RESUMEN
The microtubule associated protein tau is an intrinsically disordered phosphoprotein that accumulates under pathological conditions leading to formation of neurofibrillary tangles, a hallmark of Alzheimer's disease (AD). The mechanisms that initiate the accumulation of phospho-tau aggregates and filamentous deposits are largely unknown. In the past, our work and others' have shown that molecular chaperones play a crucial role in maintaining protein homeostasis and that imbalance in their levels or activity can drive tau pathogenesis. We have found two co-chaperones of the 90 kDa heat shock protein (Hsp90), FK506-binding protein 52 (FKBP52) and the activator of Hsp90 ATPase homolog 1 (Aha1), promote tau aggregation in vitro and in the brains of tau transgenic mice. Based on this, we hypothesized that increased levels of these chaperones could promote tau misfolding and accumulation in the brains of aged wild-type mice. We tested this hypothesis by overexpressing Aha1, FKBP52, or mCherry (control) proteins in the hippocampus of 9-month-old wild-type mice. After 7 months of expression, mice were evaluated for cognitive and pathological changes. Our results show that FKBP52 overexpression impaired spatial reversal learning, while Aha1 overexpression impaired associative learning in aged wild-type mice. FKBP52 and Aha1 overexpression promoted phosphorylation of distinct AD-relevant tau species. Furthermore, FKBP52 activated gliosis and promoted neuronal loss leading to a reduction in hippocampal volume. Glial activation and phospho-tau accumulation were also detected in areas adjacent to the hippocampus, including the entorhinal cortex, suggesting that after initiation these pathologies can propagate through other brain regions. Overall, our findings suggest a role for chaperone imbalance in the initiation of tau accumulation in the aging brain.
Asunto(s)
Encéfalo/patología , Chaperonas Moleculares/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Tauopatías/patología , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Proteínas HSP90 de Choque Térmico/metabolismo , Ratones , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Tauopatías/metabolismoRESUMEN
Tauopathies display a spectrum of phenotypes from cognitive to affective behavioral impairments; however, mechanisms promoting tau pathology and how tau elicits behavioral impairment remain unclear. We report a unique interaction between polyamine metabolism, behavioral impairment, and tau fate. Polyamines are ubiquitous aliphatic molecules that support neuronal function, axonal integrity, and cognitive processing. Transient increases in polyamine metabolism hallmark the cell's response to various insults, known as the polyamine stress response (PSR). Dysregulation of gene transcripts associated with polyamine metabolism in Alzheimer's disease (AD) brains were observed, and we found that ornithine decarboxylase antizyme inhibitor 2 (AZIN2) increased to the greatest extent. We showed that sustained AZIN2 overexpression elicited a maladaptive PSR in mice with underlying tauopathy (MAPT P301S; PS19). AZIN2 also increased acetylpolyamines, augmented tau deposition, and promoted cognitive and affective behavioral impairments. Higher-order polyamines displaced microtubule-associated tau to facilitate polymerization but also decreased tau seeding and oligomerization. Conversely, acetylpolyamines promoted tau seeding and oligomers. These data suggest that tauopathies launch an altered enzymatic signature that endorses a feed-forward cycle of disease progression. Taken together, the tau-induced PSR affects behavior and disease continuance, but may also position the polyamine pathway as a potential entry point for plausible targets and treatments of tauopathy, including AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Poliaminas Biogénicas/metabolismo , Carboxiliasas/metabolismo , Proteínas Portadoras/metabolismo , Hipocampo/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Carboxiliasas/genética , Proteínas Portadoras/genética , Femenino , Hipocampo/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Effective SARS-CoV-2 antiviral drugs are desperately needed. The SARS-CoV-2 main protease (Mpro) appears as an attractive target for drug development. We show that the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of SARS-CoV-2 Mpro. We screened a collection of ~6,070 drugs with a previous history of use in humans for compounds that inhibit the activity of Mpro in vitro and found ~50 compounds with activity against Mpro. Subsequent dose validation studies demonstrated 8 dose responsive hits with an IC50 ≤ 50 µM. Hits from our screen are enriched with hepatitis C NS3/4A protease targeting drugs including boceprevir, ciluprevir. narlaprevir, and telaprevir. This work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than boceprevir and suitable for rapid repurposing.
