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BACKGROUND: Proteostasis is a critical aging hallmark responsible for removing damaged or misfolded proteins and their aggregates by improving proteasomal degradation through the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS). Research on the impact of heat-killed probiotic bacteria and their structural components on aging hallmarks and innate immune responses is scarce, yet enhancing these effects could potentially delay age-related diseases. RESULTS: This study introduces a novel heat-killed Levilactobacillus brevis strain MKAK9 (HK MKAK9), along with its exopolysaccharide (EPS), demonstrating their ability to extend longevity by improving proteostasis and immune responses in wild-type Caenorhabditis elegans. We elucidate the underlying mechanisms through a comprehensive approach involving mRNA- and small RNA sequencing, proteomic analysis, lifespan assays on loss-of-function mutants, and quantitative RT-PCR. Mechanistically, HK MKAK9 and its EPS resulted in downregulation of the insulin-like signaling pathway in a DAF-16-dependent manner, enhancing protein ubiquitination and subsequent proteasomal degradation through activation of the ALP pathway, which is partially mediated by microRNA mir-243. Importantly, autophagosomes engulf ubiquitinylated proteins, as evidenced by increased expression of the autophagy receptor sqst-3, and subsequently fuse with lysosomes, facilitated by increased levels of the lysosome-associated membrane protein (LAMP) lmp-1, suggesting the formation of autolysosomes for degradation of the selected cargo. Moreover, HK MKAK9 and its EPS activated the p38 MAPK pathway and its downstream SKN-1 transcription factor, which are known to regulate genes involved in innate immune response (thn-1, ilys-1, cnc-2, spp-9, spp-21, clec-47, and clec-266) and antioxidation (sod-3 and gst-44), thereby reducing the accumulation of reactive oxygen species (ROS) at both cellular and mitochondrial levels. Notably, SOD-3 emerged as a transcriptional target of both DAF-16 and SKN-1 transcription factors. CONCLUSION: Our research sets a benchmark for future investigations by demonstrating that heat-killed probiotic and its specific cellular component, EPS, can downregulate the insulin-signaling pathway, potentially improving the autophagy-lysosome pathway (ALP) for degrading ubiquitinylated proteins and promoting organismal longevity. Additionally, we discovered that increased expression of microRNA mir-243 regulates insulin-like signaling and its downstream ALP pathway. Our findings also indicate that postbiotic treatment may bolster antioxidative and innate immune responses, offering a promising avenue for interventions in aging-related diseases.

RESUMEN

INTRODUCTION: Nav1.6 is closely related to the pathology of Alzheimer's Disease (AD), and astrocytes have recently been identified as a significant source of ß-amyloid (Aß). However, little is known about the connection between Nav1.6 and astrocyte-derived Aß. OBJECTIVE: This study explored the crucial role of Nav1.6 in mediated astrocyte-derived Aß in AD and knockdown astrocytic Nav1.6 alleviates AD progression by promoting autophagy and lysosome-APP fusion. METHODS: A mouse model for astrocytic Nav1.6 knockdown was constructed to study the effects of astrocytic Nav1.6 on amyloidosis. The role of astrocytic Nav1.6 on autophagy and lysosome-APP(amyloid precursor protein) fusion was used by transmission electron microscope, immunostaining, western blot and patch clamp. Glial cell activation was detected using immunostaining. Neuroplasticity and neural network were assessed using patch-clamp, Golgi stain and EEG recording. Behavioral experiments were performed to evaluate cognitive defects. RESULTS: Astrocytic Nav1.6 knockdown reduces amyloidosis, alleviates glial cell activation and morphological complexity, improves neuroplasticity and abnormal neural networks, as well as promotes learning and memory abilities in APP/PS1 mice. Astrocytic Nav1.6 knockdown reduces itself-derived Aß by promoting lysosome- APP fusion, which is related to attenuating reverse Na+-Ca2+ exchange current thus reducing intracellular Ca2+ to facilitate autophagic through AKT/mTOR/ULK pathway. CONCLUSION: Our findings unveil the crucial role of astrocyte-specific Nav1.6 in reducing astrocyte-derived Aß, highlighting its potential as a cell-specific target for modulating AD progression.

