RESUMEN
Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor-related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose-response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.
Asunto(s)
Proteínas Tirosina Quinasas Receptoras , Proteínas de Unión al GTP rab , Agrina/genética , Agrina/metabolismo , Animales , Ratones , Fosforilación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Sinucleinopatías , Ratones , Animales , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Sinucleinopatías/patología , Neuronas Dopaminérgicas/metabolismoRESUMEN
beta-dystroglycan (DG) and the dystrophin-glycoprotein complex (DGC) are localized at costameres and neuromuscular junctions in the sarcolemma of skeletal muscle. We present evidence for an ankyrin-based mechanism for sarcolemmal localization of dystrophin and beta-DG. Dystrophin binds ankyrin-B and ankyrin-G, while beta-DG binds ankyrin-G. Dystrophin and beta-DG require ankyrin-G for retention at costameres but not delivery to the sarcolemma. Dystrophin and beta-DG remain intracellular in ankyrin-B-depleted muscle, where beta-DG accumulates in a juxta-TGN compartment. The neuromuscular junction requires ankyrin-B for localization of dystrophin/utrophin and beta-DG and for maintenance of its postnatal morphology. A Becker muscular dystrophy mutation reduces ankyrin binding and impairs sarcolemmal localization of dystrophin-Dp71. Ankyrin-B also binds to dynactin-4, a dynactin subunit. Dynactin-4 and a subset of microtubules disappear from sarcolemmal sites in ankyrin-B-depleted muscle. Ankyrin-B thus is an adaptor required for sarcolemmal localization of dystrophin, as well as dynactin-4.
Asunto(s)
Ancirinas/metabolismo , Costameras/metabolismo , Distroglicanos/metabolismo , Distrofina/metabolismo , Unión Neuromuscular/metabolismo , Secuencia de Aminoácidos , Animales , Ancirinas/química , Ancirinas/genética , Complejo Dinactina , Distrofina/genética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Sarcolema/metabolismo , Alineación de SecuenciaRESUMEN
Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-ß (Aß) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aß toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n = 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (SLC24A4, MS4A6A, HS3ST1) and progressive supranuclear palsy (MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration.
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Proteínas de Homeodominio/genética , Tauopatías/genética , Tauopatías/patología , Proteínas Supresoras de Tumor/genética , Anciano , Anciano de 80 o más Años , Envejecimiento/patología , Animales , Estudios de Cohortes , Drosophila , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.
Asunto(s)
Neuronas Motoras/enzimología , Atrofia Muscular Espinal/enzimología , Unión Neuromuscular/enzimología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Unión Neuromuscular/genética , Unión Neuromuscular/patología , Proteínas Tirosina Quinasas Receptoras/genética , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.
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Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/fisiopatología , Diafragma/fisiopatología , Unión Neuromuscular/fisiopatología , Proteínas Tirosina Quinasas Receptoras/agonistas , Esclerosis Amiotrófica Lateral/patología , Animales , Diafragma/patología , Modelos Animales de Enfermedad , Activación Enzimática/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Unión Neuromuscular/patología , Superóxido Dismutasa-1/genéticaRESUMEN
OBJECTIVE: Neurofibrillary tangles (NFTs), consisting of intracellular aggregates of the tau protein, are a pathological hallmark of Alzheimer's disease (AD). Here we report the identification and initial characterization of Genentech Tau Probe 1 ([18F]GTP1), a small-molecule PET probe for imaging tau pathology in AD patients. METHODS: Autoradiography using human brain tissues from AD donors and protein binding panels were used to determine [18F]GTP1 binding characteristics. Stability was evaluated in vitro and in vivo in mice and rhesus monkey. In the clinic, whole-body imaging was performed to assess biodistribution and dosimetry. Dynamic [18F]GTP1 brain imaging and input function measurement were performed on two separate days in 5 ß-amyloid plaque positive (Aß+) AD and 5 ß-amyloid plaque negative (Aß-) cognitive normal (CN) participants. Tracer kinetic modeling was applied and reproducibility was evaluated. SUVR was calculated and compared to [18F]GTP1-specific binding