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1.
Cell ; 184(17): 4547-4563.e17, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34314701

RESUMO

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.


Assuntos
Cérebro/patologia , Proteína Semelhante a ELAV 4/genética , Ácido Glutâmico/metabolismo , Mutação/genética , Neurônios/patologia , Organoides/metabolismo , Splicing de RNA/genética , Proteínas tau/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Biomarcadores/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Hidrazonas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Splicing de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Grânulos de Estresse/efeitos dos fármacos , Grânulos de Estresse/metabolismo , Sinapses/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
2.
Proc Natl Acad Sci U S A ; 120(16): e2217864120, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37043533

RESUMO

Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This deleterious effect is mediated by pathological cleavage of the Cdk5 activator p35 into the truncated product p25, leading to prolonged Cdk5 activation and altered substrate specificity. Elevated p25 levels have been reported in humans and rodents with neurodegeneration, and the benefit of genetically blocking p25 production has been demonstrated previously in rodent and human neurodegenerative models. Here, we report a 12-amino-acid-long peptide fragment derived from Cdk5 (Cdk5i) that is considerably smaller than existing peptide inhibitors of Cdk5 (P5 and CIP) but shows high binding affinity toward the Cdk5/p25 complex, disrupts the interaction of Cdk5 with p25, and lowers Cdk5/p25 kinase activity. When tagged with a fluorophore (FITC) and the cell-penetrating transactivator of transcription (TAT) sequence, the Cdk5i-FT peptide exhibits cell- and brain-penetrant properties and confers protection against neurodegenerative phenotypes associated with Cdk5 hyperactivity in cell and mouse models of neurodegeneration, highlighting Cdk5i's therapeutic potential.


Assuntos
Quinase 5 Dependente de Ciclina , Peptídeos , Camundongos , Animais , Humanos , Quinase 5 Dependente de Ciclina/metabolismo , Fosforilação , Peptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fenótipo
3.
Int J Mol Sci ; 21(23)2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33255694

RESUMO

Tauopathies are neurodegenerative diseases characterized by the pathological accumulation of microtubule-associated protein tau (MAPT) in the form of neurofibrillary tangles and paired helical filaments in neurons and glia, leading to brain cell death. These diseases include frontotemporal dementia (FTD) and Alzheimer's disease (AD) and can be sporadic or inherited when caused by mutations in the MAPT gene. Despite an incredibly high socio-economic burden worldwide, there are still no effective disease-modifying therapies, and few tau-focused experimental drugs have reached clinical trials. One major hindrance for therapeutic development is the knowledge gap in molecular mechanisms of tau-mediated neuronal toxicity and death. For the promise of precision medicine for brain disorders to be fulfilled, it is necessary to integrate known genetic causes of disease, i.e., MAPT mutations, with an understanding of the dysregulated molecular pathways that constitute potential therapeutic targets. Here, the growing understanding of known and proposed mechanisms of disease etiology will be reviewed, together with promising experimental tau-directed therapeutics, such as recently developed tau degraders. Current challenges faced by the fields of tau research and drug discovery will also be addressed.


Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , Tauopatias/genética , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular/genética , Citoesqueleto/genética , Citoesqueleto/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Mutação/genética , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/patologia , Neuroglia/patologia , Neurônios/patologia , Tauopatias/patologia
4.
J Neurosci ; 37(41): 9917-9924, 2017 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-28912154

RESUMO

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KI mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of P301S mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation.SIGNIFICANCE STATEMENT Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aß accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KI transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.


Assuntos
Quinase 5 Dependente de Ciclina/genética , Demência Frontotemporal/genética , Fosfotransferases/genética , Células-Tronco Pluripotentes , Tauopatias/genética , Animais , Região CA3 Hipocampal/patologia , Região CA3 Hipocampal/fisiopatologia , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Demência Frontotemporal/prevenção & controle , Humanos , Potenciação de Longa Duração/genética , Camundongos , Camundongos Transgênicos , Fibras Musgosas Hipocampais/patologia , Fosforilação , Fosfotransferases/antagonistas & inibidores , Transplante de Células-Tronco , Sinapses/patologia , Sinaptofisina/genética , Tauopatias/prevenção & controle
5.
J Biol Chem ; 292(47): 19209-19225, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-28972160

RESUMO

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.


