RESUMEN
Alzheimer's disease (AD) is a complex neurodegenerative disorder that develops over decades. AD brain proteomics reveals vast alterations in protein levels and numerous altered biologic pathways. Here, we compare AD brain proteome and network changes with the brain proteomes of amyloid ß (Aß)-depositing mice to identify conserved and divergent protein networks with the conserved networks identifying an Aß amyloid responsome. Proteins in the most conserved network (M42) accumulate in plaques, cerebrovascular amyloid (CAA), and/or dystrophic neuronal processes, and overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), increases the accumulation of Aß in plaques and CAA. M42 proteins bind amyloid fibrils in vitro, and MDK and PTN co-accumulate with cardiac transthyretin amyloid. M42 proteins appear intimately linked to amyloid deposition and can regulate amyloid deposition, suggesting that they are pathology modifiers and thus putative therapeutic targets. We posit that amyloid-scaffolded accumulation of numerous M42+ proteins is a central mechanism mediating downstream pathophysiology in AD.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Encéfalo , Placa Amiloide , Proteómica , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Proteómica/métodos , Animales , Péptidos beta-Amiloides/metabolismo , Humanos , Placa Amiloide/metabolismo , Placa Amiloide/patología , Ratones , Encéfalo/metabolismo , Encéfalo/patología , Proteoma/metabolismo , Ratones Transgénicos , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Citocinas/metabolismo , MasculinoRESUMEN
We report a highly significant correlation in brain proteome changes between Alzheimers disease (AD) and CRND8 APP695NL/F transgenic mice. However, integrating protein changes observed in the CRND8 mice with co-expression networks derived from human AD, reveals both conserved and divergent module changes. For the most highly conserved module (M42, matrisome) we find many proteins accumulate in plaques, cerebrovascular amyloid (CAA), dystrophic processes, or a combination thereof. Overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), in CRND8 mice brains leads to increased accumulation of A ß ; in plaques and in CAA; further, recombinant MDK and PTN enhance A ß ; aggregation into amyloid. Multiple M42 proteins, annotated as heparan sulfate binding proteins, bind to fibrillar A ß 42 and a non-human amyloid fibril in vitro. Supporting this binding data, MDK and PTN co-accumulate with transthyretin (TTR) amyloid in the heart and islet amyloid polypeptide (IAPP) amyloid in the pancreas. Our findings establish several critical insights. Proteomic changes in modules observed in human AD brains define an A ß ; amyloid responsome that is well conserved from mouse model to human. Further, distinct amyloid structures may serve as scaffolds, facilitating the co-accumulation of proteins with signaling functions. We hypothesize that this co-accumulation may contribute to downstream pathological sequalae. Overall, this contextualized understanding of proteomic changes and their interplay with amyloid deposition provides valuable insights into the complexity of AD pathogenesis and potential biomarkers and therapeutic targets.
RESUMEN
Aggregation and accumulation of amyloid-ß (Aß) is a defining feature of Alzheimer's disease pathology. To study microglial responses to Aß, we applied exogenous Aß peptide, in either oligomeric or fibrillar conformation, to primary mouse microglial cultures and evaluated system-level transcriptional changes and then compared these with transcriptomic changes in the brains of CRND8 APP mice. We find that primary microglial cultures have rapid and massive transcriptional change in response to Aß. Transcriptomic responses to oligomeric or fibrillar Aß in primary microglia, although partially overlapping, are distinct and are not recapitulated in vivo where Aß progressively accumulates. Furthermore, although classic immune mediators show massive transcriptional changes in the primary microglial cultures, these changes are not observed in the mouse model. Together, these data extend previous studies which demonstrate that microglia responses ex vivo are poor proxies for in vivo responses. Finally, these data demonstrate the potential utility of using microglia as biosensors of different aggregate conformation, as the transcriptional responses to oligomeric and fibrillar Aß can be distinguished.
