RESUMO
Seipin is one key mediator of lipid metabolism that is highly expressed in adipose tissues as well as in the brain. Lack of Seipin gene, Bscl2, leads to not only severe lipid metabolic disorders but also cognitive impairments and motor disabilities. Myelin, composed mainly of lipids, facilitates nerve transmission and is important for motor coordination and learning. Whether Seipin deficiency-leaded defects in learning and motor coordination is underlined by lipid dysregulation and its consequent myelin abnormalities remains to be elucidated. In the present study, we verified the expression of Seipin in oligodendrocytes (OLs) and their precursors, oligodendrocyte precursor cells (OPCs), and demonstrated that Seipin deficiency compromised OPC differentiation, which led to decreased OL numbers, myelin protein, myelinated fiber proportion and thickness of myelin. Deficiency of Seipin resulted in impaired spatial cognition and motor coordination in mice. Mechanistically, Seipin deficiency suppressed sphingolipid metabolism-related genes in OPCs and caused morphological abnormalities in lipid droplets (LDs), which markedly impeded OPC differentiation. Importantly, rosiglitazone, one agonist of PPAR-gamma, substantially restored phenotypes resulting from Seipin deficiency, such as aberrant LDs, reduced sphingolipids, obstructed OPC differentiation, and neurobehavioral defects. Collectively, the present study elucidated how Seipin deficiency-induced lipid dysregulation leads to neurobehavioral deficits via impairing myelination, which may pave the way for developing novel intervention strategy for treating metabolism-involved neurological disorders.
Assuntos
Diferenciação Celular , Disfunção Cognitiva , Subunidades gama da Proteína de Ligação ao GTP , Bainha de Mielina , Células Precursoras de Oligodendrócitos , Animais , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Camundongos , Células Precursoras de Oligodendrócitos/metabolismo , Bainha de Mielina/metabolismo , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Disfunção Cognitiva/genética , Metabolismo dos Lipídeos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo , PPAR gama/genética , Camundongos Knockout , Masculino , Rosiglitazona/farmacologiaRESUMO
Seipin is a key regulator of lipid metabolism, the deficiency of which leads to severe lipodystrophy. Hypothalamus is the pivotal center of brain that modulates appetite and energy homeostasis, where Seipin is abundantly expressed. Whether and how Seipin deficiency leads to systemic metabolic disorders via hypothalamus-involved energy metabolism dysregulation remains to be elucidated. In the present study, we demonstrated that Seipin-deficiency induced hypothalamic inflammation, reduction of anorexigenic pro-opiomelanocortin (POMC), and elevation of orexigenic agonist-related peptide (AgRP). Importantly, administration of rosiglitazone, a thiazolidinedione antidiabetic agent, rescued POMC and AgRP expression, suppressed hypothalamic inflammation, and restored energy homeostasis in Seipin knockout mice. Our findings offer crucial insights into the mechanism of Seipin deficiency-associated energy imbalance and indicates that rosiglitazone could serve as potential intervening agent towards metabolic disorders linked to Seipin.
Assuntos
Proteína Relacionada com Agouti , Metabolismo Energético , Subunidades gama da Proteína de Ligação ao GTP , Homeostase , Hipotálamo , Camundongos Knockout , Pró-Opiomelanocortina , Rosiglitazona , Animais , Camundongos , Hipotálamo/metabolismo , Metabolismo Energético/efeitos dos fármacos , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/biossíntese , Proteína Relacionada com Agouti/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Rosiglitazona/farmacologia , Masculino , Doenças Neuroinflamatórias/etiologia , Camundongos Endogâmicos C57BL , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Neuropeptídeos/genética , Neuropeptídeos/deficiência , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age-linked damage to amino acid sequence NGR (Asn-Gly-Arg) results in "gain-of-function" conformational switching to isoDGR (isoAsp-Gly-Arg). This integrin-binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age-linked cardiovascular disorders. We now report that anti-isoDGR immunotherapy mitigates lifespan reduction of Pcmt1-/- mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1-/- and naturally aged WT animals, which could also be induced via injection of isoDGR-modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti-isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR-protein levels in body tissues, decreased pro-inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1-/- mice. Mechanistically, isoDGR-mAb mediated immune clearance of damaged isoDGR-proteins via antibody-dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age-linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.