RESUMEN

Gastrodin (GAS) is the main chemical component of the traditional Chinese herb Gastrodia elata (called "Tianma" in Chinese), which has been used to treat neurological conditions, including headaches, epilepsy, stroke, and memory loss. To our knowledge, it is unclear whether GAS has a therapeutic effect on Huntington's disease (HD). In the present study, we evaluated the effect of GAS on the degradation of mutant huntingtin protein (mHtt) by using PC12 cells transfected with N-terminal mHtt Q74. We found that 0.1-100 µM GAS had no effect on the survival rate of Q23 and Q74 PC12 cells after 24-48 h of incubation. The ubiquitin-proteasome system (UPS) is the main system that clears misfolded proteins in eukaryotic cells. Mutated Htt significantly upregulated total ubiquitinated protein (Ub) expression, decreased chymotrypsin-like, trypsin-like and caspase-like peptidase activity, and reduced the colocalization of the 20S proteasome with mHtt. GAS (25 µM) attenuated all of the abovementioned pathological changes, and the regulatory effect of GAS on mHtt was found to be abolished by MG132, a proteasome inhibitor. The autophagy-lysosome pathway (ALP) is another system for misfolded protein degradation. Although GAS downregulated the expression of autophagy markers (LC3II and P62), it increased the colocalization of LC3II with lysosomal associated membrane protein 1 (LAMP1), which indicates that ALP was activated. Moreover, GAS prevented mHtt-induced neuronal damage in PC12 cells. GAS has a selective effect on mHtt in Q74 PC12 cells and has no effect on Q23 and proteins encoded by other genes containing long CAGs, such as Rbm33 (10 CAG repeats) and Hcn1 (>30 CAG repeats). Furthermore, oral administration of 100 mg/kg GAS increased grip strength and attenuated mHtt aggregates in B6-hHTT130-N transgenic mice. This is a high dose (100 mg/kg GAS) when compared with experiments on HD mice with other small molecules. We will design more doses to evaluate the dose-response relationship of the inhibition effect of GAS on mHtt in our next study. In summary, GAS can promote the degradation of mHtt by activating the UPS and ALP, making it a potential therapeutic agent for HD.

Asunto(s)

Autofagia , Alcoholes Bencílicos , Glucósidos , Proteína Huntingtina , Lisosomas , Complejo de la Endopetidasa Proteasomal , Ubiquitina , Animales , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Ratas , Complejo de la Endopetidasa Proteasomal/metabolismo , Células PC12 , Autofagia/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Ubiquitina/metabolismo , Alcoholes Bencílicos/farmacología , Glucósidos/farmacología , Ratones , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Proteolisis/efectos de los fármacos , Mutación

RESUMEN

Deoxynivalenol (DON) is a common agricultural mycotoxin that is chemically stable and not easily removed from cereal foods. When organisms consume food made from contaminated crops, it can be hazardous to their health. Numerous studies in recent years have found that hesperidin (HDN) has hepatoprotective effects on a wide range of toxins. However, few scholars have explored the potential of HDN in attenuating DON-induced liver injury. In this study, we established a low-dose DON exposure model and intervened with three doses of HDN, acting on male C57 BL/6 mice and AML12 cells, which served as in vivo and in vitro models, respectively, to investigate the protective mechanism of HDN against DON exposure-induced liver injury. The results suggested that DON disrupted hepatic autophagic fluxes, thereby impairing liver structure and function, and HDN significantly attenuated these changes. Further studies revealed that HDN alleviated DON-induced excessive autophagy through the mTOR pathway and DON-induced lysosomal dysfunction through the AKT/GSK3ß/TFEB pathway. Overall, our study suggested that HDN could ameliorate DON-induced autophagy flux disorders via the mTOR pathway and the AKT/GSK3ß/TFEB pathway, thereby reducing liver injury.

Asunto(s)

Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Glucógeno Sintasa Quinasa 3 beta , Hesperidina , Hígado , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt , Serina-Treonina Quinasas TOR , Tricotecenos , Animales , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Tricotecenos/toxicidad , Masculino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Hesperidina/farmacología , Autofagia/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Humanos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Línea Celular