Asunto(s)
Péptidos beta-Amiloides/genética , Microglía/metabolismo , Ovillos Neurofibrilares/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/fisiología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/fisiología , Cultivo Primario de Células , Transcriptoma/genéticaRESUMEN
INTRODUCTION: Passive immunotherapies targeting Aß continue to be evaluated as Alzheimer's disease (AD) therapeutics, but there remains debate over the mechanisms by which these immunotherapies work. Besides the amount of preexisting Aß deposition and the type of deposit (compact or diffuse), there is little data concerning what factors, independent of those intrinsic to the antibody, might influence efficacy. Here we (i) explored how constitutive priming of the underlying innate activation states by Il10 and Il6 might influence passive Aß immunotherapy and (ii) evaluated transcriptomic data generated in the AMP-AD initiative to inform how these two cytokines and their receptors' mRNA levels are altered in human AD and an APP mouse model. METHODS: rAAV2/1 encoding EGFP, Il6 or Il10 were delivered by somatic brain transgenesis to neonatal (P0) TgCRND8 APP mice. Then, at 2 months of age, the mice were treated bi-weekly with a high-affinity anti-Aß1-16 mAb5 monoclonal antibody or control mouse IgG until 6 months of age. rAAV mediated transgene expression, amyloid accumulation, Aß levels and gliosis were assessed. Extensive transcriptomic data was used to evaluate the mRNA expression levels of IL10 and IL6 and their receptors in the postmortem human AD temporal cortex and in the brains of TgCRND8 mice, the later at multiple ages. RESULTS: Priming TgCRND8 mice with Il10 increases Aß loads and blocks efficacy of subsequent mAb5 passive immunotherapy, whereas priming with Il6 priming reduces Aß loads by itself and subsequent Aß immunotherapy shows only a slightly additive effect. Transcriptomic data shows that (i) there are significant increases in the mRNA levels of Il6 and Il10 receptors in the TgCRND8 mouse model and temporal cortex of humans with AD and (ii) there is a great deal of variance in individual mouse brain and the human temporal cortex of these interleukins and their receptors. CONCLUSIONS: The underlying immune activation state can markedly affect the efficacy of passive Aß immunotherapy. These results have important implications for ongoing human AD immunotherapy trials, as they indicate that underlying immune activation states within the brain, which may be highly variable, may influence the ability for passive immunotherapy to alter Aß deposition.
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Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Inmunidad Innata/efectos de los fármacos , Inmunización Pasiva/métodos , Animales , Humanos , Interleucina-10/inmunología , Interleucina-6/inmunología , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS: For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aß and Aß-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT: These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION: Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term ß-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.
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Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Células HEK293 , Humanos , Presenilina-2/genética , Transducción de SeñalRESUMEN
Hypothalamic-pituitary-adrenal (HPA) axis dysfunction contributes to numerous human diseases and disorders. We developed a high-affinity monoclonal antibody, CTRND05, targeting corticotropin-releasing factor (CRF). In mice, CTRND05 blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis. CTRND05 induces skeletal muscle hypertrophy and increases lean body mass, effects not previously reported with small-molecule HPA-targeting pharmacologic agents. Multiorgan transcriptomics demonstrates broad HPA axis target engagement through altering levels of known HPA-responsive transcripts such as Fkbp5 and Myostatin and reveals novel HPA-responsive pathways such as the Apelin-Apelin receptor system. These studies demonstrate the therapeutic potential of CTRND05 as a suppressor of the HPA axis and serve as an exemplar of a potentially broader approach to target neuropeptides with immunotherapies, as both pharmacologic tools and novel therapeutics.