Asunto(s)
Antivirales/farmacología , Antivirales/uso terapéutico , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , SARS-CoV-2/efectos de los fármacos , Bioensayo , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Reproducibilidad de los ResultadosRESUMEN
Tauopathies are neurological disorders characterized by intracellular tau deposits forming neurofibrillary tangles, neuropil threads, or other disease-specific aggregates composed of the protein tau. Tauopathy disorders include frontotemporal lobar degeneration, corticobasal degeneration, Pick's disease, and the largest cause of dementia, Alzheimer's disease. The lack of disease-modifying therapeutic strategies to address tauopathies remains a critical unmet need in dementia care. Thus, novel broad-spectrum tau-targeted therapeutics could have a profound impact in multiple tauopathy disorders, including Alzheimer's disease. Here we have designed a drug discovery paradigm to identify inhibitors of the pathological tau-enabling protein, MSUT2. We previously showed that activity of the RNA-binding protein MSUT2 drives tauopathy, including tau-mediated neurodegeneration and cognitive dysfunction, in mouse models. Thus, we hypothesized that MSUT2 inhibitors could be therapeutic for tauopathy disorders. Our pipeline for MSUT2 inhibitory compound identification included a primary AlphaScreen, followed by dose-response validation, a secondary fluorescence polarization orthogonal assay, a tertiary specificity screen, and a preliminary toxicity screen. Our work here serves as a proof-of-principle methodology for finding specific inhibitors of the poly(A) RNA-binding protein MSUT2 interaction. Here we identify 4,4'-diisothiocyanostilbene-2,2'-sulfonic acid (DIDS) as a potential tool compound for future work probing the mechanism of MSUT2-induced tau pathology.
Asunto(s)
Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Proteínas Portadoras/genética , Ensayos Analíticos de Alto Rendimiento , Fármacos Neuroprotectores/farmacología , Nootrópicos/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Clonación Molecular , Descubrimiento de Drogas/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Fármacos Neuroprotectores/química , Nootrópicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Tauopatías/tratamiento farmacológico , Tauopatías/genética , Tauopatías/metabolismo , Tauopatías/patología , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Catalysis of cis/trans isomerization of prolines is important for the activity and misfolding of intrinsically disordered proteins. Catalysis is achieved by peptidylprolyl isomerases, a superfamily of molecular chaperones. Here, we provide atomic insight into a tug-of-war between cis/trans isomerization and molecular chaperone activity. Catalysis of proline isomerization by cyclophilin A lowers the energy barrier for α-synuclein misfolding, while isomerase-binding to a separate, disease-associated protein region opposes aggregation. We further show that cis/trans isomerization outpowers the holding activity of cyclophilin A. Removal of the proline isomerization barrier through posttranslational truncation of α-synuclein reverses the action of the proline isomerase and turns it into a potent molecular chaperone that inhibits protein misfolding. The data reveal a conserved mechanism of dual functionality in cis/trans isomerases and define its molecular determinants acting on intrinsically disordered proteins.
Asunto(s)
Chaperonas Moleculares/metabolismo , Prolina/química , Prolina/metabolismo , Amiloide/química , Catálisis , Ciclofilina A/química , Ciclofilina A/metabolismo , Ciclosporina/farmacología , Humanos , Isomerismo , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Enfermedad de Parkinson/metabolismo , Agregado de Proteínas , Unión Proteica , Dominios Proteicos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
Neurofibrillary tangles composed of aberrantly aggregating tau protein are a hallmark of Alzheimer's disease and related dementia disorders. Recent work has shown that mammalian suppressor of tauopathy 2 (MSUT2), also named ZC3H14 (Zinc Finger CCCH-Type Containing 14), controls accumulation of pathological tau in cultured human cells and mice. Knocking out MSUT2 protects neurons from neurodegenerative tauopathy and preserves learning and memory. MSUT2 protein functions to bind polyadenosine [poly(A)] tails of mRNA through its C-terminal CCCH type zinc finger domains, and loss of CCCH domain function suppresses tauopathy in Caenorhabditis elegans and mice. Thus, we hypothesized that inhibiting the poly(A):MSUT2 RNA-protein interaction would ameliorate pathological tau accumulation. Here we present a high-throughput screening method for the identification of small molecules inhibiting the poly(A):MSUT2 RNA-protein interaction. We employed a fluorescent polarization assay for initial small molecule discovery with the intention to repurpose hits identified from the NIH Clinical Collection (NIHCC). Our drug repurposing development workflow included validation of hits by dose-response analysis, specificity testing, orthogonal assays of activity, and cytotoxicity. Validated compounds passing through this screening funnel will be evaluated for translational effectiveness in future studies. This preclinical drug development pipeline identified diverse FDA approved drugs duloxetine, saquinavir, and clofazimine as potential repurposing candidates for reducing pathological tau accumulation.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Animales , Ratones , Poli A , ARN , Tauopatías/tratamiento farmacológico , Proteínas tauRESUMEN
Peptidylprolyl isomerases (PPIases) catalyze cis/trans isomerization of prolines. The PPIase CypA colocalizes with the Parkinson's disease (PD)-associated protein α-synuclein in cells and interacts with α-synuclein oligomers. Herein, we describe atomic insights into the molecular details of the α-synuclein/CypA interaction. NMR spectroscopy shows that CypA catalyzes isomerization of prolineâ 128 in the C-terminal domain of α-synuclein. Strikingly, we reveal a second CypA-binding site formed by the hydrophobic sequence 47 GVVHGVATVA56 , termed PreNAC. The 1.38â Å crystal structure of the CypA/PreNAC complex displays a contact between alanineâ 53 of α-synuclein and glutamineâ 111 in the catalytic pocket of CypA. Mutation of alanineâ 53 to glutamate, as found in patients with early-onset PD, weakens the interaction of α-synuclein with CypA. Our study provides high-resolution insights into the structure of the PD-associated protein α-synuclein in complex with the most abundant cellular cyclophilin.
