Alzheimer's Disease (AD) is a devastating neurodegenerative disease that currently affects more than 26 million people worldwide and is projected to triple in prevalence by 2015. As such it represents an enormous economic, medical, scientific, and emotional burden on society. Despite extensive efforts, there is no cure and current treatments are only effective at ameloriating symptoms without delaying the progression of disease. Part of the challenge may stem from the use of model systems that incompletely recapitulate human disease. The recently discovered technology of human induced pluripotent stem cells (hIPSCS) has reinvigorated investigators and brought hope to the fields of AD. The power of hIPSC technology is the ability to capture the genomes of individual with complex genetic diseases in a pluripotent cell with the ability to become any one of the body. By driving hIPSC down a neuronal lineage, we can grow human neurons and model AD in a dish in the relevant cell type. A hallmark of the polarized structure of a neuron is it long thin axon where a wide variety of cargo essential for proper neuronal viability must be transported correctly to and from the axon terminal. Perturbations of this complex neuronal process can have drastic consequences and, in fact, many neurological diseases are associated with defects in neuronals novel, neuron-specific sorting defects in hIPSC-derived neurons with familial AD mutations. Specifically, we see that toxic, intracellular accumulations of [Beta]CTFs,proteolytic products of the amyloid precursor protein (APP), mediate decreased endocytosis and transcytosis of APP itself and lipoproteins. That this is a common phenotype of many fAD mutations suggests that impaired axonal delivery of lipoproteins compromises synaptic maintenance in fAD. In a specific fAD mutation, PS1[Delta]E9, we analyzed the complex axonal transport behavior of a number of AD- relevant cargo including APP, BACE1, Rab5a, mitochondria, and lysosomes. We report that PS1[Delta]E9 mutant neurons have enhanced anterograde transport of APP, possibly because of increased phosphorylated JIP1, while having no differences in Rab5a, BACE1, or mitochondrial transport behavior. Importantly, these transport changes accompanied by decreased density of axonal acidic vesicles and implicates impaired clearance of toxic axonal cargo in AD pathogenesis

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.

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.

Axonal injury and stress have long been thought to play a pathogenic role in a variety of neurodegenerative diseases. However, a model for studying single-cell axonal injury in mammalian cells and the processes of repair has not been established. The purpose of this study was to examine the response of neuronal growth cones to laser-induced axonal damage in cultures of embryonic rat hippocampal neurons and induced pluripotent stem cell (iPSC) derived human neurons. A 532-nm pulsed [Formula: see text] picosecond laser was focused to a diffraction limited spot at a precise location on an axon using a laser energy/power that did not rupture the cell membrane (subaxotomy). Subsequent time series images were taken to follow axonal recovery and growth cone dynamics. After laser subaxotomy, axons thinned at the damage site and initiated a dynamic cytoskeletal remodeling process to restore axonal thickness. The growth cone was observed to play a role in the repair process in both hippocampal and iPSC-derived neurons. Immunofluorescence staining confirmed structural tubulin damage and revealed initial phases of actin-based cytoskeletal remodeling at the damage site. The results of this study indicate that there is a repeatable and cross-species repair response of axons and growth cones after laser-induced damage.

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.

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.

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.