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Anticuerpos Monoclonales/farmacología , Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Corticosterona/inmunología , Corticosterona/metabolismo , Hormona Liberadora de Corticotropina/inmunología , Perfilación de la Expresión Génica/métodos , Humanos , Sistema Hipotálamo-Hipofisario/inmunología , Sistema Hipotálamo-Hipofisario/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Sistema Hipófiso-Suprarrenal/inmunología , Sistema Hipófiso-Suprarrenal/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Estrés Fisiológico/inmunologíaRESUMEN
Cardiac contractility is enhanced by phosphorylation of myosin light chain 2 (MLC2) by cardiac-specific MLC kinase (cMLCK), located at the neck region of myosin heavy chain. In normal mouse and human hearts, the level of phosphorylation is maintained relatively constant, at around 30-40% of total MLC2, likely by well-balanced phosphorylation and phosphatase-dependent dephosphorylation. Overexpression of cMLCK promotes sarcomere organization, while the loss of cMLCK leads to cardiac atrophy in vitro and in vivo. In this study, we showed that cMLCK is predominantly expressed at the Z-disc with additional diffuse cytosolic expression in normal adult mouse and human hearts. cMLCK interacts with the Z-disc protein, α-actinin2, with a high-affinity kinetic value of 13.4 ± 0.1 nM through the N-terminus region of cMLCK unique to cardiac-isoform. cMLCK mutant deficient for interacting with α-actinin2 did not promote sarcomeric organization and reduced cardiomyocyte cell size. In contrast, a cMLCK kinase-deficient mutant showed effects similar to wild-type cMLCK on sarcomeric organization and cardiomyocyte cell size. Our results suggest that cMLCK plays a role in sarcomere organization, likely distinct from its role in phosphorylating MLC2, both of which will contribute to the enhancement of cardiac contractility.
Asunto(s)
Actinina/metabolismo , Miosinas Cardíacas/metabolismo , Miocardio/enzimología , Cadenas Ligeras de Miosina/metabolismo , Adulto , Animales , Miosinas Cardíacas/química , Miosinas Cardíacas/genética , Humanos , Recién Nacido , Ratones , Mutación , Miocitos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Especificidad por SustratoRESUMEN
Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular ß-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.
Asunto(s)
Presenilina-1/fisiología , Presenilina-2/fisiología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Sistemas CRISPR-Cas , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Hidrólisis , Ratones , Presenilina-1/genética , Presenilina-2/genética , Especificidad por SustratoRESUMEN
Amyloid precursor protein (APP) and its metabolites play key roles in Alzheimer's disease (AD) pathophysiology. Whereas short amyloid-ß (Aß) peptides derived from APP are pathogenic, the APP holoprotein serves multiple purposes in the nervous system through its cell adhesion and receptor-like properties. Our studies focused on the signaling mediated by the APP cytoplasmic tail. We investigated whether sustained APP signaling during brain development might favor neuronal plasticity and memory process through a direct interaction with the heterotrimeric G-protein subunit GαS (stimulatory G-protein alpha subunit). Our results reveal that APP possesses autonomous regulatory capacity within its intracellular domain that promotes APP cell surface residence, precludes Aß production, facilitates axodendritic development, and preserves cellular substrates of memory. Altogether, these events contribute to strengthening cognitive functions and are sufficient to modify the course of AD pathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Memoria , Neurogénesis , Transducción de Señal , Precursor de Proteína beta-Amiloide/química , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Células Cultivadas , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal , Dominios ProteicosRESUMEN
Rare coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with increased risk for Alzheimer's disease (AD), but how they confer this risk remains uncertain. We assessed binding of TREM2, AD-associated TREM2 variants to various forms of Aß and APOE in multiple assays. TREM2 interacts directly with various forms of Aß, with highest affinity interactions observed between TREM2 and soluble Aß42 oligomers. High-affinity binding of TREM2 to Aß oligomers is characterized by very slow dissociation. Pre-incubation with Aß is shown to block the interaction of APOE In cellular assays, AD-associated variants of TREM2 reduced the amount of Aß42 internalized, and in NFAT assay, the R47H and R62H variants decreased NFAT signaling activity in response to Aß42. These studies demonstrate i) a high-affinity interaction between TREM2 and Aß oligomers that can block interaction with another TREM2 ligand and ii) that AD-associated TREM2 variants bind Aß with equivalent affinity but show loss of function in terms of signaling and Aß internalization.
