RESUMEN
Deposition of extracellular Amyloid Beta (Aß) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer's Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aß and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aß42 oligomers (AßOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Encéfalo , Violeta de Genciana , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/química , Humanos , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/patología , Ratones , Violeta de Genciana/química , Amiloide/metabolismo , Amiloide/química , Tomografía de Emisión de Positrones , FemeninoRESUMEN
Arrestins are multifunctional proteins that regulate G-protein-coupled receptor (GPCR) desensitization, signaling, and internalization. The arrestin family consists of four subtypes: visual arrestin1, ß-arrestin1, ß-arrestin2, and visual arrestin-4. Recent studies have revealed the multifunctional roles of ß-arrestins beyond GPCR signaling, including scaffolding and adapter functions, and physically interacting with non-GPCR receptors. Increasing evidence suggests that ß-arrestins are involved in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia (FTD), and Parkinson's disease (PD). ß-arrestins physically interact with γ-secretase, leading to increased production and accumulation of amyloid-beta in AD. Furthermore, ß-arrestin oligomers inhibit the autophagy cargo receptor p62/SQSTM1, resulting in tau accumulation and aggregation in FTD. In PD, ß-arrestins are upregulated in postmortem brain tissue and an MPTP model, and the ß2AR regulates SNCA gene expression. In this review, we aim to provide an overview of ß-arrestin1 and ß-arrestin2, and describe their physiological functions and roles in neurodegenerative diseases. The multifaceted roles of ß-arrestins and their involvement in neurodegenerative diseases suggest that they may serve as promising therapeutic targets.
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Enfermedad de Alzheimer , Demencia Frontotemporal , Enfermedades Neurodegenerativas , Humanos , beta-Arrestinas/metabolismo , Arrestina/metabolismo , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/terapia , Receptores Acoplados a Proteínas G/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/etiologíaRESUMEN
Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) is a nuclear-encoded mitochondrial protein which is primarily mutated in the spectrum of familial and sporadic amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD). Endogenous CHCHD10 levels decline in the brains of ALS-FTD patients, and the CHCHD10S59L mutation in Drosophila induces dominant toxicity together with PTEN-induced kinase 1 (PINK1), a protein critical for the induction of mitophagy. However, whether and how CHCHD10 variants regulate mitophagy flux in the mammalian brain is unknown. Here, we demonstrate through in vivo and in vitro models, as well as human FTD brain tissue, that ALS/FTD-linked CHCHD10 mutations (R15L and S59L) impair mitophagy flux and mitochondrial Parkin recruitment, whereas wild-type CHCHD10 (CHCHD10WT) normally enhances these measures. Specifically, we show that CHCHD10R15L and CHCHD10S59L mutations reduce PINK1 levels by increasing PARL activity, whereas CHCHD10WT produces the opposite results through its stronger interaction with PARL, suppressing its activity. Importantly, we also demonstrate that FTD brains with TAR DNA-binding protein-43 (TDP-43) pathology demonstrate disruption of the PARL-PINK1 pathway and that experimentally impairing mitophagy promotes TDP-43 aggregation. Thus, we provide herein new insights into the regulation of mitophagy and TDP-43 aggregation in the mammalian brain through the CHCHD10-PARL-PINK1 pathway.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Animales , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Mitofagia/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Quinasas/genética , Mamíferos/metabolismo , Metaloproteasas/genéticaRESUMEN
Oxidative damage in the brain is one of the earliest drivers of pathology in Alzheimer's disease (AD) and related dementias, both preceding and exacerbating clinical symptoms. In response to oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2) is normally activated to protect the brain from oxidative damage. However, Nrf2-mediated defense against oxidative stress declines in AD, rendering the brain increasingly vulnerable to oxidative damage. Although this phenomenon has long been recognized, its mechanistic basis has been a mystery. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that Slingshot homolog-1 (SSH1) drives this effect by acting as a counterweight to neuroprotective Nrf2 in response to oxidative stress and disease. Specifically, oxidative stress-activated SSH1 suppresses nuclear Nrf2 signaling by sequestering Nrf2 complexes on actin filaments and augmenting Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 interaction, independently of SSH1 phosphatase activity. We also show that Ssh1 elimination in AD models increases Nrf2 activation, which mitigates tau and amyloid-ß accumulation and protects against oxidative injury, neuroinflammation, and neurodegeneration. Furthermore, loss of Ssh1 preserves normal synaptic function and transcriptomic patterns in tauP301S mice. Importantly, we also show that human AD brains exhibit highly elevated interactions of Nrf2 with both SSH1 and Keap1. Thus, we demonstrate here a unique mode of Nrf2 blockade that occurs through SSH1, which drives oxidative damage and ensuing pathogenesis in AD. Strategies to inhibit SSH1-mediated Nrf2 suppression while preserving normal SSH1 catalytic function may provide new neuroprotective therapies for AD and related dementias.
