Observed Improvement in Cognition During a Personalized Lifestyle Intervention in People with Cognitive Decline.

BACKGROUND: Alzheimer's disease (AD) is a chronic condition marked by progressive objective cognitive impairment (OCI). No monotherapy has substantially altered disease progression, suggesting the disease is multifactorial and may require a multimodal therapeutic approach. OBJECTIVE: We sought to determine if cognitive function in a sample with OCI would change in response to a multimodal, individualized care plan based on potential contributors to cognitive decline (e.g., nutritional status, infection, etc.). METHODS: Participants (n = 34) were recruited from the San Diego, CA area. The multimodal intervention included lifestyle changes (i.e., movement, diet, and stress management), nutraceutical support, and medications. It was delivered pragmatically over four clinical visits, and outcome measures were gathered at four study visits, occurring at baseline, one, three, and six months (primary endpoint). Study participants received weekly phone calls for nutrition support throughout study participation. Outcome measures included the Cambridge Brain Sciences (CBS) battery, and the Montreal Cognitive Assessment (MoCA). RESULTS: At 6 months, mean MoCA scores improved from 19.6±3.1 to 21.7±6.2 (p = 0.013). Significant improvement was observed in mean scores of the CBS memory domain [25.2 (SD 23.3) to 35.8 (SD 26.9); p < 0.01] and CBS overall composite cognition score [24.5 (SD 16.1) to 29.7 (SD 20.5); p = 0.02]. All CBS domains improved. CONCLUSION: Multiple measures of cognitive function improved after six months of intervention. Our results support the feasibility and impact of a multimodal, individualized treatment approach to OCI, warranting further research.

Rationale for a Multi-Factorial Approach for the Reversal of Cognitive Decline in Alzheimer's Disease and MCI: A Review.

Alzheimer's disease (AD) is a multifactorial, progressive, neurodegenerative disease typically characterized by memory loss, personality changes, and a decline in overall cognitive function. Usually manifesting in individuals over the age of 60, this is the most prevalent type of dementia and remains the fifth leading cause of death among Americans aged 65 and older. While the development of effective treatment and prevention for AD is a major healthcare goal, unfortunately, therapeutic approaches to date have yet to find a treatment plan that produces long-term cognitive improvement. Drugs that may be able to slow down the progression rate of AD are being introduced to the market; however, there has been no previous solution for preventing or reversing the disease-associated cognitive decline. Recent studies have identified several factors that contribute to the progression and severity of the disease: diet, lifestyle, stress, sleep, nutrient deficiencies, mental health, socialization, and toxins. Thus, increasing evidence supports dietary and other lifestyle changes as potentially effective ways to prevent, slow, or reverse AD progression. Studies also have demonstrated that a personalized, multi-therapeutic approach is needed to improve metabolic abnormalities and AD-associated cognitive decline. These studies suggest the effects of abnormalities, such as insulin resistance, chronic inflammation, hypovitaminosis D, hormonal deficiencies, and hyperhomocysteinemia, in the AD process. Therefore a personalized, multi-therapeutic program based on an individual's genetics and biochemistry may be preferable over a single-drug/mono-therapeutic approach. This article reviews these multi-therapeutic strategies that identify and attenuate all the risk factors specific to each affected individual. This article systematically reviews studies that have incorporated multiple strategies that target numerous factors simultaneously to reverse or treat cognitive decline. We included high-quality clinical trials and observational studies that focused on the cognitive effects of programs comprising lifestyle, physical, and mental activity, as well as nutritional aspects. Articles from PubMed Central, Scopus, and Google Scholar databases were collected, and abstracts were reviewed for relevance to the subject matter. Epidemiological, pathological, toxicological, genetic, and biochemical studies have all concluded that AD represents a complex network insufficiency. The research studies explored in this manuscript confirm the need for a multifactorial approach to target the various risk factors of AD. A single-drug approach may delay the progression of memory loss but, to date, has not prevented or reversed it. Diet, physical activity, sleep, stress, and environment all contribute to the progression of the disease, and, therefore, a multi-factorial optimization of network support and function offers a rational therapeutic strategy. Thus, a multi-therapeutic program that simultaneously targets multiple factors underlying the AD network may be more effective than a mono-therapeutic approach.