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Péptidos beta-Amiloides/metabolismo , Glicoproteínas de Membrana/metabolismo , Multimerización de Proteína , Receptores Inmunológicos/metabolismo , Transducción de Señal , Enfermedad de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Endocitosis , Células HEK293 , Humanos , Factores de Transcripción NFATC/metabolismo , Unión Proteica , SolubilidadRESUMEN
Phosphorylation of the microtubule associated protein tau is an important modulator of its normal physiological functioning; however, it may also contribute to tau mis-folding and aggregation in neurodegenerative diseases, which are collectively termed tauopathies. As such, the investigations of tau phosphorylation and kinases that modify tau are important in trying to elucidate tau function and the mechanisms involved in the development of tauopathies. We have recently demonstrated that the putative tau kinase leucine-rich repeat kinase 2 is capable of phosphorylating tau at threonines 169 and 175 in vitro, and it has been previously shown that hyperphosphorylation at threonine 175 occurs in filamentous tau species from Alzheimer's brain tissue. These prior findings suggest that further studies of phosphorylation of tau at these epitopes may shed light on the pathogenesis of tauopathies. There is, however, a lack of tools available to analyze phosphorylation of tau at these sites. This study aimed to bridge that resource gap by generating monoclonal antibodies against tau phosphorylated at either threonine 169 or 175. While we did not succeed in generating a phospho-specific antibody, we did generate an antibody, MHT2, which is specific for human tau encompassing the threonine 169/175 epitope region. Immunostaining of transgenic rTg4510 mouse tissue as well as human tauopathy cases with MHT2 indicates that this antibody selectively detects cytoplasmic tau in the form of neurofibrillary tangles, and that it may have a further specificity pertaining to severity of disease progression, either because of phosphorylation or conformational bias.
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Anticuerpos Monoclonales/inmunología , Epítopos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteínas tau/inmunología , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Escherichia coli , Femenino , Células HEK293 , Humanos , Inmunohistoquímica , Ratones Transgénicos , Fosforilación , Proteínas Recombinantes/inmunología , Tauopatías/metabolismo , Tauopatías/patología , Treonina/metabolismoRESUMEN
There is considerable interest in harnessing innate immunity to treat Alzheimer's disease (AD). Here, we explore whether a decoy receptor strategy using the ectodomain of select TLRs has therapeutic potential in AD. AAV-mediated expression of human TLR5 ectodomain (sTLR5) alone or fused to human IgG4 Fc (sTLR5Fc) results in robust attenuation of amyloid ß (Aß) accumulation in a mouse model of Alzheimer-type Aß pathology. sTLR5Fc binds to oligomeric and fibrillar Aß with high affinity, forms complexes with Aß, and blocks Aß toxicity. Oligomeric and fibrillar Aß modulates flagellin-mediated activation of human TLR5 but does not, by itself, activate TLR5 signaling. Genetic analysis shows that rare protein coding variants in human TLR5 may be associated with a reduced risk of AD. Further, transcriptome analysis shows altered TLR gene expression in human AD. Collectively, our data suggest that TLR5 decoy receptor-based biologics represent a novel and safe Aß-selective class of biotherapy in AD.
Asunto(s)
Enfermedad de Alzheimer , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Inmunoglobulina G/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Receptor Toll-Like 5/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/inmunología , Animales , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/inmunologíaRESUMEN
Processing of amyloid-ß (Aß) precursor protein (APP) by γ-secretase produces multiple species of Aß: Aß40, short Aß peptides (Aß37-39), and longer Aß peptides (Aß42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aß42 but increase the relative abundance of short Aß peptides. To evaluate the pathological relevance of these peptides, we expressed Aß36-40 and Aß42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aß42 toxicity. In contrast to Aß42, the short Aß peptides were not toxic and, when coexpressed with Aß42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aß38 and Aß40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aß42 deposition, Aß38 and Aß40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aß42 by raising the levels of short Aß peptides could attenuate the toxic effects of Aß42.