Assuntos
Embrião de Mamíferos/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Neurônios/metabolismo , Pirróis/farmacologia , Ubiquitina Tiolesterase/fisiologia , Proteínas tau/metabolismo , Animais , Células Cultivadas , Citoplasma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Pirróis/síntese química , Ratos Sprague-Dawley , Ubiquitina/metabolismo , Ubiquitinação
6.
Mol Cell Neurosci ; 73: 104-15, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26826498

RESUMO

Compelling clinical, social, and economic reasons exist to innovate in the process of drug discovery for neuropsychiatric disorders. The use of patient-specific, induced pluripotent stem cells (iPSCs) now affords the ability to generate neuronal cell-based models that recapitulate key aspects of human disease. In the context of neuropsychiatric disorders, where access to physiologically active and relevant cell types of the central nervous system for research is extremely limiting, iPSC-derived in vitro culture of human neurons and glial cells is transformative. Potential applications relevant to early stage drug discovery, include support of quantitative biochemistry, functional genomics, proteomics, and perhaps most notably, high-throughput and high-content chemical screening. While many phenotypes in human iPSC-derived culture systems may prove adaptable to screening formats, addressing the question of which in vitro phenotypes are ultimately relevant to disease pathophysiology and therefore more likely to yield effective pharmacological agents that are disease-modifying treatments requires careful consideration. Here, we review recent examples of studies of neuropsychiatric disorders using human stem cell models where cellular phenotypes linked to disease and functional assays have been reported. We also highlight technical advances using genome-editing technologies in iPSCs to support drug discovery efforts, including the interpretation of the functional significance of rare genetic variants of unknown significance and for the purpose of creating cell type- and pathway-selective functional reporter assays. Additionally, we evaluate the potential of in vitro stem cell models to investigate early events of disease pathogenesis, in an effort to understand the underlying molecular mechanism, including the basis of selective cell-type vulnerability, and the potential to create new cell-based diagnostics to aid in the classification of patients and subsequent selection for clinical trials. A number of key challenges remain, including the scaling of iPSC models to larger cohorts and integration with rich clinicopathological information and translation of phenotypes. Still, the overall use of iPSC-based human cell models with functional cellular and biochemical assays holds promise for supporting the discovery of next-generation neuropharmacological agents for the treatment and ultimately prevention of a range of severe mental illnesses.


Assuntos
Técnicas de Cultura de Células/métodos , Descoberta de Drogas/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Transtornos Mentais/patologia , Medicina de Precisão/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/genética , Modelos Biológicos , Neurônios/citologia , Neurônios/efeitos dos fármacos
7.
PLoS Genet ; 9(8): e1003711, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24009518

RESUMO

Protein quality control requires constant surveillance to prevent misfolding, aggregation, and loss of cellular function. There is increasing evidence in metazoans that communication between cells has an important role to ensure organismal health and to prevent stressed cells and tissues from compromising lifespan. Here, we show in C. elegans that a moderate increase in physiological cholinergic signaling at the neuromuscular junction (NMJ) induces the calcium (Ca(2+))-dependent activation of HSF-1 in post-synaptic muscle cells, resulting in suppression of protein misfolding. This protective effect on muscle cell protein homeostasis was identified in an unbiased genome-wide screening for modifiers of protein aggregation, and is triggered by downregulation of gei-11, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR). This, in-turn, activates the voltage-gated Ca(2+) channel, EGL-19, and the sarcoplasmic reticulum ryanodine receptor in response to acetylcholine signaling. The release of calcium into the cytoplasm of muscle cells activates Ca(2+)-dependent kinases and induces HSF-1-dependent expression of cytoplasmic chaperones, which suppress misfolding of metastable proteins and stabilize the folding environment of muscle cells. This demonstrates that the heat shock response (HSR) can be activated in muscle cells by neuronal signaling across the NMJ to protect proteome health.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Resposta ao Choque Térmico/genética , Homeostase/genética , Junção Neuromuscular/genética , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cálcio/metabolismo , Calmodulina/genética , Humanos , Chaperonas Moleculares/metabolismo , Células Musculares/metabolismo , Neurônios/metabolismo , Receptores Colinérgicos/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
8.
PLoS Genet ; 7(12): e1002438, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22242008