ReCODE: A Personalized, Targeted, Multi-Factorial Therapeutic Program for Reversal of Cognitive Decline.

BACKGROUND: Alzheimer's disease (AD) is the major cause of age-associated cognitive decline, and in the absence of effective therapeutics is progressive and ultimately fatal, creating a dire need for successful prevention and treatment strategies. We recently reported results of a successful proof-of-concept trial, using a personalized, precision medicine protocol, but whether such an approach is readily scalable is unknown. OBJECTIVE: In the case of AD, there is not a single therapeutic that exerts anything beyond a marginal, unsustained, symptomatic effect. This suggests that the monotherapeutic approach of drug development for AD may not be an optimal one, at least when used alone. Using a novel, comprehensive, and personalized therapeutic system called ReCODE (reversal of cognitive decline), which proved successful in a small, proof-of-concept trial, we sought to determine whether the program could be scaled to improve cognitive and metabolic function in individuals diagnosed with subjective cognitive impairment, mild cognitive impairment, and early-stage AD. METHODS: 255 individuals submitted blood samples, took the Montreal Cognitive Assessment (MoCA) test, and answered intake questions. Individuals who enrolled in the ReCODE program had consultations with clinical practitioners, and explanations of the program were provided. Participants had follow-up visits that included education regarding diet, lifestyle choices, medications, supplements, repeat blood sample analysis, and MoCA testing between 2 and 12 months after participating in the ReCODE program. Pre- and post-treatment measures were compared using the non-parametric Wilcoxon signed rank test. RESULTS AND CONCLUSIONS: By comparing baseline to follow-up testing, we observed that MoCA scores either significantly improved or stabilized in the entire participant pool-results that were not as successful as those in the proof-of-concept trial, but more successful than anti-amyloid therapies-and other risk factors including blood glucose, high-sensitivity C-reactive protein, HOMA-IR, and vitamin D significantly improved in the participant pool. Our findings provide evidence that a multi-factorial, comprehensive, and personalized therapeutic program designed to mitigate AD risk factors can improve risk factor scores and stabilize or reverse the decline in cognitive function. Since superior results were obtained in the proof-of-concept trial, which was conducted by a small group of highly trained and experienced physicians, it is possible that results from the use of this personalized approach would be enhanced by further training and experience of the practicing physicians. Nonetheless, the current results provide further support indicating the potential of such an approach for the prevention and reversal of cognitive decline.

Neuroprotective Herbs for the Management of Alzheimer's Disease.

Background-Alzheimer's disease (AD) is a multifactorial, progressive, neurodegenerative disease that is characterized by memory loss, personality changes, and a decline in cognitive function. While the exact cause of AD is still unclear, recent studies point to lifestyle, diet, environmental, and genetic factors as contributors to disease progression. The pharmaceutical approaches developed to date do not alter disease progression. More than two hundred promising drug candidates have failed clinical trials in the past decade, suggesting that the disease and its causes may be highly complex. Medicinal plants and herbal remedies are now gaining more interest as complementary and alternative interventions and are a valuable source for developing drug candidates for AD. Indeed, several scientific studies have described the use of various medicinal plants and their principal phytochemicals for the treatment of AD. This article reviews a subset of herbs for their anti-inflammatory, antioxidant, and cognitive-enhancing effects. Methods-This article systematically reviews recent studies that have investigated the role of neuroprotective herbs and their bioactive compounds for dementia associated with Alzheimer's disease and pre-Alzheimer's disease. PubMed Central, Scopus, and Google Scholar databases of articles were collected, and abstracts were reviewed for relevance to the subject matter. Conclusions-Medicinal plants have great potential as part of an overall program in the prevention and treatment of cognitive decline associated with AD. It is hoped that these medicinal plants can be used in drug discovery programs for identifying safe and efficacious small molecules for AD.