Asunto(s)
Ciclofilina A/metabolismo , alfa-Sinucleína/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Ciclofilina A/química , Isomerismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Prolina/química , Unión Proteica , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
BACKGROUND: Tau stabilizes microtubules; however, in Alzheimer's disease (AD) and tauopathies, tau becomes hyperphosphorylated, aggregates, and results in neuronal death. Our group recently uncovered a unique interaction between polyamine metabolism and tau fate. Polyamines exert an array of physiological effects that support neuronal function and cognitive processing. Specific stimuli can elicit a polyamine stress response (PSR), resulting in altered central polyamine homeostasis. Evidence suggests that elevations in polyamines following a short-term stressor are beneficial; however, persistent stress and subsequent PSR activation may lead to maladaptive polyamine dysregulation, which is observed in AD, and may contribute to neuropathology and disease progression. METHODS: Male and female mice harboring tau P301L mutation (rTg4510) were examined for a tau-induced central polyamine stress response (tau-PSR). The direct effect of tau-PSR byproducts on tau fibrillization and oligomerization were measured using a thioflavin T assay and a N2a split superfolder GFP-Tau (N2a-ssGT) cell line, respectively. To therapeutically target the tau-PSR, we bilaterally injected caspase 3-cleaved tau truncated at aspartate 421 (AAV9 Tau ΔD421) into the hippocampus and cortex of spermidine/spermine-N1-acetyltransferase (SSAT), a key regulator of the tau-PSR, knock out (SSAT-/-), and wild type littermates, and the effects on tau neuropathology, polyamine dysregulation, and behavior were measured. Lastly, cellular models were employed to further examine how SSAT repression impacted tau biology. RESULTS: Tau induced a unique tau-PSR signature in rTg4510 mice, notably in the accumulation of acetylated spermidine. In vitro, higher-order polyamines prevented tau fibrillization but acetylated spermidine failed to mimic this effect and even promoted fibrillization and oligomerization. AAV9 Tau ΔD421 also elicited a unique tau-PSR in vivo, and targeted disruption of SSAT prevented the accumulation of acetylated polyamines and impacted several tau phospho-epitopes. Interestingly, SSAT knockout mice presented with altered behavior in the rotarod task, the elevated plus maze, and marble burying task, thus highlighting the impact of polyamine homeostasis within the brain. CONCLUSION: These data represent a novel paradigm linking tau pathology and polyamine dysfunction and that targeting specific arms within the polyamine pathway may serve as new targets to mitigate certain components of the tau phenotype.
