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.

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Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δ mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation. Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aβ accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δ transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

A genome-wide screen of phosphatases that control mitosis identified CDKN3, which acts through the CDC2 signaling axis.

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Heparan sulfate proteoglycans modified by human glucosaminyl 3-O-sulfotransferase-3 (3-OST-3) isoform generates the cellular receptor for herpes simplex virus type 1 (HSV-1). Interestingly, the ability of zebrafish (ZF)-encoded 3-OST-3 isoform to modify heparan sulfate to mediate HSV-1 entry and cell-cell fusion has not been determined although it is predominantly expressed in ZF, a popular model organism to study viral infections. Here, we demonstrate that expression of ZF-encoded 3-OST-3 isoform renders the resistant Chinese hamster ovary (CHO-K1) cells to become susceptible for HSV-1 entry. The following lines of evidence support the important role of ZF-encoded 3-OST-3 isoform as the mediator of HSV-1 entry into CHO-K1 cells: (1) ZF 3-OST-3-expressing CHO-K1 cells were able to preferentially bind HSV-1 glycoprotein D, and (2) CHO-K1 cells expressing ZF-encoded 3-OST-3 acquire the ability to fuse with cells expressing HSV-1 glycoproteins. Finally, knocking down 3-OST-3 receptor by siRNA in ZF fibroblasts cells significantly reduced HSV-1 entry and glycoprotein D binding to cells. Taken together, our results provide novel insight into the significance of ZF 3-OST-3 isoform as an HSV-1 entry and fusion receptor and its potential involvement in the HSV-1 disease model of ZF.

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/ WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G 1 -to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.The tumor suppressor p53 plays a pivotal role in carcinogenesis and is the most frequently mutated gene in human cancers (31,34). Most DNA tumor viruses have mechanisms to inactivate p53. The large T antigen (large T) of simian virus 40 (SV40) binds p53 and inactivates at least some of its functions (44). The E6 proteins of the highly oncogenic human papillomavirus type 16 (HPV-16) and HPV-18 promote the rapid degradation of p53 through a ubiquitin-dependent proteolytic pathway (47). The oncogenic human adenoviruses act to block p53 through dual functions of the E1B proteins acting directly on p53 and on its downstream targets (16).Thus far, none of these mechanisms have been ascribed to the mouse polyomavirus. This is surprising in view of the efficiency and rapidity with which this virus induces tumors. p53 is not stably upregulated in polyomavirus tumors, and most tumor cell lines examined show a normal p53 response to DNA damage (18). Regulation of p53 occurs through a variety of mechanisms that may operate differently in different cell types. The possibility that polyomavirus may have some way of counteracting p53 in various target cells was tested by determining the effect of the absence of p53 on tumor induction by the virus. Tumors arose significantly more rapidly in p53 Ϫ/Ϫ than in p53 ϩ/Ϫ or p53 ϩ/ϩ mice, supporting the generally held view that this virus has no effective way of blocking p53 functions during the course of tumor development (18). This result contrasts with those in the SV40 system, where the large T antigen clearly binds p53 and where the absence of p53 in the host can retard rather than accelerate tumor development (28).A single cycle of polyomavirus growth in mouse cells requires roughly 48 h and is dependent on cell cycle progression from G 0 /G 1 into S. In the absence of a counteracting mechanism(s) by the virus, induction of a p53 response leading either to cell cycle arrest or apoptosis would be expected to block virus growth. Expressio...

Laboratory strains of the mouse polyoma virus differ markedly in their abilities to replicate and induce tumors in newborn mice. Major determinants of pathogenicity lie in the sialic binding pocket of the major capsid protein Vp1 and dictate receptor-binding properties of the virus. Substitutions at two sites in Vp1 define three prototype strains, which vary greatly in pathogenicity. These strains replicate in a limited fashion and induce few or no tumors, cause a disseminated infection leading to the development of multiple solid tumors, or replicate and spread acutely causing early death. This investigation was undertaken to determine the Vp1 type(s) of new virus isolates from naturally infected mice. Compared with laboratory strains, truly wild-type viruses are constrained with respect to their selectivity and avidity of binding to cell receptors. Fifteen of 15 new isolates carried the Vp1 type identical to that of highly tumorigenic laboratory strains. Upon injection into newborn laboratory mice, the new isolates induced a broad spectrum of tumors, including ones of epithelial as well as mesenchymal origin. Though invariant in their Vp1 coding sequences, these isolates showed considerable variation in their regulatory sequences. The common Vp1 type has two essential features: 1) failure to recognize “pseudoreceptors” with branched chain sialic acids binding to which would attenuate virus spread, and 2) maintenance of a hydrophobic contact with true receptors bearing a single sialic acid, which retards virus spread and avoids acute and potentially lethal infection of the host. Conservation of these receptor-binding properties under natural selection preserves the oncogenic potential of the virus. These findings emphasize the importance of immune protection of neonates under conditions of natural transmission.