The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aβ secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aβ uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aβ phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.

P-element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8,000 bp of nonhomologous sequence could be copied from an ectopic template at essentially the same rate as a single-base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila melanogaster, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as a template. This synthesis-dependent strand annealing (SDSA) model as a possible general mechanism in complex organisms is discussed.We used a transposable P-element insertion allele of the white gene to study the repair of a double-stranded DNA break in Drosophila melanogaster. Unrepaired double-stranded DNA breaks can be cell lethal after mitosis. Consequently, organisms have developed efficient methods for their repair. Double strand break repair is thought to occur by a process in which the broken ends search for a homologous sequence, invade it, and serve as primers for DNA synthesis to reconstitute the broken chromosome (29, 32, 39).P elements are thought to transpose by a cut-and-paste process in which excision of the element breaks both DNA strands of the chromosome (6,12,19). Experiments have shown that the break is usually repaired by copying the corresponding sequences from the sister strand (6), thus restoring a copy of the P element to the break site. Alternatively, sequence may be copied from a template on the homolog (18,25) or one inserted in the genome at an ectopic site (12). The DNA sequence flanking the P excision site is usually converted as a continuous block with an average tract length of about 1,400 nucleotides. Recent results have also shown that small insertions and deletions from the homolog can be converted into the excision site (18).These observations suggested to us that the gap repair process could be adapted for use as an efficient gene targeting method in D. melanogaster (Fig. 1). We tested this possibility by using a P-element insertion in the white gene, whd (whd80KJ7), which is known to excise at high frequencies in the presence of P transposase. We then recovered gap repair products that occurred in the presence of one of five ectopi- cally located templates. In four of these templates, the white gene had been altered by the addition of unrelated sequences of lengths up to approximately 7,970 bp. We found that each of the nonhomologous sequences could be copied into the white locus almo...

Alzheimer's disease is a progressive loss of memory and cognition, for which there is no cure. Although genetic studies initially suggested a primary role for amyloid-in Alzheimer's disease, treatment strategies targeted at reducing amyloid-have failed to reverse cognitive symptoms. These clinical findings suggest that cognitive decline is the result of a complex pathophysiology and that targeting amyloid-alone may not be sufficient to treat Alzheimer's disease. Instead, a broad outlook on neural-circuit-damaging processes may yield insights into new therapeutic strategies for curing memory loss in the disease.

Alzheimer's disease is a devastating neurodegenerative disorder with no cure. Countless promising therapeutics have shown efficacy in rodent Alzheimer's disease models yet failed to benefit human patients. While hope remains that earlier intervention with existing therapeutics will improve outcomes, it is becoming increasingly clear that new approaches to understand and combat the pathophysiology of Alzheimer's disease are needed. Human induced pluripotent stem cell (iPSC) technologies have changed the face of preclinical research and iPSC-derived cell types are being utilized to study an array of human conditions, including neurodegenerative disease. All major brain cell types can now be differentiated from iPSCs, while increasingly complex co-culture systems are being developed to facilitate neuroscience research. Many cellular functions perturbed in Alzheimer's disease can be recapitulated using iPSC-derived cells in vitro, and co-culture platforms are beginning to yield insights into the complex interactions that occur between brain cell types during neurodegeneration. Further, iPSC-based systems and genome editing tools will be critical in understanding the roles of the numerous new genes and mutations found to modify Alzheimer's disease risk in the past decade. While still in their relative infancy, these developing iPSC-based technologies hold considerable promise to push forward efforts to combat Alzheimer's disease and other neurodegenerative disorders.

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