Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells.

Human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells, hold great promise for cell-based therapies, but safety concerns that complicate consideration for routine clinical use remain. Installing a "safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg, AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy, we precisely installed the system into a genomic safe harbor, the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1α promoter is silenced in iPSCs, and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However, the CAG promoter induces strong and stable iCASP9 expression in iPSCs, and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives, including MSCs and chondrocytes, in vitro. Furthermore, iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies.

A call for comparative effectiveness research to learn whether routine clinical care decisions can protect from dementia and cognitive decline.

Common diseases like diabetes, hypertension, and atrial fibrillation are probable risk factors for dementia, suggesting that their treatments may influence the risk and rate of cognitive and functional decline. Moreover, specific therapies and medications may affect long-term brain health through mechanisms that are independent of their primary indication. While surgery, benzodiazepines, and anti-cholinergic drugs may accelerate decline or even raise the risk of dementia, other medications act directly on the brain to potentially slow the pathology that underlies Alzheimer's and other dementia. In other words, the functional and cognitive decline in vulnerable patients may be influenced by the choice of treatments for other medical conditions. Despite the importance of these questions, very little research is available. The Alzheimer's Drug Discovery Foundation convened an advisory panel to discuss the existing evidence and to recommend strategies to accelerate the development of comparative effectiveness research on how choices in the clinical care of common chronic diseases may protect from cognitive decline and dementia.

Expert consensus document: Mind the gaps­advancing research into short-term and long-term neuropsychological outcomes of youth sports-related concussions.

Sports-related concussions and repetitive subconcussive exposure are increasingly recognized as potential dangers to paediatric populations, but much remains unknown about the short-term and long-term consequences of these events, including potential cognitive impairment and risk of later-life dementia. This Expert Consensus Document is the result of a 1-day meeting convened by Safe Kids Worldwide, the Alzheimer's Drug Discovery Foundation, and the Andrews Institute for Orthopaedics and Sports Medicine. The goal is to highlight knowledge gaps and areas of critically needed research in the areas of concussion science, dementia, genetics, diagnostic and prognostic biomarkers, neuroimaging, sports injury surveillance, and information sharing. For each of these areas, we propose clear and achievable paths to improve the understanding, treatment and prevention of youth sports-related concussions.

Overcoming obstacles to repurposing for neurodegenerative disease.

Repurposing Food and Drug Administration (FDA)-approved drugs for a new indication may offer an accelerated pathway for new treatments to patients but is also fraught with significant commercial, regulatory, and reimbursement challenges. The Alzheimer's Drug Discovery Foundation (ADDF) and the Michael J. Fox Foundation for Parkinson's Research (MJFF) convened an advisory panel in October 2013 to understand stakeholder perspectives related to repurposing FDA-approved drugs for neurodegenerative diseases. Here, we present opportunities on how philanthropy, industry, and government can begin to address these challenges, promote policy changes, and develop targeted funding strategies to accelerate the potential of FDA-approved repurposed drugs.

Dementia Prevention: optimizing the use of observational data for personal, clinical, and public health decision-making.

Worldwide, over 35 million people suffer from Alzheimer's disease and related dementias. This number is expected to triple over the next 40 years. How can we improve the evidence supporting strategies to reduce the rate of dementia in future generations? The risk of dementia is likely influenced by modifiable factors such as exercise, cognitive activity, and the clinical management of diabetes and hypertension. However, the quality of evidence is limited and it remains unclear whether specific interventions to reduce these modifiable risk factors can, in turn, reduce the risk of dementia. Although randomized controlled trials are the gold-standard for causality, the majority of evidence for long-term dementia prevention derives from, and will likely continue to derive from, observational studies. Although observational research has some unavoidable limitations, its utility for dementia prevention might be improved by, for example, better distinction between confirmatory and exploratory research, higher reporting standards, investment in effectiveness research enabled by increased data-pooling, and standardized exposure and outcome measures. Informed decision-making by the general public on low-risk health choices that could have broad potential benefits could be enabled by internet-based tools and decision-aids to communicate the evidence, its quality, and the estimated magnitude of effect.

Progress in novel cognitive enhancers for cognitive aging and Alzheimer's disease.

Increased knowledge of the biology of synaptic function has led to the development of novel cognitive-enhancing therapeutic strategies with the potential for increased efficacy and safety. This editorial highlights a diverse array of approaches currently being explored to target cognitive dysfunction due to aging and/or Alzheimer's disease.

Adenosine receptor signaling modulates permeability of the blood-brain barrier.

The blood-brain barrier (BBB) is comprised of specialized endothelial cells that form the capillary microvasculature of the CNS and is essential for brain function. It also poses the greatest impediment in the treatment of many CNS diseases because it commonly blocks entry of therapeutic compounds. Here we report that adenosine receptor (AR) signaling modulates BBB permeability in vivo. A(1) and A(2A) AR activation facilitated the entry of intravenously administered macromolecules, including large dextrans and antibodies to ß-amyloid, into murine brains. Additionally, treatment with an FDA-approved selective A(2A) agonist, Lexiscan, also increased BBB permeability in murine models. These changes in BBB permeability are dose-dependent and temporally discrete. Transgenic mice lacking A(1) or A(2A) ARs showed diminished dextran entry into the brain after AR agonism. Following treatment with a broad-spectrum AR agonist, intravenously administered anti-ß-amyloid antibody was observed to enter the CNS and bind ß-amyloid plaques in a transgenic mouse model of Alzheimer's disease (AD). Selective AR activation resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules. These results suggest that AR signaling can be used to modulate BBB permeability in vivo to facilitate the entry of potentially therapeutic compounds into the CNS. AR signaling at brain endothelial cells represents a novel endogenous mechanism of modulating BBB permeability. We anticipate these results will aid in drug design, drug delivery and treatment options for neurological diseases such as AD, Parkinson's disease, multiple sclerosis and cancers of the CNS.

The fungal pathogen Candida albicans autoinduces hyphal morphogenesis by raising extracellular pH.

UNLABELLED: pH homeostasis is critical for all organisms; in the fungal pathogen Candida albicans, pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation, C. albicans actively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes, STP2, a transcription factor regulating amino acid permeases, ACH1 (acetyl-coenzyme A [acetyl-CoA] hydrolase), DUR1,2 (urea amidolyase), and ATO5, a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprived C. albicans cells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis. IMPORTANCE: Candida albicans is the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here that C. albicans can actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability of C. albicans to cause disease.

Role of acetyl coenzyme A synthesis and breakdown in alternative carbon source utilization in Candida albicans.

Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and beta-oxidation, are required for full virulence in the fungal pathogen Candida albicans. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (ACH1) and acetyl-CoA synthetase (ACS1 and ACS2), would regulate alternative carbon utilization and virulence. We show that C. albicans strains depleted for ACS2 are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of Saccharomyces cerevisiae acs2 mutants. In contrast, deletion of ACS1 confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why acs2 mutants can utilize fatty acids. Strains lacking ACH1 have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both ACH1 and ACS2 complement mutations in their S. cerevisiae homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of C. albicans on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in S. cerevisiae and C. albicans support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.