RESUMEN

Twenty-five years have passed since the causative gene for familial Parkinson's disease (PD), Parkin (now PRKN), was identified in 1998; PRKN is the most common causative gene in young-onset PD. Parkin encodes a ubiquitin-protein ligase, and Parkin is involved in mitophagy, a type of macroautophagy, in concert with PTEN-induced kinase 1 (PINK1). Both gene products are also involved in mitochondrial quality control. Among the many genetic PD-causing genes discovered, discovering PRKN as a cause of juvenile-onset PD has significantly impacted other neurodegenerative disorders. This is because the involvement of proteolytic systems has been suggested as a common mechanism in neurodegenerative diseases in which inclusion body formation is observed. The discovery of the participation of PRKN in PD has brought attention to the involvement of the proteolytic system in neurodegenerative diseases. Our research group has successfully isolated and identified CHCHD2, which is involved in the mitochondrial electron transfer system, and prosaposin (PSAP), which is involved in the lysosomal system, in this Parkin mechanism. Hereditary PD is undoubtedly an essential clue to solitary PD, and at least 25 or so genes and loci have been reported so far. This number of genes indicates that PD is a very diverse group of diseases. Currently, the diagnosis of PD is based on clinical symptoms and imaging studies. Although highly accurate diagnostic criteria have been published, early diagnosis is becoming increasingly important in treatment strategies for neurodegenerative diseases. Here, we also describe biomarkers that our group is working on.

Asunto(s)

Biomarcadores , Enfermedad de Parkinson , Ubiquitina-Proteína Ligasas , Humanos , Enfermedad de Parkinson/genética , Ubiquitina-Proteína Ligasas/genética , Biomarcadores/metabolismo

RESUMEN

Intraneuronal dysproteostasis and extraneuronal microenvironmental abnormalities in Alzheimer's disease (AD) collectively culminate in neuronal deterioration. In the context of AD, autophagy dysfunction, a multi-link obstacle involving autophagy downregulation and lysosome defects in neurons/microglia is highly implicated in intra/extraneuronal pathological processes. Therefore, multidimensional autophagy regulation strategies co-manipulating "autophagy induction" and "lysosome degradation" in dual targets (neuron and microglia) are more reliable for AD treatment. Accordingly, we designed an RP-1 peptide-modified reactive oxygen species (ROS)-responsive micelles (RT-NM) loading rapamycin or gypenoside XVII. Guided by RP-1 peptide, the ligand of receptor for advanced glycation end products (RAGE), RT-NM efficiently targeted neurons and microglia in AD-affected region. This nano-combination therapy activated the whole autophagy-lysosome pathway by autophagy induction (rapamycin) and lysosome improvement (gypenoside XVII), thus enhancing autophagic degradation of neurotoxic aggregates and inflammasomes, and promoting Aß phagocytosis. Resultantly, it decreased aberrant protein burden, alleviated neuroinflammation, and eventually ameliorated memory defects in 3 × Tg-AD transgenic mice. Our research developed a multidimensional autophagy nano-regulator to boost the efficacy of autophagy-centered AD therapy.

RESUMEN

Although there is increasing evidence for the involvement of Hippo signaling in Alzheimer's disease (AD), the detailed functions and regulatory mechanisms are not fully understood, given the diverse biological effects of this pathway. In the present work, we used Caenorhabditis elegans and mammalian cell models to investigate changes in the Hippo signaling pathway in response to Aß and the downstream effects on AD development. Aß1-42 production in the AD models decreased phosphorylation of the upstream CST-1/WTS-1 kinase cascade and promoted an interaction between LIN-10 and YAP-1, leading to the nuclear translocation of YAP-1 and inducing gene transcription in conjunction with the transcription factor EGL-44. The YAP-1/EGL-44 complex suppressed the autophagy-lysosome pathway by modulating mTOR signaling, which enhanced Aß1-42 accumulation and promoted AD progression. These results demonstrate for the first time that crosstalk between Hippo and mTOR signaling contributes to AD development by enhancing Aß production, resulting in inhibition of Hippo signaling and autophagy-lysosome pathway and Aß accumulation, suggesting potential therapeutic targets for the treatment or prevention of AD.