Antiviral and Immunomodulation Effects of Artemisia.

Background and Objectives: Artemisia is one of the most widely distributed genera of the family Astraceae with more than 500 diverse species growing mainly in the temperate zones of Europe, Asia and North America. The plant is used in Chinese and Ayurvedic systems of medicine for its antiviral, antifungal, antimicrobial, insecticidal, hepatoprotective and neuroprotective properties. Research based studies point to Artemisia's role in addressing an entire gamut of physiological imbalances through a unique combination of pharmacological actions. Terpenoids, flavonoids, coumarins, caffeoylquinic acids, sterols and acetylenes are some of the major phytochemicals of the genus. Notable among the phytochemicals is artemisinin and its derivatives (ARTs) that represent a new class of recommended drugs due to the emergence of bacteria and parasites that are resistant to quinoline drugs. This manuscript aims to systematically review recent studies that have investigated artemisinin and its derivatives not only for their potent antiviral actions but also their utility against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Materials andMethods: PubMed Central, Scopus and Google scholar databases of published articles were collected and abstracts were reviewed for relevance to the subject matter. Conclusions: The unprecedented impact that artemisinin had on public health and drug discovery research led the Nobel Committee to award the Nobel Prize in Physiology or Medicine in 2015 to the discoverers of artemisinin. Thus, it is clear that Artemisia's importance in indigenous medicinal systems and drug discovery systems holds great potential for further investigation into its biological activities, especially its role in viral infection and inflammation.

Transcriptional Effects of ApoE4: Relevance to Alzheimer's Disease.

The major genetic risk factor for sporadic Alzheimer's disease (AD) is the lipid binding and transporting carrier protein apolipoprotein E, epsilon 4 allele (ApoE4). One of the unsolved mysteries of AD is how the presence of ApoE4 elicits this age-associated, currently incurable neurodegenerative disease. Recently, we showed that ApoE4 acts as a transcription factor and binds to the promoters of genes involved in a range of processes linked to aging and AD disease pathogenesis. These findings point to novel therapeutic strategies for AD and aging, resulting in an extension of human healthspan, the disease-free and functional period of life. Here, we review the effects and implications of the putative transcriptional role of ApoE4 and propose a model of Alzheimer's disease that focuses on the transcriptional nature of ApoE4 and its downstream effects, with the aim that this knowledge will help to define the role ApoE4 plays as a risk factor for AD, aging, and other processes such as inflammation and cardiovascular disease.

Ayurveda and the science of aging.

Since time immemorial, humanity has been concerned with developing and preserving youthful vigor, and extending longevity by stopping or delaying the aging process. By 2030, one in five of the world population will be over 65 years old. Longevity and old age are accompanied with a variety of health challenges and population studies indicate that the elderly will use between three to five times more healthcare services compared to the younger population. Modern medicine has made a great deal of progress in understanding the aging process and in controlling age-associated health issues including heart attacks, strokes, diabetes, cancer, senility, and arthritis. Thus, every individual is now looking forward to a youthful, productive lifespan of 100 or more years filled with unlimited health and opportunity. Research by aging experts is focused on ways to go against the natural order of the aging process in order to delay it. Interventions include among other things anti-aging pills, restricted food consumption and cloning body parts to stay young and delay biological aging. Ayurveda, one of the world's most authoritative mind-body-spirit medicinal systems, offers various concepts of the aging process. This system of medicine includes therapies for healthy aging so as to create an optimal health and lengthen an individual's healthspan by living in harmony with nature. This review will explore various aspects of aging and longevity by comparing the science of aging as defined by modern medicine with the Ayurvedic treatise of Jara and Vriddhavastha.

Downregulation of protein phosphatase 2A by apolipoprotein E: Implications for Alzheimer's disease.