Assuntos
Citocinas , Longevidade , Humanos , Animais , Camundongos , Idoso , Sequência de Aminoácidos , Ligação ProteicaRESUMO
Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol", contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to monitor accessible cholesterol in live cells remain limited. Here, we engineer a highly sensitive accessible cholesterol biosensor by taking advantage of the cholesterol-sensing element (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this cholesterol biosensor, which we call GRAM-W, we successfully visualize in real time the distribution of accessible cholesterol in many different cell types, including human keratinocytes and iPSC-derived neurons, and show differential dependencies on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Furthermore, we combine GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and establish a strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These tools will allow us to obtain important insights into the molecular mechanisms by which the distribution of cellular cholesterol is regulated.
Assuntos
Técnicas Biossensoriais , Colesterol , Humanos , Membrana Celular/metabolismo , Colesterol/metabolismo , Transporte Biológico , HomeostaseRESUMO
BACKGROUND: Lipodystrophy-associated metabolic disorders caused by Seipin deficiency lead to not only severe lipodystrophy but also neurological disorders. However, the underlying mechanism of Seipin deficiency-induced neuropathy is not well elucidated, and the possible restorative strategy needs to be explored. METHODS: In the present study, we used Seipin knockout (KO) mice, combined with transcriptome analysis, mass spectrometry imaging, neurobehavior test, and cellular and molecular assay to investigate the systemic lipid metabolic abnormalities in lipodystrophic mice model and their effects on adult neurogenesis in the subventricular zone (SVZ) and olfactory function. After subcutaneous adipose tissue (AT) transplantation, metabolic and neurological function was measured in Seipin KO mice to clarify whether restoring lipid metabolic homeostasis would improve neurobehavior. RESULTS: It was found that Seipin KO mice presented the ectopic accumulation of lipids in the lateral ventricle, accompanied by decreased neurogenesis in adult SVZ, diminished new neuron formation in the olfactory bulb, and impaired olfactory-related memory. Transcriptome analysis showed that the differentially expressed genes (DEGs) in SVZ of adult Seipin KO mice were significantly enriched in lipid metabolism. Mass spectrometry imaging showed that the levels of glycerophospholipid and diglyceride (DG) were significantly increased. Furthermore, we found that AT transplantation rescued the abnormality of peripheral metabolism in Seipin KO mice and ameliorated the ectopic lipid accumulation, concomitant with restoration of the SVZ neurogenesis and olfactory function. Mechanistically, PKCα expression was up-regulated in SVZ tissues of Seipin KO mice, which may be a potential mediator between lipid dysregulation and neurological disorder. DG analogue (Dic8) can up-regulate PKCα and inhibit the proliferation and differentiation of neural stem cells (NSCs) in vitro, while PKCα inhibitor can block this effect. CONCLUSION: This study demonstrates that Seipin deficiency can lead to systemic lipid disorder with concomitant SVZ neurogenesis and impaired olfactory memory. However, AT restores lipid homeostasis and neurogenesis. PKCα is a key mediator mediating Seipin KO-induced abnormal lipid metabolism and impaired neurogenesis in the SVZ, and inhibition of PKCα can restore the impaired neurogenesis. This work reveals the underlying mechanism of Seipin deficiency-induced neurological dysfunction and provides new ideas for the treatment of neurological dysfunction caused by metabolic disorders.
Assuntos
Metabolismo dos Lipídeos , Lipodistrofia , Camundongos , Animais , Camundongos Knockout , Metabolismo dos Lipídeos/genética , Proteína Quinase C-alfa/genética , Obesidade , Neurogênese/genéticaRESUMO
Parkinson's disease (PD) is a neurodegenerative disease that affects millions of elderly people worldwide and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The precise mechanisms underlying the pathogenesis of PD are still not fully understood, but it is well accepted that the misfolding, aggregation, and abnormal degradation of proteins are the key causative factors of PD. Heat shock protein 70 (Hsp70) is a molecular chaperone that participates in the degradation of misfolded and aggregated proteins in living cells and organisms. Parkin, an E3 ubiquitin ligase, participates in the degradation of proteins via the proteasome pathway. Recent studies have indicated that both Hsp70 and Parkin play pivotal roles in PD pathogenesis. In this review, we focus on discussing how dysregulation of Hsp70 and Parkin leads to PD pathogenesis, the interaction between Hsp70 and Parkin in the context of PD and their therapeutic applications in PD.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Idoso , Humanos , Proteínas de Choque Térmico HSP70 , Ubiquitina-Proteína Ligases , Neurônios DopaminérgicosRESUMO
Microglia are the resident innate immune cells in the brain with a major role in orchestrating immune responses. They also provide a frontline of host defense in the central nervous system (CNS) through their active phagocytic capability. Being a professional phagocyte, microglia participate in phagocytic and autophagic clearance of cellular waste and debris as well as toxic protein aggregates, which relies on optimal lysosomal acidification and function. Defective microglial lysosomal acidification leads to impaired phagocytic and autophagic functions which result in the perpetuation of neuroinflammation and progression of neurodegeneration. Reacidification of impaired lysosomes in microglia has been shown to reverse neurodegenerative pathology in Alzheimer's disease. In this review, we summarize key factors and mechanisms contributing to lysosomal acidification impairment and the associated phagocytic and autophagic dysfunction in microglia, and how these defects contribute to neuroinflammation and neurodegeneration. We further discuss techniques to monitor lysosomal pH and therapeutic agents that can reacidify impaired lysosomes in microglia under disease conditions. Finally, we propose future directions to investigate the role of microglial lysosomal acidification in lysosome-mitochondria crosstalk and in neuron-glia interaction for more comprehensive understanding of its broader CNS physiological and pathological implications.