Asunto(s)

Enfermedad de Alzheimer , Proteínas de Caenorhabditis elegans , Animales , Vía de Señalización Hippo , Proteínas Serina-Treonina Quinasas/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Enfermedad de Alzheimer/etiología , Serina-Treonina Quinasas TOR/metabolismo , Mamíferos/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Señalizadoras YAP

RESUMEN

Accumulation of pathogenic amyloid-ß disrupts the tight junction of retinal pigment epithelium (RPE), one of its senescence-like structural alterations. In the clearance of amyloid-ß, the autophagy-lysosome pathway plays the crucial role. In this context, mammalian target of rapamycin (mTOR) inhibits the process of autophagy and lysosomal degradation, acting as a potential therapeutic target for age-associated disorders. However, efficacy of targeting mTOR to treat age-related macular degeneration remains largely elusive. Here, we validated the therapeutic efficacy of the mTOR inhibitors, Torin and PP242, in clearing amyloid-ß by inducing the autophagy-lysosome pathway in a mouse model with pathogenic amyloid-ß with tight junction disruption of RPE, which is evident in dry age-related macular degeneration. High concentration of amyloid-ß oligomers induced autophagy-lysosome pathway impairment accompanied by the accumulation of p62 and decreased lysosomal activity in RPE cells. However, Torin and PP242 treatment restored the lysosomal activity via activation of LAMP2 and facilitated the clearance of amyloid-ß in vitro and in vivo. Furthermore, clearance of amyloid-ß by Torin and PP242 ameliorated the tight junction disruption of RPE in vivo. Overall, our findings suggest mTOR inhibition as a new therapeutic strategy for the restoration of tight junctions in age-related macular degeneration.

Asunto(s)

Degeneración Macular , Epitelio Pigmentado de la Retina , Ratones , Animales , Epitelio Pigmentado de la Retina/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Péptidos beta-Amiloides/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Degeneración Macular/metabolismo , Lisosomas/metabolismo , Autofagia/fisiología , Mamíferos

RESUMEN

Dysregulated maturation and activation of dendritic cells (DCs) play a significant role in the progression of systemic lupus erythematosus (SLE). The autophagy-lysosome pathway has been identified as a potential mechanism to inhibit DC activation and maturation, but its precise workings remain unclear. We investigated the role and regulatory mechanism of TLR9 in modulating the autophagy-lysosome pathway and DCs activation. The mRNA and protein expressions were assessed using qRT-PCR and/or western blot. NZBW/F1 mice was used to construct a lupus nephritis (LN) model in vivo. Cell apoptosis was analyzed by TUNEL staining. Flow cytometry was adopted to analyze DCs surface markers. Lyso-tracker red staining was employed to analyze lysosome acidification. Levels of anti-dsDNA, cytokines, C3, C4, urine protein and urine creatinine were examined by ELISA. The results showed that TLR9 was markedly increased in SLE patients, and its expression was positively correlated with SLEDAI scores and dsDNA level. Conversely, TLR9 expression showed a negative correlation with C3 and C4 levels. Loss-of function experiments demonstrated that TLR9 depletion exerted a substantial inhibition of renal injury, inflammation, and DCs numbers. Additionally, upregulation of TLR9 promoted DCs maturation and activation through activation of autophagy and lysosome acidification. Further investigation revealed that TLR9 targeted TRAF6 to activate the cGAS-STING pathway. Rescue experiments revealed that inactivation of the cGAS/STING signaling pathway could reverse the promoting effects of TLR9 upregulation on DCs maturation, activation, and autophagy-lysosome pathway. Overall, our findings suggested that TLR9 activated the autophagy-lysosome pathway to promote DCs maturation and activation by activating TRAF6-cGAS-STING pathway, thereby promoting SLE progression.

Asunto(s)

Lupus Eritematoso Sistémico , Receptor Toll-Like 9 , Animales , Humanos , Ratones , Autofagia/genética , Células Dendríticas/metabolismo , Lupus Eritematoso Sistémico/genética , Lisosomas/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo

RESUMEN

Intervertebral disc degeneration (IVDD) is a prevalent degenerative disease with significant adverse implications for patients' quality of life and socioeconomic status. Although the precise etiology of IVDD remains elusive, the senescence of nucleus pulposus cells is recognized as the primary pathogenic factor of IVDD; however, drugs that may targetedly inhibit senescence are still lacking. In the current study, we evaluated the small-molecule active drug 20-Deoxyingenol(20-DOI) for its effects on combating senescence and delaying the progression of IVDD. In vitro experiments revealed that the administration of 20-DOI displayed inhibitory effects on senescence and the senescence-related cGAS-STING pathway of nucleus pulposus cells. Additionally, it exhibited the ability to enhance lysosome activity and promote autophagy flux within nucleus pulposus cells. Subsequent investigations elucidated that the inhibitory impact of 20-DOI on nucleus pulposus cell senescence was mediated through the autophagy-lysosome pathway. This effect was diminished in the presence of transcription factor EB (TFEB) small hairpin RNA (shRNA), thereby confirming the regulatory role of 20-DOI on the autophagy-lysosome pathway and senescence through TFEB. In vivo experiments demonstrated that 20-DOI effectively impeded the progression ofIVDD in rats. These findings collectively illustrate that 20-DOI may facilitate the autophagy-lysosomal pathway by activating TFEB, thereby suppressing the senescence in nucleus pulposus cells, thus suggesting 20-DOI as a promising therapeutic approach for IVDD.