RESUMO

A hallmark of diseases of protein conformation and aging is the appearance of protein aggregates associated with cellular toxicity. We posit that the functional properties of the proteostasis network (PN) protect the proteome from misfolding and combat the proteotoxic events leading to cellular pathology. In this study, we have identified new components of the proteostasis network that can suppress aggregation and proteotoxicity, by performing RNA interference (RNAi) genetic screens for multiple unrelated conformationally challenged cytoplasmic proteins expressed in Caenorhabditis elegans. We identified 88 suppressors of polyglutamine (polyQ) aggregation, of which 63 modifiers also suppressed aggregation of mutant SOD1(G93A). Of these, only 23 gene-modifiers suppressed aggregation and restored animal motility, revealing that aggregation and toxicity can be genetically uncoupled. Nine of these modifiers were shown to be effective in restoring the folding and function of multiple endogenous temperature-sensitive (TS) mutant proteins, of which five improved folding in a HSF-1-dependent manner, by inducing cytoplasmic chaperones. This triage screening strategy also identified a novel set of PN regulatory components that, by altering metabolic and RNA processing functions, establish alternate cellular environments not generally dependent on stress response activation and that are broadly protective against misfolded and aggregation-prone proteins.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Testes Genéticos/métodos , Peptídeos/genética , Peptídeos/metabolismo , Interferência de RNA , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Resposta ao Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Dobramento de Proteína , Deficiências na Proteostase , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
J Biol Chem ; 287(31): 26136-45, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22669943

RESUMO

Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity.


Assuntos
Caenorhabditis elegans/metabolismo , Peptídeos/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Técnicas de Silenciamento de Genes , Proteínas Luminescentes/metabolismo , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência
10.
bioRxiv ; 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38168389

RESUMO

Tauopathies are a class of neurodegenerative diseases characterized by the progressive misfolding and accumulation of pathological tau protein in focal regions of the brain, leading to insidious neurodegeneration. Abnormalities in cholesterol metabolism and homeostasis have also been implicated in various neurodegenerative diseases. However, the connection between cholesterol dysregulation and tau pathology remains largely unknown. To model and measure the impact of cholesterol dysregulation on tau, we utilized a combination of in vitro and ex vivo tau aggregation assays using an engineered tau biosensor cell line and human induced pluripotent stem cell (iPSC)-derived neuronal cultures from an individual harboring an autosomal dominant P301L tau mutation and from a healthy control. We demonstrate that excess cholesterol esters lead to an increased rate of tau aggregation in vitro and an increase in seed-dependent insoluble tau aggregates detected in the biosensor line. We observed a strong correlation between cholesterol ester concentration and the presence of high-molecular-weight, oligomeric tau species. Importantly, in tauopathy patient iPSC-derived neurons harboring a P301L tau mutation with endogenous forms of misfolded tau, we show that acute dysregulation of cholesterol homeostasis through acute exposure to human plasma-purified cholesterol esters formed by the linkage of fatty acids to the hydroxyl group of cholesterol leads to the rapid accumulation of phosphorylated tau. Conversely, treatment with the same cholesterol esters pool did not lead to subsequent accumulation of phosphorylated tau in control iPSC-derived neurons. Finally, treatment with a heterobifunctional, small-molecule degrader designed to selectively engage and catalyze the ubiquitination and proteasomal degradation of aberrant tau species prevented cholesterol ester-induced aggregation of tau in the biosensor cell line in a Cereblon E3 ligase-dependent manner. Degrader treatment also restored the resiliency of tauopathy patient-derived neurons towards cholesterol ester-induced tau aggregation phenotypes. Taken together, our study supports a key role of cholesterol dysregulation in tau aggregation. Moreover, it provides further pre-clinical validation of the therapeutic strategy of targeted protein degradation with heterobifunctional tau degraders for blocking tau seeding.