The apolipoprotein E ε4 allele is the single most important genetic risk factor associated with Alzheimer's disease (AD). Tau phosphorylation and hyperphosphorylation is an underlying feature of AD and is regulated by specific kinases and phosphatases. Among phosphatases, protein phosphatase 2A (PP2A) is the principal tau dephosphorylating enzyme in the brain. Several abnormalities of PP2A have been reported in AD, including among others decreased protein levels of PP2A, decreased mRNA and protein levels of the catalytic subunit PP2AC and variable regulatory B subunits and reduced methylation of the catalytic subunit, all of which results in disruption of the PP2A phosphatase activity. In earlier studies we described a novel mechanism for ApoE as a transcription factor that binds regions of double-stranded DNA with high affinity, including the promoter regions of ~3000 different genes. The list of genes also included PPP2R5E (B56ε), a regulatory B' subunit of protein phosphatase 2A. Using a combination of A172 human glioblastoma cells, ApoE3/4 and ApoE-/- NSC and human postmortem tissue, we now demonstrate that ApoE not only binds to the PPP2R5E promoter but also triggers a significant reduction in PP2A activity by two mechanisms: 1) ApoE transcriptionally represses PPP2R5E and reduces protein expression, and 2) ApoE triggers demethylation of the catalytic subunit (PP2AC) of PP2A, resulting in the disruption of the PPP2R5E-PP2AC complex. Our results indicated a significant down-regulation of PPP2R5E gene expression and reduction in PP2A activity by ApoE4 compared with ApoE3. This may also explain an elevated Tau phosphorylation in AD human brains that featured at least one ApoE4 allele. Thus, our present work links ApoE and PPP2R5E expression to a reduction in the PP2A catalytic activity that has implications for Alzheimer's disease.

Ayurvedic Profiling of Alzheimer's Disease.

Alzheimer's disease (AD) is an age-associated, progressive neurodegenerative disease that is characterized by severe memory loss, personality changes, and an overall decline in cognitive function. The cause of AD is not yet completely defined and efforts to find a cure for it have so far been disappointing. AD is one of the most significant health care problems nationally and globally. Recently, we described a personalized therapeutic approach called metabolic enhancement for neurodegeneration (MEND) that successfully reversed the cognitive decline in patients with early AD. The magnitude of the improvement was exceptional, providing testimony to the fact that a personalized and programmatic approach to cognitive decline is highly effective. Ayurveda is a personalized system of traditional medicine native to India and the Indian subcontinent. Although a direct reference to AD in the ancient Ayurvedic literature is missing, concepts including forgetfulness, memory loss, and brain cell loss have been described. Using the clinical information and the metabolic profiling of AD individuals we recently reported using the MEND program, we now describe in this commentary, 3 subtypes of AD based on the Ayurvedic interpretation. Ayurvedic profiling of patients with AD reveals 3 readily distinguishable subtypes, namely Vata, Pitta, and Krimi, which will prove useful in patients with cognitive decline and those at risk for such decline from the standpoint of specific subtype-based Ayurvedic intervention.

Increased intermediate M1-M2 macrophage polarization and improved cognition in mild cognitive impairment patients on ω-3 supplementation.