Asunto(s)

Degeneración del Disco Intervertebral , Núcleo Pulposo , Humanos , Ratas , Animales , Degeneración del Disco Intervertebral/tratamiento farmacológico , Degeneración del Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , Calidad de Vida , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo

RESUMEN

One of the hallmarks of Parkinson's disease (PD) is the progressive loss of dopaminergic neurons and associated dopamine depletion. Several mechanisms, previously considered in isolation, have been proposed to contribute to the pathophysiology of dopaminergic degeneration: dopamine oxidation-mediated neurotoxicity, high dopamine transporter (DAT) expression density per neuron, and autophagy-lysosome pathway (ALP) dysfunction. However, the interrelationships among these mechanisms remained unclear. Our recent research bridges this gap, recognizing autophagy as a novel dopamine homeostasis regulator, unifying these concepts. I propose that autophagy modulates dopamine reuptake by selectively degrading DAT. In PD, ALP dysfunction could increase DAT density per neuron, and enhance dopamine reuptake, oxidation, and neurotoxicity, potentially contributing to the progressive loss of dopaminergic neurons. This integrated understanding may provide a more comprehensive view of aspects of PD pathophysiology and opens new avenues for therapeutic interventions.

RESUMEN

The mechanism underlying long-term cognitive impairment caused by neonatal hypoxic-ischemic brain injury (HIBI) remains unclear. Autophagy is a closely related mechanism and may play a role in this process. We aimed to investigate the role of lysosomal transmembrane protein 175 (TMEM175) in the autophagy-lysosome pathway in neonatal rats with HIBI. A neonatal rat model of HIBI was established, hypoxia was induced, followed by left common carotid artery ligation. Expression levels of TMEM175 and the corresponding proteins involved in autophagy flux and the endolysosomal system fusion process were measured. Rats were administered TMEM175 plasmid via intracerebroventricular injection to induce overexpression. Brain damage and cognitive function were then assessed. TMEM175 was downregulated in the hippocampal tissue, and the autophagy-lysosome pathway was impaired following HIBI in neonatal rats. Overexpression of TMEM175 significantly mitigated neuronal injury and improved long-term cognitive and memory function in neonatal rats with HIBI. We found that improvement in the autophagy-lysosome pathway and endolysosomal system homeostasis, which are TMEM175 related, occurred via regulation of lysosomal membrane dynamic fusion. TMEM175 plays a critical role in maintaining the autophagy-lysosome pathway and endolysosomal homeostasis, contributing to the amelioration of neuronal injury and impaired long-term cognitive function following neonatal HIBI.

RESUMEN

Aging is a major risk factor for many age-associated disorders, including neurodegenerative diseases. Both mitochondrial dysfunction and proteostatic decline are well-recognized hallmarks of aging and age-related neurodegeneration. Despite a lack of therapies for neurodegenerative diseases, a number of interventions promoting mitochondrial integrity and protein homeostasis (proteostasis) have been shown to delay aging-associated neurodegeneration. For example, many antioxidant polysaccharides are shown to have pharmacological potentials in Alzheimer's, Parkinson's and Huntington's diseases through regulation of mitochondrial and proteostatic pathways, including oxidative stress and heat shock responses. However, how mitochondrial and proteostatic mechanisms work together to exert the antineurodegenerative effect of the polysaccharides remains largely unexplored. Interestingly, recent studies have provided a growing body of evidence to support the crosstalk between mitostatic and proteostatic networks as well as the impact of the crosstalk on neurodegeneration. Here we summarize the recent progress of antineurodegenerative polysaccharides with particular attention in the mitochondrial and proteostatic context and provide perspectives on their implications in the crosstalk along the mitochondria-proteostasis axis.

