Efforts to develop drugs for Alzheimer’s disease (AD) have shown promise in animal studies, only to fail in human trials, suggesting a pressing need to study AD in human model systems. Using human neurons derived from induced pluripotent stem cells carrying the major genetic risk factor apolipoprotein E4 (apoE4), we demonstrate that apoE4 neurons have higher levels of tau phosphorylation unrelated to their increased Aβ production and displayed GABAergic neuron degeneration. ApoE4 increased Aβ production in human, but not in mouse, neurons. Converting apoE4 to apoE3 by gene editing rescued these phenotypes, indicating the specific effects of apoE4. Neurons lacking apoE behaved like those expressing apoE3, and introducing apoE4 expression recapitulated the pathological phenotypes, suggesting a gain of toxic effects from apoE4. Treating apoE4 neurons with a small-molecule structure corrector ameliorated the detrimental effects, providing a proof of concept that correcting the pathogenic conformation of apoE4 is a viable therapeutic approach for apoE4-related AD.

Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G 1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/C Cdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G 1 and disappearing in late M. In acm1⌬ strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/C Cdh1 -mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1⌬ cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1⌬ and hsl1⌬ resulted in a dramatic enhancement of bud elongation and G 2 /M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/C Cdh1 .

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.The three isoforms of human apoE (apoE2, apoE3, and apoE4) differ at amino acid positions 112 or 158 or both (1, 2). ApoE4 is the major genetic risk factor for Alzheimer disease (AD) 2 (3-5), and apoE4 carriers account for 65-80% of all AD cases, highlighting the importance of apoE4 in AD pathogenesis (6). Two biophysical properties that distinguish apoE4 from the other isoforms likely hold the key to a mechanistic understanding of its association with AD. First, apoE4 is more unstable and tends to form a molten globule state (7,8). Second, the amino-terminal domain (amino acids 1-191) of apoE4 interacts with its carboxyl-terminal domain (amino acids 223-299) (9, 10). This domain interaction occurs predominantly in apoE4, in which positively charged Arg-112 repels the side chain of Arg-61 in the amino-terminal domain, allowing the formation of a salt bridge between Arg-61 and the negatively charged Glu-255 in the carboxyl-terminal domain (9, 10). Domain interaction occurs to a significantly lesser extent in apoE2 and apoE3 because both have Cys-112, resulting in a different conformation of Arg-61 (11). Importantly, only human apoE has Arg-61; the 17 other species in which the apoE gene has been sequenced have 11). Mutation of Arg-61 to Thr in apoE4 prevents domain interaction, converting apoE4 to an apoE3-like molecule (9 -12). ApoE4 domain interaction occurs on lipoprotein particles in vitro in human plasma, in cultured Neuro-2a cells, and in Arg-61 knock-in mice, in wh...

The anaphase-promoting complex (APC) regulates cell division in eukaryotes by targeting specific proteins for destruction. APC substrates generally contain one or more short degron sequences that help mediate their recognition and poly-ubiquitination by the APC. The most common and well characterized degrons are the destruction box (D box) and the KEN box. The budding yeast Acm1 protein, an inhibitor of Cdh1-activated APC (APC Cdh1 ) also contains several conserved D and KEN boxes, and here we report that two of these located in the central region of Acm1 constitute a pseudosubstrate sequence required for APC Cdh1 inhibition. Acm1 interacted with and inhibited substrate binding to the WD40 repeat domain of Cdh1. Combined mutation of the central D and KEN boxes strongly reduced both binding to the Cdh1 WD40 domain and APC Cdh1 inhibition. Despite this, the double mutant, but not wild-type Acm1, was poly-ubiquitinated by APC Cdh1 in vitro. Thus, unlike substrates in which D and KEN boxes promote ubiquitination, these same elements in the central region of Acm1 prevent ubiquitination. We propose that this unique property of the Acm1 degron sequences results from an unusually high affinity interaction with the substrate receptor site on the WD40 domain of Cdh1 that may serve both to promote APC inhibition and protect Acm1 from destruction.The anaphase-promoting complex (APC), 4 is a highly conserved multisubunit ubiquitin ligase and an important regulator of eukaryotic cell division (1). It targets key cell cycle proteins for proteolysis via the ubiquitin pathway (2), including securin, an inhibitor of chromosome segregation, and the S and M phase cyclin subunits of cyclin-dependent kinase (CDK). Securin proteolysis triggers the initiation of anaphase once all sister chromatids have been properly bi-oriented on the mitotic spindle during metaphase (3-5). Cyclin proteolysis leads to inactivation of CDK, which is necessary for cells to exit from mitosis (6 -8).In addition, APC controls the destruction of numerous other proteins such as Aurora A kinase, Polo-like kinases, the APC co-activator Cdc20, the Skp2 F-box protein, various spindle components, and replication factors such as Cdc6, Dbf4, and geminin (1). Several of these substrates were recently reported to be overexpressed in a wide range of malignant cancers (9), highlighting the critical role APC plays in maintaining genomic stability and proper regulation of the cell division cycle.Although many substrates of APC have been identified, the mechanism by which they are specifically recognized is still poorly understood. Substrate recognition was originally proposed to be mediated by the Cdc20 and Cdh1 proteins, two related WD40 repeat domain proteins that are essential for APC activity at different times during the cell cycle (10, 11). The identification of direct interactions between these co-activators and several substrates (12-16) led to a model for APC activation in which Cdc20 and Cdh1 acted as substrate-recruiting factors, much like the F-box proteins of ...

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC Cdh1 inhibitor. Acm1 disappears in late mitosis when APC Cdh1 becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G 1 /S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.