Full-length amyloid precursor protein regulates lipoprotein metabolism and amyloid-ß clearance in human astrocytes.

Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer's disease (AD) pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by proteolytic processing of APP have previously been implicated in the regulation of cholesterol metabolism. However, the physiological function of APP in regulating lipoprotein homeostasis in astrocytes, which are responsible for de novo cholesterol biosynthesis and regulation in the brain, remains unclear. To address this, here we used CRISPR/Cas9 genome editing to generate isogenic APP-knockout (KO) human induced pluripotent stem cells (hiPSCs) and differentiated them into human astrocytes. We found that APP-KO astrocytes have reduced cholesterol and elevated levels of sterol regulatory element-binding protein (SREBP) target gene transcripts and proteins, which were both downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and to examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series harboring the APP Swedish and APP V717F variants. Only astrocytes homozygous for the APP Swedish (APPSwe/Swe) mutation, which had reduced full-length APP (FL APP) due to increased ß-secretase cleavage, recapitulated the APP-KO phenotypes. Astrocytic internalization of ß-amyloid (Aß), another ligand for low-density lipoprotein (LDL) receptors, was also impaired in APP-KO and APPSwe/Swe astrocytes. Finally, impairing cleavage of FL APP through ß-secretase inhibition in APPSwe/Swe astrocytes reversed the LDL and Aß endocytosis defects. In conclusion, FL APP is involved in the endocytosis of LDL receptor ligands and is required for proper cholesterol homeostasis and Aß clearance in human astrocytes.

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.

Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer's Disease Mutations.

We investigated early phenotypes caused by familial Alzheimer's disease (fAD) mutations in isogenic human iPSC-derived neurons. Analysis of neurons carrying fAD PS1 or APP mutations introduced using genome editing technology at the endogenous loci revealed that fAD mutant neurons had previously unreported defects in the recycling state of endocytosis and soma-to-axon transcytosis of APP and lipoproteins. The endocytosis reduction could be rescued through treatment with a ß-secretase inhibitor. Our data suggest that accumulation of ß-CTFs of APP, but not Aß, slow vesicle formation from an endocytic recycling compartment marked by the transcytotic GTPase Rab11. We confirm previous results that endocytosis is affected in AD and extend these to uncover a neuron-specific defect. Decreased lipoprotein endocytosis and transcytosis to the axon suggest that a neuron-specific impairment in endocytic axonal delivery of lipoproteins and other key materials might compromise synaptic maintenance in fAD.

Rat embryonic hippocampus and induced pluripotent stem cell derived cultured neurons recover from laser-induced subaxotomy.

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.

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 Aß peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aß peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aß 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.

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

Deposition of potentially neurotoxic Aß 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 Aß, sAPPα, and sAPPß 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 ß-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.

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-ß 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-ß(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3ß (aGSK-3ß). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with ß-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3ß levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-ß, in GSK-3ß 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.