Asunto(s)

Antioxidantes , Proteostasis , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Estrés Oxidativo , Mitocondrias , Polisacáridos/uso terapéutico

RESUMEN

Protein degradation is a general process to maintain cell homeostasis. The intracellular protein quality control system mainly includes the ubiquitin-proteasome system and the lysosome pathway. Inspired by the physiological process, strategies to degrade specific proteins have developed, which emerge as potent and effective tools in biological research and drug discovery. This review focuses on recent advances in targeted protein degradation techniques, summarizing the principles, advantages, and challenges. Moreover, the potential applications and future direction in biological science and clinics are also discussed.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , Ubiquitina , Proteolisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinación , Homeostasis

RESUMEN

BACKGROUND: Diabetic encephalopathy (DE) is a complication of type 2 diabetes mellitus (T2DM) that features Alzheimer's disease (AD)-like pathology, which can be degraded by the autophagy-lysosome pathway (ALP). Since transcription factor EB (TFEB) is a master regulator of ALP, TFEB-mediated ALP activation might have a therapeutic effect on DE, but this has yet to be investigated. METHODS: We established T2DM mouse models and cultured HT22 cells under high-glucose (HG) conditions to confirm the role of ALP in DE. To further investigate this, both mice and HT22 cells were treated with 3-methyladenine (3-MA). We also analyzed the content of TFEB in the nucleus and cytoplasm to evaluate its role in ALP. To confirm the effect of TFEB activation at the post-translational level in DE, we used rapamycin to inhibit the mechanistic target of rapamycin (mTOR). We transduced both mice and cells with TFEB vector to evaluate the therapeutic effect of TFEB overexpression on DE. Conversely, we conducted TFEB knockdown to verify its role in DE in another direction. RESULTS: We found that T2DM mice experienced compromised cognitive function, while HG-cultured HT22 cells exhibited increased cell apoptosis. Additionally, both T2DM mice and HG-cultured HT22 cells showed impaired ALP and heavier AD-like pathology. This pathology worsened after treatment with 3-MA. We also observed decreased TFEB nuclear translocation in both T2DM mice and HG-cultured HT22 cells. However, inhibiting mTOR with rapamycin or overexpressing TFEB increased TFEB nuclear translocation, enhancing the clearance of ALP-targeted AD-like pathology. This contributed to protection against neuronal apoptosis and alleviation of cognitive impairment. Conversely, TFEB knockdown lessened ALP-targeted AD-like pathology clearance and had a negative impact on DE. CONCLUSION: Our findings suggest that impaired ALP is responsible for the aggravation of AD-like pathology in T2DM. We propose that mTOR-dependent TFEB activation and TFEB overexpression are promising therapeutic strategies for DE, as they enhance the clearance of ALP-targeted AD-like pathology and alleviate neuronal apoptosis. Our study provides insight into the underlying mechanisms of DE and offers potential avenues for the development of new treatments for this debilitating complication of T2DM. Video abstract.

Asunto(s)

Enfermedad de Alzheimer , Diabetes Mellitus Tipo 2 , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Autofagia , Lisosomas/metabolismo

RESUMEN

Accumulation of the misfolded synaptic protein α-synuclein (αSyn*) is a hallmark of neurodegenerative disease in Parkinson's disease (PD). Recent studies suggest that the autophagy lysosome pathway (ALP) including both the Beclin1-associated and mTOR-signaling pathways is involved in the αSyn* clearance mechanism. In this study, a mathematical model is proposed for the degradation of αSyn* by ALP with the crosstalk element of mTOR. Using codimension-1 bifurcation analysis, the tri-stability of αSyn* is surveyed under three different stress signals and, in addition, consideration is given to the regulatory mechanisms for the Beclin1- and mTOR-dependent rates on αSyn* degradation using the codimension-1 and-2 bifurcation diagrams. It was found that, especially under internal and external oxidative stresses (S 1), the bistable switch of the aggregation of αSyn* can be transformed from an irreversible to a reversible condition through the ALP degradation pathways. Furthermore, the robustness of the tri-stable state for the stress S 1 to the parameters related to mTOR-mediated ALP was probed. It was confirmed that mTOR-mediated ALP is important for maintaining the essential dynamic features of the tri-stable state. This study may provide a promising avenue for conducting further experiments and simulations of the degradation mechanism of dynamic modeling in PD.