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Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Amiloide/genética , Amiloide/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Drosophila melanogaster , Ojo/metabolismo , Ojo/patología , Ojo/ultraestructura , Femenino , Locomoción , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fenotipo , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismoRESUMEN
BACKGROUND: Amyloid-ß (Aß) 42 has been implicated as the initiating molecule in the pathogenesis of Alzheimer's disease (AD); thus, therapeutic strategies that target Aß42 are of great interest. γ-Secretase modulators (GSMs) are small molecules that selectively decrease Aß42. We have previously reported that many acidic steroids are GSMs with potencies ranging in the low to mid micromolar concentration with 5ß-cholanic acid being the most potent steroid identified GSM with half maximal effective concentration (EC50) of 5.7 µM. RESULTS: We find that the endogenous cholesterol metabolite, 3ß-hydroxy-5-cholestenoic acid (CA), is a steroid GSM with enhanced potency (EC50 of 250 nM) relative to 5ß-cholanic acid. CA i) is found in human plasma at ~100-300 nM concentrations ii) has the typical acidic GSM signature of decreasing Aß42 and increasing Aß38 levels iii) is active in in vitro γ-secretase assay iv) is made in the brain. To test if CA acts as an endogenous GSM, we used Cyp27a1 knockout (Cyp27a1-/-) and Cyp7b1 knockout (Cyp7b1-/-) mice to investigate if manipulation of cholesterol metabolism pathways relevant to CA formation would affect brain Aß42 levels. Our data show that Cyp27a1-/- had increased brain Aß42, whereas Cyp7b1-/- mice had decreased brain Aß42 levels; however, peripheral dosing of up to 100 mg/kg CA did not affect brain Aß levels. Structure-activity relationship (SAR) studies with multiple known and novel CA analogs studies failed to reveal CA analogs with increased potency. CONCLUSION: These data suggest that CA may act as an endogenous GSM within the brain. Although it is conceptually attractive to try and increase the levels of CA in the brain for prevention of AD, our data suggest that this will not be easily accomplished.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Colesterol/análogos & derivados , Fragmentos de Péptidos/metabolismo , Animales , Barrera Hematoencefálica , Células CHO , Células Cultivadas , Colestanotriol 26-Monooxigenasa/deficiencia , Colestanotriol 26-Monooxigenasa/genética , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacología , Ácidos Cólicos/farmacología , Técnicas de Cocultivo , Cricetinae , Cricetulus , Familia 7 del Citocromo P450 , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Molecular , Neuroglía/metabolismo , Neuronas/metabolismo , Esteroide Hidroxilasas/deficiencia , Esteroide Hidroxilasas/genética , Relación Estructura-ActividadRESUMEN
BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder that is pathologically characterized by loss of dopaminergic neurons from the substantia nigra, the presence of aggregated α-synuclein (αS) and evidence of neuroinflammation. Experimental studies have shown that the cerebral injection of recombinant fibrillar αS, especially in αS transgenic mouse models, can induce the formation and spread of αS inclusion pathology. However, studies reporting this phenomenon did not consider the presence of lipopolysaccharide (LPS) in the injected αS, produced in E. coli, as a potential confound. The objectives of this study are to develop a method to remove the LPS contamination and investigate the differences in pathologies induced by αS containing LPS or αS highly purified of LPS. RESULTS AND CONCLUSIONS: We were able to remove >99.5% of the LPS contamination from the αS preparations through the addition of a cation exchange step during purification. The αS pathology induced by injection of fibrils produced from αS containing LPS or purified of LPS, showed a similar distribution pattern; however, there was less spread into the cortex of the mice injected with αS containing higher levels of LPS. As previously reported, injection of αS fibrils could induce astrogliosis, and αS inclusions were present within astrocytes in mice injected with fibrils comprised of αS with or without cation exchange purification. Furthermore, we identified the presence of αS pathology in ependymal cells in both groups of mice, which suggests the involvement of a novel mechanism for spread in this model of αS pathology.