Asunto(s)
Acetiltransferasas/metabolismo , Poliaminas/metabolismo , Estrés Fisiológico , Tauopatías/enzimología , Acetiltransferasas/genética , Animales , Femenino , Hipocampo/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Agregación Patológica de Proteínas/metabolismo , Proteínas tau/metabolismoRESUMEN
A microsatellite expansion mutation in C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). The expansion mutation leads to C9orf72 loss of function, RNA foci formation, and generation of five species of non-AUG RAN translated dipeptide repeat proteins (DPRs), such as poly(GA), poly(GP), poly(GR), poly(PA), and poly(PR). Although one cell can contain more than type of DPRs, information about interplay between different DPR species is limited. Here we show that the combined expression of distinct C9orf72-derived dipeptide repeat species produces cellular outcomes and structural differences that are unique compared to the expression of a single DPR species, suggesting the complex biological interactions that occur when multiple DPR variants are simultaneously expressed. Our data highlights the need for further analysis of how combined expression of different DPRs affects the disease state.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Dipéptidos , Demencia Frontotemporal , Secuencias Repetitivas de Aminoácido , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Línea Celular , Dipéptidos/genética , Dipéptidos/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , HumanosRESUMEN
The hypothalamus-pituitary-adrenal (HPA) axis directly controls the stress response. Dysregulation of this neuroendocrine system is a common feature among psychiatric disorders. Steroid hormone receptors, like glucocorticoid receptor (GR), function as transcription factors of a diverse set of genes upon activation. This activity is regulated by molecular chaperone heterocomplexes. Much is known about the structure and function of these GR/heterocomplexes. There is strong evidence suggesting altered regulation of steroid receptor hormones by chaperones, particularly the 51 kDa FK506-binding protein (FKBP51), may work with environmental factors to increase susceptibility to various psychiatric illnesses including post-traumatic stress disorder (PTSD), major depressive disorder (MDD), and anxiety. This review highlights the regulation of steroid receptor dynamics by the 90kDa heat shock protein (Hsp90)/cochaperone heterocomplexes with an in depth look at how the structural regulation and imbalances in cochaperones can cause functional effects on GR activity. Links between the stress response and circadian systems and the development of novel chaperone-targeting therapeutics are also discussed.
Asunto(s)
Trastorno Depresivo Mayor/patología , Proteínas HSP90 de Choque Térmico/metabolismo , Receptores de Esteroides/metabolismo , Ritmo Circadiano , Ciclofilinas/metabolismo , Trastorno Depresivo Mayor/metabolismo , Glicoproteínas/metabolismo , Humanos , Transducción de Señal , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer's disease-related protein Tau. We reveal that the FKBP51/Hsp90 complex, which synergizes to promote toxic Tau oligomers in vivo, is highly dynamic and stabilizes the extended conformation of the Hsp90 dimer resulting in decreased Hsp90 ATPase activity. Within the ternary Hsp90/FKBP51/Tau complex, Hsp90 serves as a scaffold that traps the PPIase and nucleates multiple conformations of Tau's proline-rich region next to the PPIase catalytic pocket in a phosphorylation-dependent manner. Our study defines a conceptual model for dynamic Hsp90/co-chaperone/client recognition.
Asunto(s)
Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/toxicidad , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/toxicidad , Proteínas tau/química , Proteínas tau/toxicidad , Biocatálisis/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Conformación Proteica , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas tau/metabolismoRESUMEN
Genetic and epigenetic alterations in FK506-binding protein 5 ( FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.
Asunto(s)
Benzotropina/farmacología , Depresión/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/metabolismo , Receptores de Glucocorticoides/metabolismo , Trastornos por Estrés Postraumático/tratamiento farmacológico , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Benzotropina/química , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Depresión/metabolismo , Descubrimiento de Drogas , Humanos , Ratones , Terapia Molecular Dirigida , Unión Proteica/efectos de los fármacos , Trastornos por Estrés Postraumático/metabolismo , Proteínas de Unión a Tacrolimus/antagonistas & inhibidoresRESUMEN
The microtubule-associated protein tau (MAPT, tau) forms neurotoxic aggregates that promote cognitive deficits in tauopathies, the most common of which is Alzheimer's disease (AD). The 90-kDa heat shock protein (Hsp90) chaperone system affects the accumulation of these toxic tau species, which can be modulated with Hsp90 inhibitors. However, many Hsp90 inhibitors are not blood-brain barrier-permeable, and several present associated toxicities. Here, we find that the cochaperone, activator of Hsp90 ATPase homolog 1 (Aha1), dramatically increased the production of aggregated tau. Treatment with an Aha1 inhibitor, KU-177, dramatically reduced the accumulation of insoluble tau. Aha1 colocalized with tau pathology in human brain tissue, and this association positively correlated with AD progression. Aha1 overexpression in the rTg4510 tau transgenic mouse model promoted insoluble and oligomeric tau accumulation leading to a physiological deficit in cognitive function. Overall, these data demonstrate that Aha1 contributes to tau fibril formation and neurotoxicity through Hsp90. This suggests that therapeutics targeting Aha1 may reduce toxic tau oligomers and slow or prevent neurodegenerative disease progression.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Agregado de Proteínas , Agregación Patológica de Proteínas/etiología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/prevención & control , Tauopatías/etiología , Tauopatías/metabolismo , Tauopatías/prevención & control , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.