RESUMEN

Targeted Protein Degradation is an emerging and rapidly developing technique for designing and treating new drugs. With the emergence of a promising class of pharmaceutical molecules, Heterobifunctional Proteolysis-targeting chimeras (PROTACs), TPD has become a powerful tool to completely tackle pathogenic proteins with traditional small molecule inhibitors. However, the conventional PROTACs have gradually exposed potential disadvantages of poor oral bioavailability and pharmacokinetic (PK) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics due to their larger molecular weight and more complex structure than the conventional small-molecule inhibitors. Therefore, 20 years after the concept of PROTAC was proposed, more and more scientists are committed to developing new TPD technology to overcome its defects. And several new technologies and means have been explored based on "PROTAC" to target "undruggable proteins". Here, we aim to comprehensively summarize and profoundly analyze the research progress of targeted protein degradation based on PROTAC targeting the degradation of "undruggable" targets. In order to clarify the significance of emerging and highly effective strategies based PROTACs in the treatment of various diseases especially in overcoming drug resistance in cancer, we will focus on the molecular structure, action mechanism, design concepts, development advantages and challenges of these emerging methods(e.g., aptamer-PROTAC conjugates, antibody-PROTACs and folate-PROTACs).

Asunto(s)

Neoplasias , Humanos , Proteolisis , Neoplasias/tratamiento farmacológico , Disponibilidad Biológica , Anticuerpos , Peso Molecular , Quimera Dirigida a la Proteólisis , Ubiquitina-Proteína Ligasas , Complejo de la Endopetidasa Proteasomal

RESUMEN

This study examined autophagy-lysosome pathway (ALP) perturbations in synovial monocytes/macrophages from patients with gouty arthritis (GA) and the associations of ALP perturbations with cell death. Synovial fluid mononuclear cells (SFMCs) and synovial tissues (STs) from patients with GA, as well as monosodium urate (MSU) crystal-exposed macrophages, underwent immunoblotting, quantitative polymerase chain reaction, and immunofluorescence analyses of markers linked to the ALP (microtubule-associated protein 1 light chain 3B [LC3B], p62, cathepsin D [CTSD], and lysosome-associated membrane protein 2 [LAMP2]) and cell death (caspase-3). GA STs underwent immunohistochemistry and immunofluorescence analyses to determine the distributions of LC3B-positive autophagosomes and macrophages. GA SFMCs and STs exhibited impaired autophagic degradation, indicated by elevated levels of LC3B and p62, along with CTSD upregulation and caspase-3 activation. Macrophages from GA STs exhibited significant accumulation of LC3B-positive autophagosomes. The temporal effects of MSU crystals on the ALP and the associations of these effects with cell death were investigated using a macrophage model of GA. MSU crystal-exposed macrophages exhibited early (2 h) autophagosome formation but later (6-24 h) autophagic flux impairment, demonstrated by p62 accumulation, lysosomal inhibitor failure to increase LC3B accumulation, and LC3B colocalization with p62. These macrophages exhibited autophagic flux impairment because of CTSD inactivation-mediated lysosomal dysfunction, which caused immature CTSD to accumulate within damaged LAMP2-positive lysosomes. This accumulation coincided with caspase-3-dependent cell death (24 h) that was unaffected by CTSD inhibition. These findings indicate that GA involves MSU crystal-induced impairment of autophagic degradation via CTSD inactivation-mediated lysosomal dysfunction, which promotes apoptosis in macrophages.

Asunto(s)

Artritis Gotosa , Humanos , Artritis Gotosa/inducido químicamente , Artritis Gotosa/metabolismo , Caspasa 3/metabolismo , Catepsina D/metabolismo , Catepsina D/farmacología , Ácido Úrico/farmacología , Ácido Úrico/metabolismo , Apoptosis , Autofagia , Macrófagos/metabolismo , Lisosomas/metabolismo