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Endotoxinas/farmacología , Trastornos Parkinsonianos/inducido químicamente , alfa-Sinucleína/toxicidad , Animales , Astrocitos/patología , Recuento de Células , Células Cultivadas , Cromatografía por Intercambio Iónico , Progresión de la Enfermedad , Contaminación de Medicamentos , Endotoxinas/aislamiento & purificación , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/patología , Escherichia coli/química , Escherichia coli/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cuerpos de Inclusión/química , Inflamación , Inyecciones , Ratones , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/patología , Trastornos Parkinsonianos/patología , Placa Amiloide/química , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/toxicidad , alfa-Sinucleína/administración & dosificación , alfa-Sinucleína/aislamiento & purificaciónRESUMEN
Altered production of ß-amyloid (Aß) from the amyloid precursor protein (APP) is closely associated with Alzheimer's disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of ß-secretase derived APP carboxyl terminal fragment (CTFß, C99) impairs processive cleavage by γ-secretase increasing production of long Aßs (e.g., Aß1-42, 43). Other studies report that APP CTFß dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aß, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aß. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aß production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aß production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Medios de Cultivo Condicionados/química , Ensayo de Inmunoadsorción Enzimática , Espectrometría de Masas , Mutación , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-ß (Aß) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer's disease modify the profile of Aß isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aß42 and in some circumstances Aß43. Compounds termed γ-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of Aß42, respectively. As GSMs lower the level of production of pathogenic forms of long Aß isoforms, they are of great interest as potential Alzheimer's disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the γ-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate γ-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Procesamiento Proteico-Postraduccional/genética , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/química , Animales , Células CHO , Cricetinae , Cricetulus , Regulación hacia Abajo/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual/genética , Especificidad por Sustrato/genética , Regulación hacia Arriba/genéticaRESUMEN
Understanding how different species of Aß are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aßx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aß fusion protein expression constructs designed to generate peptides from Aß1-38 to Aß1-55 and C99 (CTFß) were transfected into cells, and Aß production was assessed. Secreted and cell-associated Aß were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aß peptides from 1-38 to 1-55 were readily detected in the cells and as soluble full-length Aß proteins in the media. Aß peptides longer than Aß1-48 were efficiently cleaved by γ-secretase and produced varying ratios of Aß1-40:Aß1-42. γ-Secretase cleavage of Aß1-51 resulted in much higher levels of Aß1-42 than any other long Aß peptides, but the processing of Aß1-51 was heterogeneous with significant amounts of shorter Aßs, including Aß1-40, produced. Two PSEN1 variants altered Aß1-42 production from Aß1-51 but not Aß1-49. Unexpectedly, long Aß peptide substrates such as Aß1-49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aß substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aß, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Péptidos beta-Amiloides/química , Modelos Químicos , Inhibidores de Proteasas/química , Proteolisis , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Aggregation and accumulation of Aß42 play an initiating role in Alzheimer's disease (AD); thus, selective lowering of Aß42 by γ-secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aß production, we screened 170 steroids at 10 µM for effects on Aß42 secreted from human APP-overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aß42, whereas many nonacidic steroids actually raised Aß42. Studies on the more potent compounds showed that Aß42-lowering steroids were bonafide GSMs and Aß42-raising steroids were inverse GSMs. The most potent steroid GSM identified was 5ß-cholanic acid (EC50=5.7 µM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4-androsten-3-one-17ß-carboxylic acid ethyl ester (EC50=6.25 µM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aß production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/efectos de los fármacos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Esteroides/química , Esteroides/farmacología , Animales , Células CHO , Línea Celular , Colesterol/farmacología , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Estrógenos/farmacología , Humanos , Espectrometría de Masas , Progesterona/farmacologíaRESUMEN
Inspired by marine cyanobacterial natural products, we synthesized modified peptides with a central statine-core unit, characteristic for aspartic protease inhibition. A series of tasiamide B analogues inhibited BACE1, a therapeutic target in Alzheimer's disease. We probed the stereospecificity of target engagement and determined additional structure-activity relationships with respect to BACE1 and related aspartic proteases, cathepsins D and E. We cocrystallized selected inhibitors with BACE1 to reveal the structural basis for the activity. Hybrid molecules that combine features of tasiamide B and an isophthalic acid moiety-containing sulfonamide showed nanomolar cellular activity. Compounds were screened in a series of rigorous complementary cell-based assays. We measured secreted APP ectodomain (sAPPß), membrane bound carboxyl terminal fragment (CTF), levels of ß-amyloid (Aß) peptides and selectivity for ß-secretase (BACE1) over γ-secretase. Prioritized compounds showed reasonable stability in vitro and in vivo, and our most potent inhibitor showed efficacy in reducing Aß levels in the rodent brain.