parameters derived from the kinetic modeling. [18F]GTP1 performance in a larger cross-sectional group of 60 Aß+ AD participants and ten (Aß- or Aß+) CN was evaluated with images acquired 60 to 90 min post tracer administration. RESULTS: [18F]GTP1 exhibited high affinity and selectivity for tau pathology with no measurable binding to ß-amyloid plaques or MAO-B in AD tissues, or binding to other tested proteins at an affinity predicted to impede image data interpretation. In human, [18F]GTP1 exhibited favorable dosimetry and brain kinetics, and no evidence of defluorination. [18F]GTP1-specific binding was observed in cortical regions of the brain predicted to contain tau pathology in AD and exhibited low (< 4%) test-retest variability. SUVR measured in the 60 to 90-min interval post injection correlated with tracer-specific binding (slope = 1.36, r2 = 0.98). Furthermore, in a cross-sectional population, the degree of [18F]GTP1-specific binding increased with AD severity and could differentiate diagnostic cohorts. CONCLUSIONS: [18F]GTP1 is a promising PET probe for the study of tau pathology in AD.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico por imagen , Radioisótopos de Flúor/farmacocinética , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Proteínas tau/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Femenino , Radioisótopos de Flúor/administración & dosificación , Humanos , Cinética , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Ovillos Neurofibrilares/metabolismo , Tomografía de Emisión de Positrones/normas , Unión Proteica , Radiofármacos/administración & dosificación , Sensibilidad y EspecificidadRESUMEN
Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with ß2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on ß2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required ß2 spectrin. Together, these results are consistent with a functional hierarchy beginning with ß2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.
Asunto(s)
Ancirinas/metabolismo , Proteínas Portadoras/metabolismo , Distrofina/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Músculo Esquelético , Animales , Ancirinas/genética , Costameras/metabolismo , Complejo Dinactina , Matriz Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Esfuerzo Físico/fisiología , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patologíaRESUMEN
Primary age-related tauopathy (PART) is a form of Alzheimer-type neurofibrillary degeneration occurring in the absence of amyloid-beta (Aß) plaques. While PART shares some features with Alzheimer disease (AD), such as progressive accumulation of neurofibrillary tangle pathology in the medial temporal lobe and other brain regions, it does not progress extensively to neocortical regions. Given this restricted pathoanatomical pattern and variable symptomatology, there is a need to reexamine and improve upon how PART is neuropathologically assessed and staged. We performed a retrospective autopsy study in a collection (n = 174) of post-mortem PART brains and used logistic regression to determine the extent to which a set of clinical and neuropathological features predict cognitive impairment. We compared Braak staging, which focuses on hierarchical neuroanatomical progression of AD tau and Aß pathology, with quantitative assessments of neurofibrillary burden using computer-derived positive pixel counts on digitized whole slide images of sections stained immunohistochemically with antibodies targeting abnormal hyperphosphorylated tau (p-tau) in the entorhinal region and hippocampus. We also assessed other factors affecting cognition, including aging-related tau astrogliopathy (ARTAG) and atrophy. We found no association between Braak stage and cognitive impairment when controlling for age (p = 0.76). In contrast, p-tau burden was significantly correlated with cognitive impairment even when adjusting for age (p = 0.03). The strongest correlate of cognitive impairment was cerebrovascular disease, a well-known risk factor (p < 0.0001), but other features including ARTAG (p = 0.03) and hippocampal atrophy (p = 0.04) were also associated. In contrast, sex, APOE, psychiatric illness, education, argyrophilic grains, and incidental Lewy bodies were not. These findings support the hypothesis that comorbid pathologies contribute to cognitive impairment in subjects with PART. Quantitative approaches beyond Braak staging are critical for advancing our understanding of the extent to which age-related tauopathy changes impact cognitive function.