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Enfermedad de Alzheimer , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neuroprotección , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiologíaRESUMEN
TAS2Rs (bitter taste receptors) are GPCRs (G protein-coupled receptors) expressed on human airway smooth muscle (HASM) cells; when activated by receptor agonists they evoke marked airway relaxation. In both taste and HASM cells, TAS2Rs activate a canonical Gßγ-mediated stimulation of Ca2+ release from intracellular stores by activation of PLCß (phospholipase Cß). Alone, this [Ca2+]i signaling does not readily account for relaxation, particularly since bronchoconstrictive agonists acting at Gq-coupled receptors also increase [Ca2+]i. We established that TAS2R14 activation in HASM promotes relaxation through F-actin (filamentous actin) severing. This destabilization of actin was from agonist-promoted activation (dephosphorylation) of cofilin, which was pertussis toxin sensitive. Cofilin dephosphorylation was due to TAS2R-mediated deactivation of LIM domain kinase. The link between early receptor action and the distal cofilin dephosphorylation was found to be the polarity protein partitioning defective 3 (Par3), a known binding partner with PLCß that inhibits LIM kinase. The physiologic relevance of this pathway was assessed using knock-downs of cofilin and Par3 in HASM cells and in human precision-cut lung slices. Relaxation by TAS2R14 agonists was ablated with knock-down of either protein as assessed by magnetic twisting cytometry in isolated cells or intact airways in the slices. Blocking [Ca2+]i release by TAS2R14 inhibited agonist-promoted cofilin dephosphorylation, confirming a role for [Ca2+]i in actin-modifying pathways. These results further elucidate the mechanistic basis of TAS2R-mediated HASM relaxation and point toward nodal points that may act as asthma or chronic obstructive pulmonary disease response modifiers or additional targets for novel bronchodilators.
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Actinas , Asma , Receptores Acoplados a Proteínas G , Humanos , Actinas/metabolismo , Asma/metabolismo , Quinasas Lim/metabolismo , Pulmón/metabolismo , Relajación Muscular/fisiología , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Accumulating toxic protein assemblies, including Aß and tau, and dysfunctional mitochondria are associated with synaptic and neuronal loss in Alzheimer's disease (AD). Such accumulations are thought to be owing to clearance defects in the autophagy-lysosome pathway. Mitochondrial dysfunction is evident in AD brains and animal models at multiple levels, such as mitochondrial genomic mutations, disrupted bioenergetics, deregulated mitochondrial dynamics and impaired clearance of damaged mitochondria (mitophagy). Slingshot homolog-1 (SSH1) is a phosphatase activated by oxidative stress, high intracellular levels of Ca2+ and Aß42 oligomers (Aß42O), known for its function to dephosphorylate/activate cofilin through the N-terminal region. SSH1-mediated cofilin dephosphorylation results in Ab42O-induced severing of F-actin and translocation of cofilin to mitochondria, which promotes mitochondria-mediated apoptosis, synaptic loss and synaptic deficits. On the other hand, SSH1-mediated dephosphorylation/deactivation of the autophagy-cargo receptor p62 (SQSTM1), through its C-terminal region, inhibits p62 autophagy flux. However, the interplay between these two different activities of SSH1 in Aß42O-induced mitochondrial toxicity remains unclear. In this study, we assessed the role of endogenous SSH1 and different regions of SSH1 in regulating mitochondrial health, mitochondrial respiration, clearance of damaged mitochondria and synaptic integrity in vitro and in vivo. Our results indicate that SSH1 suppresses mitochondrial health and respiration through the cofilin-binding N-terminal region, whereas SSH1 impairs mitophagy through a newly identified ~ 100 residue p62-binding domain in the C-terminal region. These results indicate that both N-terminal and C-terminal regions negatively impact mitochondria by distinct and independent modalities to amplify mitochondrial abnormalities, making SSH1 an excellent target to mitigate AD pathogenesis.