11.
Nat Commun ; 14(1): 7575, 2023 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-37989753

RESUMO

MicroRNAs (miRNAs) regulate fundamental biological processes by silencing mRNA targets and are dysregulated in many diseases. Therefore, miRNA replacement or inhibition can be harnessed as potential therapeutics. However, existing strategies for miRNA modulation using oligonucleotides and gene therapies are challenging, especially for neurological diseases, and none have yet gained clinical approval. We explore a different approach by screening a biodiverse library of small molecule compounds for their ability to modulate hundreds of miRNAs in human induced pluripotent stem cell-derived neurons. We demonstrate the utility of the screen by identifying cardiac glycosides as potent inducers of miR-132, a key neuroprotective miRNA downregulated in Alzheimer's disease and other tauopathies. Coordinately, cardiac glycosides downregulate known miR-132 targets, including Tau, and protect rodent and human neurons against various toxic insults. More generally, our dataset of 1370 drug-like compounds and their effects on the miRNome provides a valuable resource for further miRNA-based drug discovery.


Assuntos
Glicosídeos Cardíacos , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Humanos , MicroRNAs/genética , RNA Mensageiro/genética , Sequenciamento de Nucleotídeos em Larga Escala
12.
Sci Transl Med ; 15(691): eabq2915, 2023 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-37043557

RESUMO

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer's disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC-derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.


Assuntos
Prolil Oligopeptidases , Tauopatias , Camundongos , Humanos , Animais , Células HEK293 , Tauopatias/metabolismo , Proteínas tau/metabolismo , Camundongos Transgênicos , Serina Endopeptidases/metabolismo , Inibidores Enzimáticos , Modelos Animais de Doenças
13.
bioRxiv ; 2023 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-37503195

RESUMO

Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1 / ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2 . Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.

14.
Methods Mol Biol ; 2429: 143-174, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507160

RESUMO

A major obstacle in studying human central nervous system (CNS) diseases is inaccessibility to the affected tissue and cells. Even in limited cases where tissue is available through surgical interventions, differentiated neurons cannot be maintained for extended time frames, which is prohibitive for experimental repetition and scalability. Advances in methodologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSC) and directed differentiation of human neurons in culture now allow access to physiological and disease relevant cell types. In particular, patient iPSC-derived neurons represent unique ex vivo neuronal networks that allow investigating disease genetic and molecular pathways in physiologically accurate cellular microenvironments, importantly recapitulating molecular and cellular phenotypic aspects of disease. Generation of functional neural cells from iPSCs relies on manipulation of culture formats in the presence of specific factors that promote the conversion of pluripotent stem cells into neurons. To this end, several experimental protocols have been developed. Direct differentiation of stem cells into post-mitotic neurons is usually associated with low throughput, low yield, and high technical variability. Instead, methods relying on expansion of the intermediate neural progenitor cells (NPCs) show incredible potential for posterior generation of suitable neuronal cultures for cellular and biochemical assays, as well as drug screening. NPCs are expandable, self-renewable multipotent cells that can differentiate into astrocytes, oligodendrocytes, and electrically active neurons. Here, we describe a protocol for generating iPSC-derived NPCs via formation of neural aggregates and selection of NPC precursor neural rosettes, followed by a simple and reproducible method for generating a mixed population of cortical-like neurons through growth factor withdrawal. Implementation of this protocol has the potential to advance the goals of precision medicine research for both neurological and psychiatric disorders.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Diferenciação Celular/fisiologia , Humanos , Neurônios/metabolismo , Medicina de Precisão
15.
Front Cell Neurosci ; 16: 801179, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35317195