Monocyte/macrophages of patients with mild cognitive impairment (MCI) and Alzheimer disease (AD) are defective in phagocytosis and degradation amyloid ß1-42 (Aß1-42), but are improved by ω-3 fatty acids (ω-3s). The hypothesis of this study was that active Aß1-42 phagocytosis by macrophages prevents brain amyloidosis and thus maintains cognition. We studied the effects of self-supplementation with a drink with ω-3s, antioxidants, and resveratrol on Mini-Mental State Examination (MMSE) scores, macrophage M1M2 phenotype [the ratio of inflammatory cluster of differentiation (CD)54+CD80 and proresolution markers CD163+CD206], and Aß1-42 phagocytosis in patients initially diagnosed as having MCI or subjective cognitive impairment (SCI). At baseline, the median MMSE score in patients in both the apolipoprotein E (ApoE) ε3/ε3 and ApoE ε3/ε4 groups was 26.0 and macrophage Aß1-42 phagocytosis was defective. The MMSE rate of change increased in the ApoE ε3/ε3 group a median 2.2 points per year (P = 0.015 compared to 0) but did not change in the ApoE ε3/ε4 group (P = 0.014 between groups). In the ApoE ε3/ε3 group, all patients remained cognitively stable or improved; in the ApoE ε3/ε4 group, 1 recovered from dementia, but 3 lapsed into dementia. The macrophage phenotype polarized in patients bearing ApoE ε3/ε3 to an intermediate (green zone) M1-M2 type at the rate of 0.226 U/yr, whereas in patients bearing ApoE ε3/ε4, polarization was negative (P = 0.08 between groups). The baseline M1M2 type in the extreme M1 (red zone) or M2 (white zone) was unfavorable for cognitive outcome. Aß1-42 phagocytosis increased in both ApoE groups (P = 0.03 in each groups). In vitro, the lipidic mediator resolvin D1 (RvD1) down regulated the M1 type in patients with ApoE ε3/ε3 but in some patients with ε3/ε4, paradoxically up-regulated the M1 type. Antioxidant/ω-3/resveratrol supplementation was associated with favorable immune and cognitive responses in ApoE ε3/ε3 and individual patients bearing ApoE ε3/ε4, and brings into personalized clinical practice the immune benefits expected from ω-3 mediators called resolvins. The validity of this study is limited by its small size and uncontrolled design.-Famenini, S., Rigali, E. A., Olivera-Perez, H. M., Dang, J., Chang, M T., Halder, R., Rao, R. V., Pellegrini, M., Porter, V., Bredesen, D., Fiala, M. Increased intermediate M1-M2 macrophage polarization and improved cognition in mild cognitive impairment patients on ω-3 supplementation.

Direct Transcriptional Effects of Apolipoprotein E.

A major unanswered question in biology and medicine is the mechanism by which the product of the apolipoprotein E ε4 allele, the lipid-binding protein apolipoprotein E4 (ApoE4), plays a pivotal role in processes as disparate as Alzheimer's disease (AD; in which it is the single most important genetic risk factor), atherosclerotic cardiovascular disease, Lewy body dementia, hominid evolution, and inflammation. Using a combination of neural cell lines, skin fibroblasts from AD patients, and ApoE targeted replacement mouse brains, we show in the present report that ApoE4 undergoes nuclear translocation, binds double-stranded DNA with high affinity (low nanomolar), and functions as a transcription factor. Using chromatin immunoprecipitation and high-throughput DNA sequencing, our results indicate that the ApoE4 DNA binding sites include ∼1700 gene promoter regions. The genes associated with these promoters provide new insight into the mechanism by which AD risk is conferred by ApoE4, because they include genes associated with trophic support, programmed cell death, microtubule disassembly, synaptic function, aging, and insulin resistance, all processes that have been implicated in AD pathogenesis. Significance statement: This study shows for the first time that apolipoprotein E4 binds DNA with high affinity and that its binding sites include 1700 promoter regions that include genes associated with neurotrophins, programmed cell death, synaptic function, sirtuins and aging, and insulin resistance, all processes that have been implicated in Alzheimer's disease pathogenesis.

Neuroprotective Sirtuin ratio reversed by ApoE4.

The canonical pathogenesis of Alzheimer's disease links the expression of apolipoprotein E ε4 allele (ApoE) to amyloid precursor protein (APP) processing and Aß peptide accumulation by a set of mechanisms that is incompletely defined. The development of a simple system that focuses not on a single variable but on multiple factors and pathways would be valuable both for dissecting the underlying mechanisms and for identifying candidate therapeutics. Here we show that, although both ApoE3 and ApoE4 associate with APP with nanomolar affinities, only ApoE4 significantly (i) reduces the ratio of soluble amyloid precursor protein alpha (sAPPα) to Aß; (ii) reduces Sirtuin T1 (SirT1) expression, resulting in markedly differing ratios of neuroprotective SirT1 to neurotoxic SirT2; (iii) triggers Tau phosphorylation and APP phosphorylation; and (iv) induces programmed cell death. We describe a subset of drug candidates that interferes with the APP-ApoE interaction and returns the parameters noted above to normal. Our data support the hypothesis that neuronal connectivity, as reflected in the ratios of critical mediators such as sAPPα:Aß, SirT1:SirT2, APP:phosphorylated (p)-APP, and Tau:p-Tau, is programmatically altered by ApoE4 and offer a simple system for the identification of program mediators and therapeutic candidates.