RESUMEN

The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are two major protein degradation pathways in eukaryotic cells. In the present study, we investigated the role of two systems and their interaction after Brucella.suis (B.suis) infected RAW264.7 murine macrophage. We demonstrated that B.suis activated ALP by upregulating LC3-Ⅱlevels as well as incomplete inhibition of P62 expression in RAW264.7 cells. On the other hand, we used pharmacological agents to confirm that ALP contributed the intracellular proliferation of B.suis. At present, the studies on the relationship between UPS and Brucella remain less understanding. In the study, we demonstrated that UPS machinery was also activated by promoting expression of 20 s proteasome after B.suis infected RAW264.7 cells, and that, the UPS could also promote intracellular proliferation of B.suis. Many recent studies propose the close link and dynamic interconversion between UPS and ALP. Currently, the experiments demonstrated that after RAW264.7 cells infected B.suis, ALP was activated following UPS inhibition, while the UPS was not effectively activated after ALP inhibition. Last, we compared the ability to promote intracellular proliferation of B.suis between UPS and ALP. The results displayed that the ability of UPS to promote intracellular proliferation of B.suis was stronger than that of ALP, and simultaneous inhibition of UPS and ALP led to seriously affection on intracellular proliferation of B.suis. All above, our research provides a better understanding on the interaction between Brucella and both systems.

Asunto(s)

Brucella suis , Complejo de la Endopetidasa Proteasomal , Ratones , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Autofagia/fisiología , Lisosomas/metabolismo

RESUMEN

Objective:To explore the regulatory mechanism of α-synuclein in the degradation of autophagy-lysosome pathway(ALP) in Parkinson disease(PD) model cells after interference or overexpression of dynein heavy chain(Dynhc) gene.Methods:SH-SY5Y cells were divided into control group, PD group, Dynhc interference group, Dynhc overexpression group, and Dynhc interference+ rapamycin group according to experimental requirements.Using Western blot to detect Dynhc, α-synuclein, microtubule-associated protein l light chain 3 (LC3), lysosome-associated membrane protein 2 (LAMP2), tubulin, dynein activator protein p150, and kinesin KIF5B.Flow cytometry was used to detect the level of cell apoptosis.Immunoconfocal microscopy was used to observe the structure of tubulin and the co-localization of LC3 and LAMP.SPSS 23.0 software was used for statistical analysis.One-way ANOVA was used for inter group comparisons, and further pairwise comparisons were conducted by LSD- t test. Results:There were statistically significant differences in the expression of α-synuclein, autophagy-related proteins, microtubules, and microtubule-related proteins among cells in the 5 groups(all P<0.001). The protein expression levels of Dynhc, α-synuclein, LC3, LAMP2, p150, and KIF5B in the PD group were higher than those in the control group (all P<0.05). The protein levels of Dynhc, LAMP2, tubulin and p150 in the Dynhc interference group were lower than those in the PD group (all P<0.05), while the protein levels of α-synuclein, LC3 and KIF5B were higher than those in the PD group (all P<0.05). The protein levels of α-synuclein, LC3, and KIF5B in the Dynhc overexpression group were lower than those in the PD group (all P<0.05), while the protein levels of Dynhc, LAMP2 and p150 were higher than those in the PD group (all P<0.05). The protein level of LC3 in the Dynhc interference+ rapamycin group was higher than that in the Dynhc interference group ( P<0.05). There were no statistically significant differences in the protein levels of Dynhc, α-synuclein, LAMP2, microtubule protein, p150 and KIF5B compared to the Dynhc interference group (all P>0.05). Compared with the control group, the cell apoptosis rate in PD group increased((12.77±1.66)%, (7.64±1.45)%), the microtubule morphology remained unchanged, and autophagosomes fused more with lysosomes. Compared with the PD group, the cell apoptosis rate of Dynhc overexpression group decreased, and there was no significant change in microtubule structure, and there was more fusion between autophagosomes and lysosomes.Compared with the PD group, the cell apoptosis rat of Dynhc interference group increased((18.45±1.91)%), and the microtubule morphology was sparse, and there was less fusion between autophagosomes and lysosomes. Compared with the PD group, the Dynhc overexpression group showed a decrease in cell apoptosis rate ((9.95±1.56)%), no significant changes in microtubule structure, and more fusion between autophagosomes and lysosomes.Compared with the Dynhc interference group, the Dynhc interference+ rapamycin group showed no significant changes in cell apoptosis rate ((19.05±2.46)%), microtubule morphology, and fusion of autophagosomes and lysosomes. Conclusion:Dynhc can reduce cell apoptosis by enhancing cell ALP function, increasing the degradation of α-synuclein and maintaining of microtubule structure integrity.