Probing the secrets of Alzheimer's disease using human-induced pluripotent stem cell technology.

Our understanding of Alzheimer's disease (AD) is still incomplete and, as a result, we lack effective therapies. Reprogramming to generate human-induced pluripotent stem cells provides a new approach to the generation of human neurons that carry the genomes of people with familial or sporadic AD. Differentiation of such stem cells to human neurons is already providing new insights into AD and molecular pathways that may provide new targets for effective therapy. These pathways include typical amyloid response pathways, as well as pathways leading from altered behavior of amyloid precursor protein to the elevated phosphorylation of tau protein. There is also a need for standardization of models so that isogenic lines differing only in the familial AD mutation can be compared

Axonal amyloid precursor protein and its fragments undergo somatodendritic endocytosis and processing.

Deposition of potentially neurotoxic Aβ fragments derived from amyloid precursor protein (APP) at synapses may be a key contributor to Alzheimer's disease. However, the location(s) of proteolytic processing and subsequent secretion of APP fragments from highly compartmentalized, euploid neurons that express APP and processing enzymes at normal levels is not well understood. To probe the behavior of endogenous APP, particularly in human neurons, we developed a system using neurons differentiated from human embryonic stem cells, cultured in microfluidic devices, to enable direct biochemical measurements from axons. Using human or mouse neurons in these devices, we measured levels of Aβ, sAPPα, and sAPPβ secreted solely from axons. We found that a majority of the fragments secreted from axons were processed in the soma, and many were dependent on somatic endocytosis for axonal secretion. We also observed that APP and the β-site APP cleaving enzyme were, for the most part, not dependent on endocytosis for axonal entry. These data establish that axonal entry and secretion of APP and its proteolytic processing products traverse different pathways in the somatodendritic compartment before axonal entry

A role for kinesin heavy chain in controlling vesicle transport into dendrites in Drosophila.

The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based transport to deliver essential cargo into dendrites. To test different models of differential motor protein regulation and to understand how different compartments in neurons are supplied with necessary functional proteins, we studied mechanisms of dendritic transport, using Drosophila as a model system. Our data suggest that dendritic targeting systems in Drosophila and mammals are evolutionarily conserved, since mammalian cargoes are moved into appropriate domains in Drosophila. In a genetic screen for mutants that mislocalize the dendritic marker human transferrin receptor (hTfR), we found that kinesin heavy chain (KHC) may function as a dendritic motor. Our analysis of dendritic and axonal phenotypes of KHC loss-of-function clones revealed a role for KHC in maintaining polarity of neurons, as well as ensuring proper axonal outgrowth. In addition we identified adenomatous polyposis coli 1 (APC1) as an interaction partner of KHC in controlling directed transport and modulating kinesin function in neurons

Stabilizing the Retromer Complex in a Human Stem Cell Model of Alzheimer's Disease Reduces TAU Phosphorylation Independently of Amyloid Precursor Protein.

Developing effective therapeutics for complex diseases such as late-onset, sporadic Alzheimer's disease (SAD) is difficult due to genetic and environmental heterogeneity in the human population and the limitations of existing animal models. Here, we used hiPSC-derived neurons to test a compound that stabilizes the retromer, a highly conserved multiprotein assembly that plays a pivotal role in trafficking molecules through the endosomal network. Using this human-specific system, we have confirmed previous data generated in murine models and show that retromer stabilization has a potentially beneficial effect on amyloid beta generation from human stem cell-derived neurons. We further demonstrate that manipulation of retromer complex levels within neurons affects pathogenic TAU phosphorylation in an amyloid-independent manner. Taken together, our work demonstrates that retromer stabilization is a promising candidate for therapeutic development in AD and highlights the advantages of testing novel compounds in a human-specific, neuronal system

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost

A γ-secretase inhibitor, but not a γ-secretase modulator, induced defects in BDNF axonal trafficking and signaling: evidence for a role for APP.

Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative

Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells.

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients

ImpulsePal: The systematic development of a smartphone app to manage food temptations using intervention mapping

This is the final version. Available on open access from SAGE Publications via the DOI in this recordBackground: Impulsive processes driving eating behaviour can often undermine peoples' attempts to change their behaviour, lose weight and maintain weight loss. Aim: To develop an impulse management intervention to support weight loss in adults. Methods: Intervention Mapping (IM) was used to systematically develop the "ImpulsePal" intervention. The development involved: (1) a needs assessment including a qualitative study, Patient and Public advisory group and expert group consultations, and a systematic review of impulse management techniques; (2) specification of performance objectives, determinants, and change objectives; (3) selection of intervention strategies (mapping of change techniques to the determinants of change); (4) creation of programme materials; (5) specification of adoption and implementation plans; (6) devising an evaluation plan. Results: Application of the IM Protocol resulted in a smartphone app that could support reductions in unhealthy (energy dense) food consumption, overeating, and alcoholic and sugary drink consumption. ImpulsePal includes inhibition training, mindfulness techniques, implementation intentions (if-then planning), visuospatial loading, use of physical activity for craving management, and context-specific reminders. An "Emergency Button" was also included to provide access to in-the-moment support when temptation is strong. Conclusions: ImpulsePal is a novel, theory- and evidence-informed, person-centred app that aims to support impulse management for healthier eating. Intervention Mapping facilitated the incorporation of app components that are practical operationalisations of change techniques targeting our specific change objectives and their associated theoretical determinants. Using IM enabled transparency and provided a clear framework for evaluation, and enhances replicability and the potential of the intervention to accomplish the desired outcome of facilitating weight loss through dietary change.University of ExeterNational Institute for Health Research (NIHR

Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells.

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation

App-based food Go/No-Go training: User engagement and dietary intake in an opportunistic observational study.

This is the final version. Available from Elsevier via the DOI in this record. Food Go/No-Go training aims to alter implicit food biases by creating associations between perceiving unhealthy foods and withholding a dominant response. Asking participants to repeatedly inhibit an impulse to approach unhealthy foods can decrease unhealthy food intake in laboratory settings. Less is known about how people engage with app-based Go/No-Go training in real-world settings and how this might relate to dietary outcomes. This pragmatic observational study investigated associations between the number of completed app-based food Go/No-Go training trials and changes in food intake (Food Frequency Questionnaire; FFQ) for different healthy and unhealthy food categories from baseline to one-month follow-up. In total, 1234 participants (m(BMI) = 29 kg/m2, m(age) = 43years, 69% female) downloaded the FoodT app and completed food-Go/No-Go training at their own discretion (mean number of completed sessions = 10.7, sd = 10.3, range: 1-122). In pre-registered analyses, random-intercept linear models predicting intake of different foods, and controlled for baseline consumption, BMI, age, sex, smoking, metabolic syndrome, and dieting status, revealed small, significant associations between the number of completed training trials and reductions in unhealthy food intake (b = -0.0005, CI95= [-0.0007;-0.0003]) and increases in healthy food intake (b = 0.0003, CI95 = [0.0000; 0.0006]). These relationships varied by food category, and exploratory analyses suggest that more temporally spaced training was associated with greater changes in dietary intake. Taken together, these results imply a positive association between the amount of training completed and beneficial changes in food intake. However, the results of this pragmatic study should be interpreted cautiously, as self-selection biases, motivation and other engagement-related factors that could underlie these associations were not accounted for. Experimental research is needed to rule out these possible confounds and establish causal dose-response relationships between patterns of engagement with food Go/No-Go training and changes in dietary intake.Finnish Cultural FoundationSigne and Ane Gyllenberg FoundationAlfred Kordelin FoundationUniversity of Helsink