Dimethyl sulfoxide and dimethyl formamide increase lifespan of C. elegans in liquid.

Lifespan extension through pharmacological intervention may provide valuable tools to understanding the mechanisms of aging and could uncover new therapeutic approaches for the treatment of age-related disease. Although the nematode Caenorhabditis elegans is well known as a particularly suitable model for genetic manipulations, it has been recently used in a number of pharmacological studies searching for compounds with anti-aging activity. These compound screens are regularly performed in amphipathic solvents like dimethyl sulfoxide (DMSO), the solvent of choice for high-throughput drug screening experiments performed throughout the world. In this work, we report that exposing C. elegans to DMSO in liquid extends lifespan up to 20%. Interestingly, another popular amphipathic solvent, dimethyl formamide (DMF), produces a robust 50% increase in lifespan. These compounds work through a mechanism independent of insulin-like signaling and dietary restriction (DR). Additionally, the mechanism does not involve an increased resistance to free radicals or heat shock suggesting that stress resistance does not play a major role in the lifespan extension elicited by these compounds. Interestingly, we found that DMSO and DMF are able to decrease the paralysis associated with amyloid-ß3-42 aggregation, suggesting a role of protein homeostasis for the mechanism elicited by these molecules to increase lifespan.

The small co-chaperone p23 overexpressing transgenic mouse.

Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we described the role of p23 a small co-chaperone protein in preventing ER stress-induced cell death. p23 is cleaved by caspases at D142 to yield p19 (a 19 kDa product) during ER stress-induced cell death. Mutation of the caspase cleavage site not only blocks formation of the 19 kDa product but also attenuates the cell death process triggered by various ER stressors. Thus, uncleavable p23 (p23D142N) emerges as a reasonable candidate to test for potential inhibition of neurodegenerative disease phenotype that features misfolded proteins and ER stress. In the present work we report the generation of transgenic mouse lines that overexpress wild-type p23 or uncleavable p23 under the control of a ROSA promoter. These mice should prove useful for studying the role of p23 and/or uncleavable p23 in cellular stress-induced cell death.

Ayurvedic medicinal plants for Alzheimer's disease: a review.

Alzheimer's disease is an age-associated, irreversible, progressive neurodegenerative disease that is characterized by severe memory loss, unusual behavior, personality changes, and a decline in cognitive function. No cure for Alzheimer's exists, and the drugs currently available to treat the disease have limited effectiveness. It is believed that therapeutic intervention that could postpone the onset or progression of Alzheimer's disease would dramatically reduce the number of cases in the next 50 years. Ayurvedic medicinal plants have been the single most productive source of leads for the development of drugs, and over a hundred new products are already in clinical development. Indeed, several scientific studies have described the use of various Ayurvedic medicinal plants and their constituents for treatment of Alzheimer's disease. Although the exact mechanism of their action is still not clear, phytochemical studies of the different parts of the plants have shown the presence of many valuable compounds, such as lignans, flavonoids, tannins, polyphenols, triterpenes, sterols, and alkaloids, that show a wide spectrum of pharmacological activities, including anti-inflammatory, anti-amyloidogenic, anti-cholinesterase, hypolipidemic, and antioxidant effects. This review gathers research on various medicinal plants that have shown promise in reversing the Alzheimer's disease pathology. The report summarizes information concerning the phytochemistry, biological, and cellular activities and clinical applications of these various plants in order to provide sufficient baseline information that could be used in drug discovery campaigns and development process, thereby providing new functional leads for Alzheimer's disease.

The small chaperone protein p23 and its cleaved product p19 in cellular stress.