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Factores Despolimerizantes de la Actina , Enfermedad de Alzheimer , Animales , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Enfermedad de Alzheimer/metabolismo , Mitocondrias/metabolismoRESUMEN
Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.
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Enfermedad de Alzheimer , Tauopatías , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Caracteres Sexuales , Tauopatías/genética , Tauopatías/patología , Tioléster Hidrolasas/genética , Proteasas Ubiquitina-Específicas , Proteínas tau/genéticaRESUMEN
Increasing evidence indicates that the accumulation misfolded proteins in Alzheimer's disease (AD) arises from clearance defects in the autophagy-lysosome pathway. Misfolded proteins such as Aß and tau are secreted in small extracellular vesicles (i.e., exosomes) and are propagated from cell to cell in part through secreted small extracellular vesicles (sEVs). Recent studies suggest that autophagic activity and exosome secretion are coregulated events, and multiple autophagy-related proteins are found in sEVs, including the cargo receptors Sqstm1/p62 and optineurin. However, whether and how autophagy cargo receptors per se regulate the secretion of sEVs is unknown. Moreover, despite the prominent role of actin dynamics in secretory vesicle release, its role in EV secretion is unknown. In this study, we leveraged the dual axes of Slingshot Homolog-1 (SSH1), which inhibits Sqstm1/p62-mediated autophagy and activates cofilin-mediated actin dynamics, to study the regulation of sEV secretion. Here we show that cargo receptors Sqstm1/p62 and optineurin inhibit sEV secretion, an activity that requires their ability to bind ubiquitinated cargo. Conversely, SSH1 increases sEV secretion by dephosphorylating Sqstm1/p62 at pSer403, the phospho-residue that allows Sqstm1/p62 to bind ubiquitinated cargo. In addition, increasing actin dynamics through the SSH1-cofilin activation pathway also increases sEV secretion, which is mimicked by latrunculin B treatment. Finally, Aß42 oligomers and mutant tau increase sEV secretion and are physically associated with secreted sEVs. These findings suggest that increasing cargo receptor engagement with autophagic cargo and reducing actin dynamics (i.e., SSH1 inhibition) represents an attractive strategy to promote misfolded protein degradation while reducing sEV-mediated cell to cell spread of pathology.
