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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 (APP(Swe/Swe)) mutation, which had reduced full-length APP (FL APP) due to increased β-secretase cleavage, recapitulated the APP-KO phenotypes. Astrocytic internalization of β-amyloid (Aβ), another ligand for low-density lipoprotein (LDL) receptors, was also impaired in APP-KO and APP(Swe/Swe) astrocytes. Finally, impairing cleavage of FL APP through β-secretase inhibition in APP(Swe/Swe) astrocytes reversed the LDL and Aβ endocytosis defects. In conclusion, FL APP is involved in the endocytosis of LDL receptor ligands and is required for proper cholesterol homeostasis and Aβ clearance in human astrocytes.

In the mammalian genome, the clustered protocadherin (cPcdh) locus is a paradigm of stochastic gene expression with the potential to generate a unique cPcdh combination in every neuron. Here, we report a chromatin-based mechanism emerging during the transition from the naive to the primed states of cell pluripotency that 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, months-old organoids, and engrafted cells in the rat spinal cord. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, unless there is a condition of delayed maturation such as Down Syndrome. We therefore propose that a pattern of limited cPcdh diversity is maintained while human neurons still retain fetal-like levels of maturation., Editorial SUMMARY: Short and long-term cultures of human stem cell-derived neurons reveal that a pattern of restricted selection of clustered protocadherin isoforms, pre-established in pluripotent cells, distinguishes immature from mature neurons.

Summary Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies.