Updates on mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the United States (US). Animal models, specifically mouse models have been developed to better elucidate disease mechanisms and test therapeutic strategies for AD. A large portion of effort in the field was focused on developing transgenic (Tg) mouse models through over-expression of genetic mutations associated with familial AD (FAD) patients. Newer generations of mouse models through knock-in (KI)/knock-out (KO) or CRISPR gene editing technologies, have been developed for both familial and sporadic AD risk genes with the hope to more accurately model proteinopathies without over-expression of human AD genes in mouse brains. In this review, we summarized the phenotypes of a few commonly used as well as newly developed mouse models in translational research laboratories including the presence or absence of key pathological features of AD such as amyloid and tau pathology, synaptic and neuronal degeneration as well as cognitive and behavior deficits. In addition, advantages and limitations of these AD mouse models have been elaborated along with discussions of any sex-specific features. More importantly, the omics data from available AD mouse models have been analyzed to categorize molecular signatures of each model reminiscent of human AD brain changes, with the hope to guide future selection of most suitable models for specific research questions to be addressed in the AD field.

Sex specific molecular networks and key drivers of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive and age-associated neurodegenerative disorder that affects women disproportionally. However, the underlying mechanisms are poorly characterized. Moreover, while the interplay between sex and ApoE genotype in AD has been investigated, multi-omics studies to understand this interaction are limited. Therefore, we applied systems biology approaches to investigate sex-specific molecular networks of AD. METHODS: We integrated large-scale human postmortem brain transcriptomic data of AD from two cohorts (MSBB and ROSMAP) via multiscale network analysis and identified key drivers with sexually dimorphic expression patterns and/or different responses to APOE genotypes between sexes. The expression patterns and functional relevance of the top sex-specific network driver of AD were further investigated using postmortem human brain samples and gene perturbation experiments in AD mouse models. RESULTS: Gene expression changes in AD versus control were identified for each sex. Gene co-expression networks were constructed for each sex to identify AD-associated co-expressed gene modules shared by males and females or specific to each sex. Key network regulators were further identified as potential drivers of sex differences in AD development. LRP10 was identified as a top driver of the sex differences in AD pathogenesis and manifestation. Changes of LRP10 expression at the mRNA and protein levels were further validated in human AD brain samples. Gene perturbation experiments in EFAD mouse models demonstrated that LRP10 differentially affected cognitive function and AD pathology in sex- and APOE genotype-specific manners. A comprehensive mapping of brain cells in LRP10 over-expressed (OE) female E4FAD mice suggested neurons and microglia as the most affected cell populations. The female-specific targets of LRP10 identified from the single cell RNA-sequencing (scRNA-seq) data of the LRP10 OE E4FAD mouse brains were significantly enriched in the LRP10-centered subnetworks in female AD subjects, validating LRP10 as a key network regulator of AD in females. Eight LRP10 binding partners were identified by the yeast two-hybrid system screening, and LRP10 over-expression reduced the association of LRP10 with one binding partner CD34. CONCLUSIONS: These findings provide insights into key mechanisms mediating sex differences in AD pathogenesis and will facilitate the development of sex- and APOE genotype-specific therapies for AD.

Multiomics Analyses Identify Proline Endopeptidase-Like Protein As a Key Regulator of Protein Trafficking, a Pathway Underlying Alzheimer's Disease Pathogenesis.

Current treatments for Alzheimer's disease (AD) help reduce symptoms for a limited time but do not treat the underlying pathology. To identify potential therapeutic targets for AD, an integrative network analysis was previously carried out using 364 human postmortem control, mild cognitive impairment, and AD brains. This analysis identified proline endopeptidase-like protein (PREPL), an understudied protein, as a downregulated protein in late-onset AD patients. In this study we investigate the role of PREPL. Analyses of data from human postmortem samples and PREPL knockdown (KD) cells suggest that PREPL expression modulates pathways associated with protein trafficking, synaptic activities, and lipid metabolism. Furthermore, PREPL KD impairs cell proliferation and modulates the structure of vesicles, levels of neuropeptide-processing enzymes, and secretion of neuropeptides. In addition, decrease in PREPL levels leads to changes in the levels of a number of synaptic proteins as well as changes in the levels of secreted amyloid beta (Aß) 42 peptide and Tau phosphorylation. Finally, we report that local decrease in PREPL levels in mouse hippocampus attenuates long-term potentiation, suggesting a role in synaptic plasticity. Together, our results indicate that PREPL affects neuronal function by modulating protein trafficking and synaptic function, an important mechanism of AD pathogenesis. SIGNIFICANCE STATEMENT: Integrative network analysis reveals proline endopeptidase-like protein (PREPL) to be downregulated in human sporadic late-onset Alzheimer's disease brains. Down regulation of PREPL leads to increases in amyloid beta secretion, Tau phosphorylation, and decreases in protein trafficking and long-term potentiation.

Exploring Morphine-Triggered PKC-Targets and Their Interaction with Signaling Pathways Leading to Pain via TrkA.

It is well accepted that treatment of chronic pain with morphine leads to µ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, ß-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain.

Protein folding creates structure-based, noncontiguous consensus phosphorylation motifs recognized by kinases.

Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C ßI (PKCßI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCßI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.

Activation of protein kinase C delta by ψδRACK peptide promotes embryonic stem cell proliferation through ERK 1/2.

The protein kinase C (PKC) family of serine/threonine kinases participate in embryonic stem cell (ESC) proliferation/self-renewal. A few stimuli that induce ESC proliferation activate several PKC isoenzymes including δPKC, however, the role of this isoenzyme under basal conditions that maintain undifferentiated ESCs remains to be determined. Herewith, we aimed to characterize signaling events that occur in undifferentiated ESCs upon δPKC activation. Using phosphoproteomics and a δPKC specific activator peptide, ψδRACK, it was seen that the majority of proteins whose phosphorylation increased upon δPKC activation participate in cell proliferation. Network analysis of these proteins directly connected δPKC to Raf1 and 14-3-3. Experimental validation studies showed that activation of δPKC increased its binding to 14-3-3, transiently activated ERK1/2 and increased ESC proliferation. Independently inhibiting MEK or PI3 kinase both led to a decrease in proliferation of approximately 50%, but δPKC activation only recovered the effect of PI3 kinase inhibition suggesting that ERK1/2 activation via δPKC is probably a parallel pathway to PI3 kinase and that both pathways are necessary for undifferentiated ESC proliferation. BIOLOGICAL SIGNIFICANCE: The use of embryonic stem cells and induced pluripotent stem cells for regenerative therapies is still a challenge. Understanding the underlying mechanisms that keep these cells proliferating with the ability to differentiate in more than 200 cell types (self-renewal) will aid in the future use of these cells therapeutically. Using a targeted phosphoproteomics study, insights into signaling pathways involved in ESC proliferation can be obtained. Modulating these pathways will aid the obtention of a larger number of self-renewing stem cells and induced pluripotent stem cells that can be used therapeutically.

Identificação e validação funcional de novos alvos das PKCs em célula tronco embrionária / Identification and functional validation of new targets of PKC in embryonic stem cell

Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCßI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCßI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCßI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a ß-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCßI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e ß-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na ß-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCßI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCßI em CTE indiferenciadas

Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCßI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCßI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCßI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and ß-tubulin, as a phosphorylation site of α-tubulin by PKCßI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and ß-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in ß-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCßI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCßI in undifferentiated ESC

Phosphoproteomics profiling suggests a role for nuclear ßΙPKC in transcription processes of undifferentiated murine embryonic stem cells.

Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, ßIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of ßΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one ßIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect ßΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting ßΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express ßΙPKC and ßΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that ßIPKC takes part in the processes that maintain ESCs in their undifferentiated state.

Role of p53 in the induction of cyclooxygenase-2 by cisplatin or paclitaxel in non-small cell lung cancer cell lines.

Non-small cell lung Cancer (NSCLC) is extremely resistant to chemotherapeutic agents, such as cisplatin. High expression of the inflammatory enzyme cyclooxygenase-2 (COX-2) has been shown to inhibit chemotherapy-induced apoptosis, but little is known about COX-2 regulation upon drug treatment. Recent data indicate the tumor suppressor protein p53 as an important regulator of COX-2. Therefore, TP53 status could change tumor sensitivity to chemotherapy through induction of the anti-apoptotic protein COX-2. The main objective of this work was to analyze the effect of chemotherapy on the expression of COX-2, according to TP53 status. We report herein that lung cancer cell lines expressing wild-type p53, when exposed to cisplatin treatment, induced COX-2 (mRNA and protein), with concurrent synthesis of prostaglandins (PGE(2)). In contrast, COX-2 expression was not changed after cisplatin treatment of cells containing an inactive form of p53. Further, after silencing of wild-type p53 expressed in A549 cells by RNA interference, cisplatin was no longer able to induce COX-2 expression. Therefore, we suggest that induction of COX-2 by cisplatin in NSCLC cell lines is dependent on p53. For paclitaxel treatment, an increase in COX-2 mRNA expression was observed in H460 and A549 (wild-type p53 cell lines). Moreover, paclitaxel treatment increased COX-2 expression in ACC-LC-319 cell lines (p53 null), showing a p53-independent effect. These data may have therapeutic implications in the selection of patients and strategy for future COX-2 inhibition trials.