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Coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) is a mitochondrial protein that plays important roles in cristae structure, oxidative phosphorylation and apoptosis. Multiple mutations in CHCHD2 have been associated with Lewy body disorders (LBDs), such as Parkinson's disease (PD) and dementia with Lewy bodies, with the CHCHD2-T61I mutation being the most widely studied. However, at present, only CHCHD2 knockout or CHCHD2/CHCHD10 double knockout mouse models have been investigated. They do not recapitulate the pathology seen in patients with CHCHD2 mutations. We generated the first transgenic mouse model expressing the human PD-linked CHCHD2-T61I mutation driven by the mPrP promoter. We show that CHCHD2-T61I Tg mice exhibit perinuclear mitochondrial aggregates, neuroinflammation, and have impaired long-term synaptic plasticity associated with synaptic dysfunction. Dopaminergic neurodegeneration, a hallmark of PD, is also observed along with α-synuclein pathology. Significant motor dysfunction is seen with no changes in learning and memory at 1 year of age. A minor proportion of the CHCHD2-T61I Tg mice (~10%) show a severe motor phenotype consistent with human Pisa Syndrome, an atypical PD phenotype. Unbiased proteomics analysis reveals surprising increases in many insoluble proteins predominantly originating from mitochondria and perturbing multiple canonical biological pathways as assessed by ingenuity pathway analysis, including neurodegenerative disease-associated proteins such as tau, cofilin, SOD1 and DJ-1. Overall, CHCHD2-T61I Tg mice exhibit pathological and motor changes associated with LBDs, indicating that this model successfully captures phenotypes seen in human LBD patients with CHCHD2 mutations and demonstrates changes in neurodegenerative disease-associated proteins, which delineates relevant pathological pathways for further investigation.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , Animales , Ratones , Enfermedad de Parkinson/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas Mitocondriales/genética , Mutación , Modelos Animales de EnfermedadRESUMEN
Mutations in CHCHD10, a gene coding for a mitochondrial intermembrane space protein, are associated with Frontotemporal dementia (FTD)-Amyotrophic lateral sclerosis (ALS) spectrum disorders, which are pathologically characterized by cytoplasmic inclusions containing TDP-43. FTD/ALS-linked CHCHD10 mutations and TDP-43 inclusions similarly induce mitochondrial defects in respiration, fusion/fission, mtDNA stability, and cristae structure, while sizeable amounts of cytoplasmic TDP-43 aggregates are found in mitochondria. However, the mechanistic link between CHCHD10 and TDP-43 pathogenesis remains unclear. In this study, we present immunohistochemical and biochemical evidence demonstrating that insoluble CHCHD10 aggregates accumulate and colocalize with phospho-TDP-43 inclusions in brains of FTLD-TDP and AD patients, and that insoluble CHCHD10 levels tightly correlate with insoluble TDP-43 levels in control and FTLD-TDP brains. In an experimental exploration of this pathological phenotype, transgenic mice neuronally expressing FTD/ALS-linked CHCHD10R15L or CHCHDS59L mutations but not CHCHD10WT transgenic mice exhibit significantly increased CHCHD10 aggregation and phospho-TDP-43 pathology, which often colocalize within the same inclusions. Such pathologies are reflected in poor functional outcomes in long-term synaptic plasticity, motor unit physiology, and behavior in CHCHD10R15L and CHCHDS59L transgenic mice. In contrast, expression of CHCHD10WT in hTDP-43 transgenic mice (TAR4;CHCHD10WT) significantly mitigates phospho-TDP-43 pathology and rescues TDP-43-induced impairments in synaptic integrity and long-term synaptic plasticity. In isolated mitochondria, the S59L mutation induces the aggregation of resident CHCHD10S59L protein as well as the aggregation and slower turnover of recombinant TDP-43 imported into mitochondria. Likewise, in an in vitro cell-free system, the S59L mutation induces the aggregation of CHCHD10S59L protein while simultaneously enhancing the aggregation of recombinant TDP-43, as evidenced by filter trap assays and atomic force microscopy. In contrast, recombinant CHCHD10WT inhibits the growth of TDP-43 aggregates. These results in human brains, transgenic mice, and in vitro systems substantiate the role of wild type and mutant CHCHD10 in modulating mitochondrial CHCHD10 and TDP-43 pathogenesis together with associated phenotypes in long-term synaptic plasticity and motor unit physiology in mice and humans.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Humanos , Ratones , Ratones Transgénicos , Proteínas Mitocondriales/genética , Plasticidad NeuronalRESUMEN
G protein coupled receptors (GPCRs) are a superfamily of transmembrane-spanning receptors that are activated by multiple endogenous ligands and are the most common target for agonist or antagonist therapeutics across a broad spectrum of diseases. Initial characterization within the superfamily suggested that a receptor activated a single intracellular pathway, depending on the G protein to which it coupled. However, it has become apparent that a given receptor can activate multiple different pathways, some being therapeutically desirable, while others are neutral or promote deleterious signaling. The activation of pathways that limit effectiveness of a primary pathway or promote unwanted signals has led to abandonment of some GPCRs as drug targets. However, it is now recognized that the conformation of the receptor in its ligand-bound state can be altered by the structure of the agonist or antagonist to achieve pathway selectivity, a property termed biased signaling. Biased ligands could dramatically expand the number of novel drugs acting at GPCRs for new indications. However, the field struggles with the complexity and uncertainty of these structure-functions relationships. In this review we define the theoretical underpinnings of the biased effect, discuss the methods for measuring bias, and the pitfalls that can lead to incorrect assignments of bias. Using the recent elucidation of a ß2-adrenergic receptor agonist that is biased in favor of Gs coupling over ß-arrestin binding, we provide an example of how large libraries of compounds that are impartial to preconceived notions of agonist binding can be utilized to discover pathway-specific agonists. In this case, an agonist that lacks tachyphylaxis for the treatment of obstructive lung diseases was uncovered, with a structure that was distinctly different from other agonists. We show how biased characteristics were ascertained analytically, and how molecular modeling and simulations provide a structural basis for a restricted signaling repertoire.