Assuntos
Doença de Alzheimer , Microglia , Humanos , Microglia/metabolismo , Doenças Neuroinflamatórias , Doença de Alzheimer/metabolismo , Lisossomos/metabolismo , Concentração de Íons de HidrogênioRESUMO
Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.
Assuntos
Degeneração Macular , Transcriptoma , Adulto , Animais , Humanos , Coelhos , Degeneração Macular/genética , Degeneração Macular/terapia , Células-Tronco , Células Epiteliais , Pigmentos da RetinaRESUMO
Parkinson's Disease (PD) is a prevalent neurodegenerative disorder that is characterized pathologically by the loss of A9-specific dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Despite intensive research, the etiology of PD is currently unresolved, and the disease remains incurable. This, in part, is due to the lack of an experimental disease model that could faithfully recapitulate the features of human PD. However, the recent advent of induced pluripotent stem cell (iPSC) technology has allowed PD models to be created from patient-derived cells. Indeed, DA neurons from PD patients are now routinely established in many laboratories as monolayers as well as 3D organoid cultures that serve as useful toolboxes for understanding the mechanism underlying PD and also for drug discovery. At the same time, the iPSC technology also provides unprecedented opportunity for autologous cell-based therapy for the PD patient to be performed using the patient's own cells as starting materials. In this review, we provide an update on the molecular processes underpinning the development and differentiation of human pluripotent stem cells (PSCs) into midbrain DA neurons in both 2D and 3D cultures, as well as the latest advancements in using these cells for drug discovery and regenerative medicine. For the novice entering the field, the cornucopia of differentiation protocols reported for the generation of midbrain DA neurons may seem daunting. Here, we have distilled the essence of the different approaches and summarized the main factors driving DA neuronal differentiation, with the view to provide a useful guide to newcomers who are interested in developing iPSC-based models of PD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Células-Tronco Pluripotentes , Humanos , Doença de Parkinson/terapia , Mesencéfalo , Neurônios Dopaminérgicos , OrganoidesRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. The etiology of PD has yet to be elucidated, and the disease remains incurable. Increasing evidence suggests that oxidative stress is the key causative factor of PD. Due to their capacity to alleviate oxidative stress, antioxidants hold great potential for the treatment of PD. Vitamins are essential organic substances for maintaining the life of organisms. Vitamin deficiency is implicated in the pathogenesis of various diseases, such as PD. In the present study, we investigated whether administration of vitamin B12 (VB12) could ameliorate PD phenotypes in vitro and in vivo. Our results showed that VB12 significantly reduced the generation of reactive oxygen species (ROS) in the rotenone-induced SH-SY5Y cellular PD model. In a Parkin gene knockout C. elegans PD model, VB12 mitigated motor dysfunction. Moreover, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse PD model, VB12 also displayed protective effects, including the rescue of mitochondrial function, dopaminergic neuron loss, and movement disorder. In summary, our results suggest that vitamin supplementation may be a novel method for the intervention of PD, which is safer and more feasible than chemical drug treatment.