Asunto(s)
Amiloide/metabolismo , Ciclofilinas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Amiloide/genética , Amiloide/ultraestructura , Animales , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/fisiopatología , Peptidil-Prolil Isomerasa F , Ciclofilinas/genética , Ciclosporina/farmacología , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Masculino , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Enfermedades Neurodegenerativas/genética , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas , Tauopatías/genética , Tauopatías/metabolismo , alfa-Sinucleína/genética , Proteínas tau/genéticaRESUMEN
Three scaffolds with inhibitory activity against the heat shock protein 70 (Hsp70) family of chaperones have been found to enhance the degradation of the microtubule associated protein tau in cells, neurons, and brain tissue. This is important because tau accumulation is linked to neurodegenerative diseases including Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Here, we expanded upon this study to investigate the anti-tau efficacy of additional scaffolds with Hsp70 inhibitory activity. Five of the nine scaffolds tested lowered tau levels, with the rhodacyanine and phenothiazine scaffolds exhibiting the highest potency as previously described. Because phenothiazines also inhibit tau aggregation in vitro, we suspected that this activity might be a more accurate predictor of tau lowering. Interestingly, the rhodacyanines did inhibit in vitro tau aggregation to a similar degree as phenothiazines, correlating well with tau-lowering efficacy in cells and ex vivo slices. Moreover, other Hsp70 inhibitor scaffolds with weaker tau-lowering activity in cells inhibited tau aggregation in vitro, albeit at lower potencies. When we tested six well-characterized tau aggregation inhibitors, we determined that this mechanism of action was not a better predictor of tau-lowering than Hsp70 inhibition. Instead, we found that compounds possessing both activities were the most effective at promoting tau clearance. Moreover, cytotoxicity and PAINS activity are critical factors that can lead to false-positive lead identification. Strategies designed around these principles will likely yield more efficacious tau-lowering compounds.
Asunto(s)
Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas tau/antagonistas & inhibidores , Células HEK293 , Humanos , Técnicas In VitroRESUMEN
Neuronal inclusions of poly(GA), a protein unconventionally translated from G4C2 repeat expansions in C9ORF72, are abundant in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) caused by this mutation. To investigate poly(GA) toxicity, we generated mice that exhibit poly(GA) pathology, neurodegeneration and behavioral abnormalities reminiscent of FTD and ALS. These phenotypes occurred in the absence of TDP-43 pathology and required poly(GA) aggregation. HR23 proteins involved in proteasomal degradation and proteins involved in nucleocytoplasmic transport were sequestered by poly(GA) in these mice. HR23A and HR23B similarly colocalized to poly(GA) inclusions in C9ORF72 expansion carriers. Sequestration was accompanied by an accumulation of ubiquitinated proteins and decreased xeroderma pigmentosum C (XPC) levels in mice, indicative of HR23A and HR23B dysfunction. Restoring HR23B levels attenuated poly(GA) aggregation and rescued poly(GA)-induced toxicity in neuronal cultures. These data demonstrate that sequestration and impairment of nuclear HR23 and nucleocytoplasmic transport proteins is an outcome of, and a contributor to, poly(GA) pathology.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuronas/patología , Proteínas/toxicidad , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Atrofia/patología , Conducta Animal , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Proteína C9orf72 , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Expresión Génica/genética , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Ratones , Mutación , Degeneración Nerviosa/patología , Neuronas/metabolismo , Cultivo Primario de Células , Proteínas/genética , Proteínas/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMEN
Peptidyl-prolyl cis/trans isomerases (PPIases), a unique family of molecular chaperones, regulate protein folding at proline residues. These residues are abundant within intrinsically disordered proteins, like the microtubule-associated protein tau. Tau has been shown to become hyperphosphorylated and accumulate as one of the two main pathological hallmarks in Alzheimer's disease, the other being amyloid beta (Ab). PPIases, including Pin1, FK506-binding protein (FKBP) 52, FKBP51, and FKBP12, have been shown to interact with and regulate tau biology. This interaction is particularly important given the numerous proline-directed phosphorylation sites found on tau and the role phosphorylation has been found to play in pathogenesis. This regulation then affects downstream aggregation and oligomerization of tau. However, many PPIases have yet to be explored for their effects on tau biology, despite the high likelihood of interaction based on proline content. Moreover, Pin1, FKBP12, FKBP52, cyclophilin (Cyp) A, CypB, and CypD have been shown to also regulate Ab production or the toxicity associated with Ab pathology. Therefore, PPIases directly and indirectly regulate pathogenic protein multimerization in Alzheimer's disease and represent a family rich in targets for modulating the accumulation and toxicity.