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Receptores Acoplados a Proteínas G , Transducción de Señal , Desarrollo de Medicamentos , Humanos , Ligandos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.
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Agonistas de Receptores Adrenérgicos beta 2/farmacología , Relajación Muscular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 2/química , Animales , Línea Celular , Simulación por Computador , Cricetinae , Descubrimiento de Drogas , Epinefrina/química , Epinefrina/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Estructura Molecular , Músculo Liso/efectos de los fármacos , Unión Proteica , Conformación Proteica , Sistema Respiratorio , Bibliotecas de Moléculas PequeñasRESUMEN
Rare mutations in the mitochondrial protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) are associated with Parkinson's disease (PD) and other Lewy body disorders. CHCHD2 is a bi-organellar mediator of oxidative phosphorylation, playing crucial roles in regulating electron flow in the mitochondrial electron transport chain and acting as a nuclear transcription factor for a cytochrome c oxidase subunit (COX4I2) and itself in response to hypoxic stress. CHCHD2 also regulates cell migration and differentiation, mitochondrial cristae structure, and apoptosis. In this review, we summarize the known disease-associated mutations of CHCHD2 in Asian and Caucasian populations, the physiological functions of CHCHD2, how CHCHD2 mutations contribute to α-synuclein pathology, and current animal models of CHCHD2. Further, we discuss the necessity of continued investigation into the divergent functions of CHCHD2 and CHCHD10 to determine how mutations in these similar mitochondrial proteins contribute to different neurodegenerative diseases.
RESUMEN
For most G protein-coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein-coupled receptor kinase (GRK)-mediated phosphorylation, leading to ß-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:ß-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the ß-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and ß-arrestin binding. However, ß-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.
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Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Miocitos del Músculo Liso/metabolismo , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sustitución de Aminoácidos , Bronquios/citología , Bronquios/metabolismo , Calcio/metabolismo , Difenhidramina/farmacología , Endosomas/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/química , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Mutación , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica , ARN Interferente Pequeño/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Taquifilaxis/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , beta-Arrestinas/genética , beta-Arrestinas/metabolismoRESUMEN
Accumulation of toxic protein assemblies and damaged mitochondria are key features of neurodegenerative diseases, which arise in large part from clearance defects in the Macroautophagy/autophagy-lysosome system. The autophagy cargo receptor SQSTM1/p62 plays a major role in the clearance of ubiquitinated cargo through Ser403 phosphorylation by multiple kinases. However, no phosphatase is known to physiologically dephosphorylate SQSTM1 on this activating residue. RNAi-mediated knockdown and overexpression experiments using genetically encoded fluorescent reporters and defined mutant constructs in cell lines, primary neurons, and brains show that SSH1, the canonical CFL (cofilin) phosphatase, mediates the dephosphorylation of phospho-Ser403-SQSTM1, thereby impairing SQSTM1 flux and phospho-MAPT/tau clearance. The inhibitory action of SSH1 on SQSTM1 is fully dependent on SQSTM1 Ser403 phosphorylation status and is separable from SSH1-mediated CFL activation. These findings reveal a unique action of SSH1 on SQSTM1 independent of CFL and implicate an inhibitory role of SSH1 in SQSTM1-mediated clearance of autophagic cargo, including phospho-MAPT/tau. Abbreviations: AAV: adeno-associated virus; Aß42O: amyloid ß1-42 oligomers; AD: Alzheimer disease; CA3: cornu Ammonis 3; CSNK2/CK2: casein kinase 2; FCCP: 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile; FTLD: frontotemporal lobar degeneration; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; SQSTM1/p62: sequestosome-1; PLA: proximity ligation assay; RFP: red fluorescent protein; RIPA: radioimmunoprecipitation assay; shRNA: short hairpin RNA; siRNA: small interfering RNA; Ser403: Serine403; SSH1: slingshot protein phosphatase 1; TBK1: TANK-binding kinase 1; ULK: unc-51 like kinase 1.