RESUMO
BACKGROUND: Considerable evidence links dietary salt intake with the development of hypertension, left ventricular hypertrophy, and increased risk of stroke and coronary heart disease. Despite extensive epidemiological and basic science interrogation of the relationship between high salt (HS) intake and blood pressure, it remains unclear how HS impacts endothelial cell (EC) and vascular structure in vivo. This study aims to elucidate HS-induced vascular pathology using a differential systemic decellularization in vivo approach. METHODS: We performed systematic molecular characterization of the endothelial glycocalyx and EC proteomes in mice with HS (8%) diet-induced hypertension versus healthy control animals. Isolation of eGC and EC compartments was achieved using differential systemic decellularization in vivo methodology. Altered protein expression in hypertensive compared to normal mice was characterized by liquid chromatography tandem mass spectrometry. Proteomic results were validated using functional assays, microscopic imaging, and histopathologic evaluation. RESULTS: Proteomic analysis revealed a significant downregulation of eGC and associated proteins in HS diet-induced hypertensive mice (among 1696 proteins identified in this group, 723 were markedly decreased in abundance, while only 168 were increased in abundance. Bioinformatic analysis indicated substantial derangement of the eGC layer, which was subsequently confirmed by fluorescent and electron microscopy assessment of vessel damage ex vivo. In the EC fraction, HS-induced hypertension significantly altered protein mediators of contractility, metabolism, mechanotransduction, renal function, and the coagulation cascade. In particular, we observed dysregulation of integrin subunits α2, α2b, and α5, which was associated with arterial wall inflammation and substantial infiltration of CD68+ monocyte-macrophages. Consequently, HS-induced hypertensive mice also displayed reduced vascular integrity of multiple organs including lungs, kidneys, and heart. CONCLUSIONS: These findings provide novel molecular insight into HS-induced structural changes in eGC and EC composition that may increase cardiovascular risk and potentially guide the development of new diagnostics and therapeutic interventions.
Assuntos
Hipertensão , Cloreto de Sódio na Dieta , Camundongos , Animais , Cloreto de Sódio na Dieta/efeitos adversos , Proteômica , Mecanotransdução Celular , Pressão Sanguínea/fisiologiaRESUMO
Abnormal lipid homeostasis has been observed in the brain of Parkinson's disease (PD) patients and experimental models, although the mechanism underlying this phenomenon is unclear. Notably, previous studies have reported that the PD-linked protein Parkin functionally interacts with important lipid regulators, including Sterol Regulatory Element-Binding Proteins (SREBPs) and cluster of differentiation 36 (CD36). Here, we demonstrate a functional relationship between Parkin and lipoprotein lipase (LPL), a triglyceride lipase that is widely expressed in the brain. Using a human neuroblastoma cell line and a Parkin knockout mouse model, we demonstrate that Parkin expression level positively correlates with neuronal LPL protein level and activity. Importantly, our study identified SREBP2, a major regulator of sterol and fatty acid synthesis, as a potential mediator between Parkin and LPL. Supporting this, SREBP2 genetic ablation abolished Parkin effect on LPL expression. We further demonstrate that Parkin-LPL pathway regulates the formation of intracellular lipid droplets, and that this pathway is upregulated upon exposure to PD-linked oxidative stress induced by rotenone. Finally, we show that inhibition of either LPL or SREBP2 exacerbates rotenone-induced cell death. Taken together, our findings reveal a novel pathway linking Parkin, SREBP2 and LPL in neuronal lipid homeostasis that may be relevant to the pathogenesis of PD.
Assuntos
Lipase Lipoproteica , Doença de Parkinson , Proteína de Ligação a Elemento Regulador de Esterol 2 , Ubiquitina-Proteína Ligases , Animais , Humanos , Camundongos , Homeostase , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Camundongos Knockout , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Rotenona/efeitos adversos , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Alzheimer's disease (AD) is the most common cause of dementia that affects millions of predominantly elderly individuals worldwide. Despite intensive research over several decades, controversies still surround the etiology of AD and the disease remains incurable. Meanwhile, new molecular players of the central amyloid cascade hypothesis have emerged and among these is a protease known as ß-site APP cleavage enzyme 2 (BACE2). Unlike BACE1, BACE2 cleaves the amyloid-ß protein precursor within the Aß domain that accordingly prevents the generation of Aß42 peptides, the aggregation of which is commonly regarded as the toxic entity that drives neurodegeneration in AD. Given this non-amyloidogenic role of BACE2, it is attractive to position BACE2 as a therapeutic target for AD. Indeed, several groups including ours have demonstrated a neuroprotective role for BACE2 in AD. In this review, we discuss emerging evidence supporting the ability of BACE2 in mitigating AD-associated pathology in various experimental systems including human pluripotent stem cell-derived cerebral organoid disease models. Alongside this, we also provide an update on the identification of single nucleotide polymorphisms occurring in the BACE2 gene that are linked to increased risk and earlier disease onset in the general population. In particular, we highlight a recently identified point mutation on BACE2 that apparently leads to sporadic early-onset AD. We believe that a better understanding of the role of BACE2 in AD would provide new insights for the development of viable therapeutic strategies for individuals with dementia.
