Current understanding of amyloid-β (Aβ) metabolism and toxicity provides an extensive list of potential targets for developing drugs for treating Alzheimer's disease. We took two independent approaches, including synaptic-plasticity-based analysis and behavioral screening of synthetic compounds, for identifying single compounds that are capable of rescuing the Aβ-induced memory loss in both transgenic fruit fly and transgenic mouse models. Two clinically available drugs and three synthetic compounds not only showed positive effects in behavioral tests but also antagonized the Aβ oligomers-induced activation of the epidermal growth factor receptor (EGFR). Such surprising converging outcomes from two parallel approaches lead us to conclude that EGFR is a preferred target for treating Aβ-induced memory loss.A myloid-β (Aβ) oligomers-induced memory loss is thought to be a hallmark of Alzheimer's disease (AD) progression (1-3). Aβ peptides are cleaved from a membrane protein APP via β-and γ-secretases' activities (4). They can be removed through activities of neprilysin, insulin-degradation enzyme, and possibly other mechanisms (5-7). Aβ is also able to bind with a large array of target proteins, such as EphB2, TNF-R1, RAGE1, and NMDA receptor and prion (8)(9)(10)(11)(12) to exert a wide range of effects, including synaptic transmission, protein transportation, mitochondrial functions, and others (13-15). Thus, there are a large number of potential targets for developing AD treatment based on the Aβ hypothesis, for example, the mechanisms either reducing the production of Aβ peptides or enhancing the degradation process. It remains, however, an open question as to whether some of these targets are, at the organism level, better suited for drug development than others. One important reason is that manipulating activities of such production and degradation enzymes may affect many other physiological proteins. Thus, reported failures in a number of Aβ-based drug efforts (16) stress the necessity of identifying such preferred targets.To evaluate the possibility of finding preferred targets, we looked for overlapping and converging effects of identified targets with multiple independent approaches. First, following the direction of a synaptic-plasticity-based, mechanism-guided study, we continued to work on the signal transduction pathway of PI3-kinase that has been shown to mediate an Aβ-induced change in long-term synaptic depression as well as the Aβ-induced memory loss in Aβ42-expressing Drosophila, which recapitulates several ADlike symptoms (17). These efforts led us to find Aβ42 oligomersinduced activation of the epidermal growth factor receptor (EGFR) and the rescue of Aβ-induced memory loss in transgenic Drosophila and APP/PS1 double transgenic mouse models through treatments with clinically available EGFR inhibitors. Second, we worked to identify single compounds capable of rescuing Aβ-induced memory loss through large-scale behavioral screening with Aβ42-expressing transgenic fruit flies, followed by a confirma...

Accelerated forgetting has been identified as a feature of Alzheimer’s disease (AD), but the therapeutic efficacy of the manipulation of biological mechanisms of forgetting has not been assessed in AD animal models. Ras-related C3 botulinum toxin substrate 1 (Rac1), a small GTPase, has been shown to regulate active forgetting in Drosophila and mice. Here, we showed that Rac1 activity is aberrantly elevated in the hippocampal tissues of AD patients and AD animal models. Moreover, amyloid-beta 42 could induce Rac1 activation in cultured cells. The elevation of Rac1 activity not only accelerated 6-hour spatial memory decay in 3-month-old APP/PS1 mice, but also significantly contributed to severe memory loss in aged APP/PS1 mice. A similar age-dependent Rac1 activity-based memory loss was also observed in an AD fly model. Moreover, inhibition of Rac1 activity could ameliorate cognitive defects and synaptic plasticity in AD animal models. Finally, two novel compounds, identified through behavioral screening of a randomly selected pool of brain permeable small molecules for their positive effect in rescuing memory loss in both fly and mouse models, were found to be capable of inhibiting Rac1 activity. Thus, multiple lines of evidence corroborate in supporting the idea that inhibition of Rac1 activity is effective for treating AD-related memory loss.Electronic supplementary materialThe online version of this article (10.1007/s13238-019-0641-0) contains supplementary material, which is available to authorized users.

Nek2 is a cell cycle-regulated serine/threonine protein kinase that is up-regulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic C termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic, and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A having a weak NLS, and Nek2B having no NLS. Finally, we identified a 28-kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization, enabling it to have both nuclear and cytoplasmic functions.

Mammalian sperm were previously shown to express the PP1gamma2 isoform of protein phosphatase 1 (PP1) as well as its regulatory proteins inhibitor 2 and glycogen synthase kinase 3. Furthermore, the development of sperm motility during transit through the epididymis correlates with changes in PP1 activity. Thus, since PP1 cellular activity is determined by the partners it binds, we embarked on a study aimed at defining the specific interactomes of PP1gamma1 and PP1gamma2 (the two known alternatively spliced variants of PP1gamma). To this end, exhaustive screens were performed on a human testis cDNA library using the yeast two-hybrid method. Among the various proteins detected, the most abundant interactors with PP1gamma2 were Nek2A and R15B. Closer sequence analysis revealed novel alternatively spliced variants of Nek2A and NIPP1, which we designated Nek2A-T and NIPP1-T, respectively. They were shown to be highly expressed in rat and human testis by Northern analysis and to result from alternative splicing events by RT-PCR. Thus, both the previously known Nek2A isoform and the novel Nek2A-T and NIPP1-T variants appear to bind PP1gamma2 in vitro (blot overlays) and in vivo by coexpression in yeast. The usefulness of testis-specific alternatively spliced proteins as targets for the development of novel therapeutic strategies for male infertility and contraception is discussed. PP1gamma2, Nek2A-T, and NIPP1-T are currently being investigated as alternatively spliced targets for signal transduction therapeutics.