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Factores Despolimerizantes de la Actina , Autofagia , Factores Despolimerizantes de la Actina/metabolismo , Autofagia/genética , Lisosomas/metabolismo , Macroautofagia , Proteína Sequestosoma-1/metabolismoRESUMEN
Diabetic neuropathy is a major complication of diabetes. Current treatment options alleviate pain but do not stop the progression of the disease. At present, there are no approved disease-modifying therapies. Thus, developing more effective therapies remains a major unmet medical need. Seeking to better understand the molecular mechanisms driving peripheral neuropathy, as well as other neurological complications associated with diabetes, we performed spatiotemporal lipidomics, biochemical, ultrastructural, and physiological studies on PNS and CNS tissue from multiple diabetic preclinical models. We unraveled potentially novel molecular fingerprints underlying nerve damage in obesity-induced diabetes, including an early loss of nerve mitochondrial (cardiolipin) and myelin signature (galactosylceramide, sulfatide, and plasmalogen phosphatidylethanolamine) lipids that preceded mitochondrial, myelin, and axonal structural/functional defects; started in the PNS; and progressed to the CNS at advanced diabetic stages. Mechanistically, we provided substantial evidence indicating that these nerve mitochondrial/myelin lipid abnormalities are (surprisingly) not driven by hyperglycemia, dysinsulinemia, or insulin resistance, but rather associate with obesity/hyperlipidemia. Importantly, our findings have major clinical implications as they open the door to novel lipid-based biomarkers to diagnose and distinguish different subtypes of diabetic neuropathy (obese vs. nonobese diabetics), as well as to lipid-lowering therapeutic strategies for treatment of obesity/diabetes-associated neurological complications and for glycemic control.
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Diabetes Mellitus/patología , Neuropatías Diabéticas/patología , Hiperlipidemias/complicaciones , Lípidos/análisis , Mitocondrias/patología , Vaina de Mielina/patología , Obesidad/complicaciones , Animales , Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Neuropatías Diabéticas/etiología , Neuropatías Diabéticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Mitocondrias/metabolismo , Vaina de Mielina/metabolismoRESUMEN
Mutations in CHCHD10, a gene coding for a mitochondrial protein, are implicated in ALS-FTD spectrum disorders, which are pathologically characterized by transactive response DNA binding protein 43 kDa (TDP-43) accumulation. While both TDP-43 and CHCHD10 mutations drive mitochondrial pathogenesis, mechanisms underlying such phenotypes are unclear. Moreover, despite the disruption of the mitochondrial mitofilin protein complex at cristae junctions in patient fibroblasts bearing the CHCHD10S59L mutation, the role of CHCHD10 variants in mitofilin-associated protein complexes in brain has not been examined. Here, we utilized novel CHCHD10 transgenic mouse variants (WT, R15L, & S59L), TDP-43 transgenic mice, FTLD-TDP patient brains, and transfected cells to assess the interplay between CHCHD10 and TDP-43 on mitochondrial phenotypes. We show that CHCHD10 mutations disrupt mitochondrial OPA1-mitofilin complexes in brain, associated with impaired mitochondrial fusion and respiration. Likewise, CHCHD10 levels and OPA1-mitofilin complexes are significantly reduced in brains of FTLD-TDP patients and TDP-43 transgenic mice. In cultured cells, CHCHD10 knockdown results in OPA1-mitofilin complex disassembly, while TDP-43 overexpression also reduces CHCHD10, promotes OPA1-mitofilin complex