Assuntos
Doença de Alzheimer , Humanos , Idoso , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismoRESUMO
Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium-some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD.
Assuntos
Ácido Glutâmico , Compostos de Lítio , Sinapses , Vesículas Sinápticas , Compostos de Lítio/farmacologia , Ácido Glutâmico/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Transtorno Bipolar/metabolismo , Transtorno Bipolar/patologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Hipocampo/patologia , Exocitose/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Animais , Ratos , Células CultivadasRESUMO
α-Synuclein (α-syn) is important in synucleinopathies such as Parkinson's disease (PD). While genome-wide association studies (GWASs) of synucleinopathies have identified many risk loci, the underlying genes have not been shown for most loci. Using Drosophila, we screened 3,471 mutant chromosomes for genetic modifiers of α-synuclein and identified 12 genes. Eleven modifiers have human orthologs associated with diseases, including MED13 and CDC27, which lie within PD GWAS loci. Drosophila Skd/Med13 and glycolytic enzymes are co-upregulated by α-syn-associated neurodegeneration. While elevated α-syn compromises mitochondrial function, co-expressing skd/Med13 RNAi and α-syn synergistically increase the ratio of oxidized-to-reduced glutathione. The resulting neurodegeneration can be suppressed by overexpressing a glycolytic enzyme or treatment with deferoxamine, suggesting that compensatory glycolysis is neuroprotective. In addition, the functional relationship between α-synuclein, MED13, and glycolytic enzymes is conserved between flies and mice. We propose that hypoxia-inducible factor and MED13 are part of a druggable pathway for PD.
Assuntos
Proteínas de Drosophila , Doença de Parkinson , Sinucleinopatias , Animais , Camundongos , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Estudo de Associação Genômica Ampla , Doença de Parkinson/metabolismo , Glicólise , Drosophila/metabolismo , Complexo Mediador/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
BACKGROUND: Parkinson's disease (PD) is characterized by selective and progressive dopamine (DA) neuron loss in the substantia nigra and other brain regions, with the presence of Lewy body formation. Most PD cases are sporadic, whereas monogenic forms of PD have been linked to multiple genes, including Leucine kinase repeat 2 (LRRK2) and PTEN-induced kinase 1 (PINK1), two protein kinase genes involved in multiple signaling pathways. There is increasing evidence to suggest that endogenous DA and DA-dependent neurodegeneration have a pathophysiologic role in sporadic and familial PD. METHODS: We generated patient-derived dopaminergic neurons and human midbrain-like organoids (hMLOs), transgenic (TG) mouse and Drosophila models, expressing both mutant and wild-type (WT) LRRK2 and PINK1. Using these models, we examined the effect of LRRK2 and PINK1 on tyrosine hydroxylase (TH)-DA pathway. RESULTS: We demonstrated that PD-linked LRRK2 mutations were able to modulate TH-DA pathway, resulting in up-regulation of DA early in the disease which subsequently led to neurodegeneration. The LRRK2-induced DA toxicity and degeneration were abrogated by wild-type (WT) PINK1 (but not PINK1 mutations), and early treatment with a clinical-grade drug, α-methyl-L-tyrosine (α-MT), a TH inhibitor, was able to reverse the pathologies in human neurons and TG Drosophila models. We also identified opposing effects between LRRK2 and PINK1 on TH expression, suggesting that functional balance between these two genes may regulate the TH-DA pathway. CONCLUSIONS: Our findings highlight the vital role of the TH-DA pathway in PD pathogenesis. LRRK2 and PINK1 have opposing effects on the TH-DA pathway, and its balance affects DA neuron survival. LRRK2 or PINK1 mutations can disrupt this balance, promoting DA neuron demise. Our findings provide support for potential clinical trials using TH-DA pathway inhibitors in early or prodromic PD.