The presence of misfolded proteins elicits cellular responses including an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Accumulation of these proteins in excessive amounts, however, overwhelms the "cellular quality control" system and impairs the protective mechanisms designed to promote correct folding and degrade misfolded proteins, ultimately leading to organelle dysfunction and cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we reported the role of the small co-chaperone protein p23 in preventing ER stress-induced cell death. p23 undergoes caspase-dependent cleavage to yield a 19-kD product (p19), and mutation of this caspase cleavage site not only blocks the formation of the 19-kD product but also attenuates the ER stress-induced cell death process triggered by various stressors. Thus, a critical question is whether p23 and/or p19 could serve as an in vivo marker for neurodegenerative diseases featuring misfolded proteins and cellular stress. In the present study, we used an antibody that recognizes both p23 and p19 as well as a specific neo-epitope antibody that detects only the p19 fragment. These antibodies were used to detect the presence of both these proteins in cells, primary neurons, brain samples from a mouse model of Alzheimer's disease (AD), and fixed human AD brain samples. While patients with severe AD did display a consistent reduction in p23 levels, our inability to observe p19 in mouse or human AD brain samples suggests that the usefulness of the p23 neo-epitope antibody is restricted to cells and primary neurons undergoing cellular stress.

Endogenously EGFP-Labeled Mouse Embryonic Stem Cells.

Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping ß-actin promoter The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759-769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies.

Valosin-containing protein gene mutations: cellular phenotypes relevant to neurodegeneration.

Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.

Endoplasmic reticulum stress-induced cell death in dopaminergic cells: effect of resveratrol.

Resveratrol, a naturally occurring polyphenol, exhibits antioxidant, antiaging, and anticancer activity. Resveratrol has also been shown to inhibit tumor initiation, promotion, and progression in a variety of cell culture systems. Earlier, we showed that paraquat, a bipyridyl herbicide, triggers endoplasmic reticulum stress, cell dysfunction, and dopaminergic cell death. Due to its antioxidant activity, we assessed the ability of resveratrol to rescue cells from the toxic effects of paraquat. While resveratrol did not have any protective effect at low concentrations, it triggered endoplasmic reticulum (ER) stress-induced cell death at higher concentrations (50-250 microM). The present study was carried out to determine the mechanism by which resveratrol triggers ER stress and cell death in dopaminergic N27 cells. Our studies demonstrate that resveratrol triggers ER stress and cell dysfunction, caspase activation, p23 cleavage and inhibition of proteasomal activity in dopaminergic N27 cells. While over expression of uncleavable p23 was associated with decreased cell death, downregulation of p23 protein expression by siRNA resulted in enhancement of ER stress-induced cell death triggered by resveratrol indicating a protective role for the small co-chaperone p23 in dopaminergic cell death.

Coupling endoplasmic reticulum stress to the cell death program in dopaminergic cells: effect of paraquat.

Parkinson's disease (PD) features oxidative stress and accumulation of misfolded (unfolded, alternatively folded, or mutant) proteins with associated loss of dopaminergic neurons. Oxidative stress and the accumulated misfolded proteins elicit cellular responses that include an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Chronic ER stress and accumulation of misfolded proteins in excessive amounts, however, overwhelm the cellular 'quality control' system and impair the protective mechanisms designed to promote correct folding and degrade faulty proteins, ultimately leading to organelle dysfunction and neuronal cell death. Paraquat belongs to a class of bipyridyl herbicides and triggers oxidative stress and dopaminergic cell death. Epidemiological studies suggest an increased risk for developing PD following chronic exposure to paraquat. The present study was carried out to determine the role of paraquat in triggering cellular stress particularly ER stress and to elucidate the pathways that couple ER stress to dopaminergic cell death. We demonstrate that paraquat triggers ER stress, cell dysfunction, and dopaminergic cell death. p23, a small co-chaperone protein, is cleaved during ER stress-induced cell death triggered by paraquat and blockage of the caspase cleavage site of p23 was associated with decreased cell death. Paraquat also inhibits proteasomal activity that may further trigger accumulation of misfolded proteins resulting in ER stress. Our results indicate a protective role for p23 in PD-related programmed cell death. The data also underscore the involvement of ER, caspases, and the proteasomal system in ER stress-induced cell death process.