RESUMO

Accumulation of misfolded, aggregating proteins concurrent with disease onset and progression is a hallmark of neurodegenerative proteinopathies. An important class of these are tauopathies, such as frontotemporal dementia (FTD) and Alzheimer's disease (AD), associated with accumulation of aberrant forms of tau protein in the brain. Pathological tau undergoes abnormal post-translational modifications, misfolding, oligomerization and changes in solubility, cellular redistribution, and spreading. Development and testing of experimental therapeutics that target these pathological tau conformers requires use of cellular models that recapitulate neuronal endogenous, non-heterologous tau expression under genomic and physiological contexts relevant to disease. In this study, we employed FTD-patient induced pluripotent stem cells (iPSC)-derived neurons, expressing a tau variant or mutation, as primary models for driving a medicinal chemistry campaign around tau targeting degrader series. Our screening goal was to establish structure-activity relationships (SAR) for the different chemical series to identify the molecular composition that most efficiently led to tau degradation in human FTD ex vivo neurons. We describe the identification of the lead compound QC-01-175 and follow-up optimization strategies for this molecule. We present three final lead molecules with tau degradation activity in mutant neurons, which establishes potential disease relevance and will drive future studies on specificity and pharmacological properties.

16.
Mol Neurobiol ; 59(1): 683-702, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34757590

RESUMO

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer's disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Sinapses/metabolismo
17.
ACS Chem Neurosci ; 12(2): 271-284, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33417763

RESUMO

Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.


Assuntos
Histona Desacetilases , Células-Tronco Pluripotentes Induzidas , Benzofenonas , Histona Desacetilase 1 , Humanos , Neurônios
18.
Sci Rep ; 11(1): 17029, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426604

RESUMO

Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.


Assuntos
Glutamatos/metabolismo , Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Neurônios/patologia , Proteômica , Tauopatias/patologia , Proteínas tau/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
19.
Ann N Y Acad Sci ; 1471(1): 18-56, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-30875083

RESUMO

Development of effective therapeutics for neurological disorders has historically been challenging partly because of lack of accurate model systems in which to investigate disease etiology and test new therapeutics at the preclinical stage. Human stem cells, particularly patient-derived induced pluripotent stem cells (iPSCs) upon differentiation, have the ability to recapitulate aspects of disease pathophysiology and are increasingly recognized as robust scalable systems for drug discovery. We review advances in deriving cellular models of human central nervous system (CNS) disorders using iPSCs along with strategies for investigating disease-relevant phenotypes, translatable biomarkers, and therapeutic targets. Given their potential to identify novel therapeutic targets and leads, we focus on phenotype-based, small-molecule screens employing human stem cell-derived models. Integrated efforts to assemble patient iPSC-derived cell models with deeply annotated clinicopathological data, along with molecular and drug-response signatures, may aid in the stratification of patients, diagnostics, and clinical trial success, shifting translational science and precision medicine approaches. A number of remaining challenges, including the optimization of cost-effective, large-scale culture of iPSC-derived cell types, incorporation of aging into neuronal models, as well as robustness and automation of phenotypic assays to support quantitative drug efficacy, toxicity, and metabolism testing workflows, are covered. Continued advancement of the field is expected to help fully humanize the process of CNS drug discovery.


Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Medicina de Precisão , Diferenciação Celular/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Descoberta de Drogas , Humanos , Neurônios/efeitos dos fármacos
20.
Nat Commun ; 11(1): 3258, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591533

RESUMO

Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.


Assuntos
Autofagia , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estresse Fisiológico , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fenótipo , Ratos Wistar , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Tauopatias/patologia , Fatores de Tempo
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