Assuntos
Proteínas de Drosophila , Doença de Parkinson , Camundongos , Animais , Humanos , Dopamina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Doença de Parkinson/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Camundongos Transgênicos , Drosophila/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
BACKGROUND: Aberrant aggregation of α-synuclein (α-syn) is a key pathological feature of Parkinson's disease (PD), but the precise role of intestinal α-syn in the progression of PD is unclear. In a number of genetic Drosophila models of PD, α-syn was frequently ectopically expressed in the neural system to investigate the pathobiology. METHOD: We investigated the potential role of intestinal α-syn in PD pathogenesis using a Drosophila model. Human α-syn was overexpressed in Drosophila guts, and life span, survival, immunofluorescence and climbing were evaluated. Immunofluorescence, Western blotting and reactive oxygen species (ROS) staining were performed to assess the effects of intestinal α-syn on intestinal dysplasia. High-throughput RNA and 16S rRNA gene sequencing, quantitative RT-PCR, immunofluorescence, and ROS staining were performed to determine the underlying molecular mechanism. RESULTS: We found that the intestinal α-syn alone recapitulated many phenotypic and pathological features of PD, including impaired life span, loss of dopaminergic neurons, and progressive motor defects. The intestine-derived α-syn disrupted intestinal homeostasis and accelerated the onset of intestinal ageing. Moreover, intestinal expression of α-syn induced dysbiosis, while microbiome depletion was efficient to restore intestinal homeostasis and ameliorate the progression of PD. Intestinal α-syn triggered ROS, and eventually led to the activation of the dual oxidase (DUOX)-ROS-Jun N-terminal Kinase (JNK) pathway. In addition, α-syn from both the gut and the brain synergized to accelerate the progression of PD. CONCLUSIONS: The intestinal expression of α-syn recapitulates many phenotypic and pathologic features of PD, and induces dysbiosis that aggravates the pathology through the DUOX-ROS-JNK pathway in Drosophila. Our findings provide new insights into the role of intestinal α-syn in PD pathophysiology.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Drosophila/genética , Drosophila/metabolismo , Oxidases Duais , Disbiose/complicações , Disbiose/genética , Humanos , Intestinos/patologia , Proteínas Quinases JNK Ativadas por Mitógeno , Doença de Parkinson/metabolismo , RNA Ribossômico 16S , Espécies Reativas de Oxigênio , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Autophagy has emerged as the prime machinery for implementing organelle quality control. In the context of mitophagy, the ubiquitin E3 ligase Parkin tags impaired mitochondria with ubiquitin to activate autophagic degradation. Although ubiquitination is essential for mitophagy, it is unclear how ubiquitinated mitochondria activate autophagosome assembly locally to ensure efficient destruction. Here, we report that Parkin activates lipid remodeling on mitochondria targeted for autophagic destruction. Mitochondrial Parkin induces the production of phosphatidic acid (PA) and its subsequent conversion to diacylglycerol (DAG) by recruiting phospholipase D2 and activating the PA phosphatase, Lipin-1. The production of DAG requires mitochondrial ubiquitination and ubiquitin-binding autophagy receptors, NDP52 and optineurin (OPTN). Autophagic receptors, via Golgi-derived vesicles, deliver an autophagic activator, EndoB1, to ubiquitinated mitochondria. Inhibition of Lipin-1, NDP52/OPTN, or EndoB1 results in a failure to produce mitochondrial DAG, autophagosomes, and mitochondrial clearance, while exogenous cell-permeable DAG can induce autophagosome production. Thus, mitochondrial DAG production acts downstream of Parkin to enable the local assembly of autophagosomes for the efficient disposal of ubiquitinated mitochondria.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitina-Proteína Ligases/genética , LipídeosRESUMO
Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.
Assuntos
Epitélio Pigmentado da Retina , Células-Tronco , Diferenciação Celular , Células Epiteliais/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismoRESUMO
The blood-brain barrier (BBB) represents a key challenge in developing brain-penetrating therapeutic molecules. BBB dysfunction is also associated with the onset and progression of various brain diseases. The BBB-on-a-chip (µBBB), an organ-on-chip technology, has emerged as a powerful in vitro platform that closely mimics the human BBB microenvironments. While the µBBB technology has seen wide application in the study of brain cancer, its utility in other brain disease models ("µBBB+") is less appreciated. Based on the advances of the µBBB technology and the evolution of in vitro models for brain diseases over the last decade, we propose the concept of a "µBBB+" system and summarize its major promising applications in pathological studies, personalized medical research, drug development, and multi-organ-on-chip approaches. We believe that such a sophisticated "µBBB+" system is a highly tunable and promising in vitro platform for further advancement of the understanding of brain diseases.