and neuroprotective ones as A2-like without directly referring to the A1/A2 original nomenclature. We have also focused on the dual nature from a functional perspective of some cytokines depending on AD-stage, highlighting a number of them as major candidates in AD therapy. Therefore, we suggest that promoting subtype-specific beneficial roles, inhibiting subtype-specific detrimental roles or targeting subtype-specific cytokines constitute a novel therapeutic approach to AD treatment.

BH3-only proteins Puma and Beclin1 regulate autophagic death in neurons in response to Amyloid-ß.

Alzheimer's disease (AD) is characterized by accumulation of senile amyloid-ß (Aß) plaques and hyperphosphorylated tau tangles causing progressive loss of synapse and neuronal death. Out of the various neuron death modalities, autophagy and apoptosis are reported to be the major death paradigms in AD. However, how these two processes lead to neuronal loss is still inconspicuous. Here we report that under Aß toxicity, aberrant autophagy is induced with inefficient autophagic flux in neurons. Simultaneous activation of both autophagy and apoptosis are seen in primary cortical neurons as well as in transgenic mice brains. We found that induction of autophagy by rapamycin is detrimental for neurons; whereas downregulation of Beclin1, an important autophagy inducing protein, provides significant protection in Aß treated neuronal cells by blocking cytochrome-c release from the mitochondria. We further report that downregulation of Puma, a BH3-only pro-apoptotic protein, inhibits the induction of aberrant autophagy and also ameliorates the autophagy flux under the influence of Aß. Notably, stereotactic administration of shRNAs against Puma and Beclin1 in adult Aß-infused rat brains inhibits both apoptotic and autophagic pathways. The regulation of both of the death processes is brought about by the direct interaction between Puma and Beclin1 upon Aß treatment. We conclude that both Beclin1 and Puma play essential roles in the neuronal death caused by the induction of aberrant autophagy in AD and targeting their interaction could be vital to understand the crosstalk of autophagy and apoptosis as well as to develop a potential therapeutic strategy in AD.

Rheb-mTOR activation rescues Aß-induced cognitive impairment and memory function by restoring miR-146 activity in glial cells.

Deposition of amyloid beta plaques in adult rat or human brain is associated with increased production of proinflammatory cytokines by associated glial cells that are responsible for degeneration of the diseased tissue. The expression of these cytokines is usually under check and is controlled at the post-transcriptional level via several microRNAs. Computational analysis of gene expression profiles of cortical regions of Alzheimer's disease patients' brain suggests ineffective target cytokine mRNA suppression by existing micro-ribonucleoproteins (miRNPs) in diseased brain. Exploring the mechanism of amyloid beta-induced cytokine expression, we have identified how the inactivation of the repressive miR-146 miRNPs causes increased production of cytokines in amyloid beta-exposed glial cells. In exploration of the cause of miRNP inactivation, we have noted amyloid beta oligomer-induced sequestration of the mTORC1 complex to early endosomes that results in decreased Ago2 phosphorylation, limited Ago2-miRNA uncoupling, and retarded Ago2-cytokine mRNA interaction in rat astrocytes. Interestingly, constitutive activation of mTORC1 by Rheb activator restricts proinflammatory cytokine production by reactivating miR-146 miRNPs in amyloid beta-exposed glial cells to rescue the disease phenotype in the in vivo rat model of Alzheimer's disease.