disassembly via CHCHD10, and impairs mitochondrial fusion and respiration, phenotypes that are rescued by wild type (WT) CHCHD10. These results indicate that disruption of CHCHD10-regulated OPA1-mitofilin complex contributes to mitochondrial abnormalities in FTLD-TDP and suggest that CHCHD10 restoration could ameliorate mitochondrial dysfunction in FTLD-TDP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/metabolismo , GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Demencia Frontotemporal/genética , GTP Fosfohidrolasas/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/genética , Dinámicas Mitocondriales/genética , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Musculares/genética , Mutación/genética , Células 3T3 NIH , FenotipoRESUMEN
Multiple G protein-coupled receptors (GPCRs) are targets in the treatment of dementia, and the arrestins are common to their signaling. ß-Arrestin2 was significantly increased in brains of patients with frontotemporal lobar degeneration (FTLD-tau), a disease second to Alzheimer's as a cause of dementia. Genetic loss and overexpression experiments using genetically encoded reporters and defined mutant constructs in vitro, and in cell lines, primary neurons, and tau P301S mice crossed with ß-arrestin2-/- mice, show that ß-arrestin2 stabilizes pathogenic tau and promotes tau aggregation. Cell and mouse models of FTLD showed this to be maladaptive, fueling a positive feedback cycle of enhanced neuronal tau via non-GPCR mechanisms. Genetic ablation of ß-arrestin2 markedly ablates tau pathology and rescues synaptic plasticity defects in tau P301S transgenic mice. Atomic force microscopy and cellular studies revealed that oligomerized, but not monomeric, ß-arrestin2 increases tau by inhibiting self-interaction of the autophagy cargo receptor p62/SQSTM1, impeding p62 autophagy flux. Hence, reduction of oligomerized ß-arrestin2 with virus encoding ß-arrestin2 mutants acting as dominant-negatives markedly reduces tau-laden neurofibrillary tangles in FTLD mice in vivo. Reducing ß-arrestin2 oligomeric status represents a new strategy to alleviate tau pathology in FTLD and related tauopathies.
Asunto(s)
Demencia Frontotemporal/patología , Arrestina beta 2/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Autofagia , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Demencia Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Transcriptoma , Arrestina beta 2/genéticaRESUMEN
The defining pathological hallmarks of Alzheimer's disease (AD) are proteinopathies marked by the amyloid-ß (Aß) peptide and hyperphosphorylated tau. In addition, Hirano bodies and cofilin-actin rods are extensively found in AD brains, both of which are associated with the actin cytoskeleton. The actin-binding protein cofilin known for its actin filament severing, depolymerizing, nucleating, and bundling activities has emerged as a significant player in AD pathogenesis. In this review, we discuss the regulation of cofilin by multiple signaling events impinging on LIM kinase-1 (LIMK1) and/or Slingshot homolog-1 (SSH1) downstream of Aß. Such pathophysiological signaling pathways impact actin dynamics to regulate synaptic integrity, mitochondrial translocation of cofilin to promote neurotoxicity, and formation of cofilin-actin pathology. Other intracellular signaling proteins, such as ß-arrestin, RanBP9, Chronophin, PLD1, and 14-3-3 also impinge on the regulation of cofilin downstream of Aß. Finally, we discuss the role of activated cofilin as a bridge between actin and microtubule dynamics by displacing tau from microtubules, thereby destabilizing tau-induced microtubule assembly, missorting tau, and promoting tauopathy.