Asunto(s)
Encéfalo/patología , Trastornos Cerebrovasculares/epidemiología , Disfunción Cognitiva/epidemiología , Ovillos Neurofibrilares/patología , Tauopatías/epidemiología , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Autopsia , Encéfalo/metabolismo , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Disfunción Cognitiva/psicología , Comorbilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Placa Amiloide/patología , Estudios Retrospectivos , Tauopatías/metabolismo , Tauopatías/patología , Tauopatías/psicología , Proteínas tau/genéticaRESUMEN
Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunización Pasiva , Ratones , Ratones Transgénicos , Tauopatías/tratamiento farmacológico , Proteínas tau/metabolismoRESUMEN
Primary age-related tauopathy (PART) is a neurodegenerative entity defined as Alzheimer-type neurofibrillary degeneration primarily affecting the medial temporal lobe with minimal to absent amyloid-ß (Aß) plaque deposition. The extent to which PART can be differentiated pathoanatomically from Alzheimer disease (AD) is unclear. Here, we examined the regional distribution of tau pathology in a large cohort of postmortem brains (n = 914). We found an early vulnerability of the CA2 subregion of the hippocampus to neurofibrillary degeneration in PART, and semiquantitative assessment of neurofibrillary degeneration in CA2 was significantly greater than in CA1 in PART. In contrast, subjects harboring intermediate-to-high AD neuropathologic change (ADNC) displayed relative sparing of CA2 until later stages of their disease course. In addition, the CA2/CA1 ratio of neurofibrillary degeneration in PART was significantly higher than in subjects with intermediate-to-high ADNC burden. Furthermore, the distribution of tau pathology in PART diverges from the Braak NFT staging system and Braak stage does not correlate with cognitive function in PART as it does in individuals with intermediate-to-high ADNC. These findings highlight the need for a better understanding of the contribution of PART to cognitive impairment and how neurofibrillary degeneration interacts with Aß pathology in AD and PART.
Asunto(s)
Envejecimiento/patología , Región CA2 Hipocampal/patología , Neuronas/patología , Tauopatías/patología , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Péptidos beta-Amiloides/metabolismo , Región CA2 Hipocampal/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Tauopatías/metabolismo , Proteínas tau/metabolismoRESUMEN
Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos/química , Asparagina/química , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Amidas/química , Animales , Afinidad de Anticuerpos , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Mutación/genética , Unión Proteica , Estabilidad ProteicaRESUMEN
Microglia, the CNS-resident immune cells, play important roles in disease, but the spectrum of their possible activation states is not well understood. We derived co-regulated gene modules from transcriptional profiles of CNS myeloid cells of diverse mouse models, including new tauopathy model datasets. Using these modules to interpret single-cell data from an Alzheimer's disease (AD) model, we identified microglial subsets-distinct from previously reported "disease-associated microglia"-expressing interferon-related or proliferation modules. We then analyzed whole-tissue RNA profiles from human neurodegenerative diseases, including a new AD dataset. Correcting for altered cellular composition of AD tissue, we observed elevated expression of the neurodegeneration-related modules, but also modules not implicated using expression profiles from mouse models alone. We provide a searchable, interactive database for exploring gene expression in all these datasets (http://research-pub.gene.com/BrainMyeloidLandscape). Understanding the dimensions of CNS myeloid cell activation in human disease may reveal opportunities for therapeutic intervention.
Asunto(s)
Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Microglía/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
The aggregation of intracellular tau protein is a major hallmark of Alzheimer's disease (AD). The extent and the stereotypical spread of tau pathology in the AD brain are correlated with cognitive decline during disease progression. Here we present an in-depth analysis of endogenous tau fragmentation in a well-characterized cohort of AD and age-matched control subjects. Using protein mass spectrometry and Edman degradation to interrogate endogenous tau fragments in the human brain, we identified two novel proteolytic sites, G323 and G326, as major tau cleavage events in both normal and AD cortex. These sites are located within the sequence recently identified as the structural core of tau protofilaments, suggesting an inhibitory mechanism of fibril formation. In contrast, a different set of novel cleavages showed a distinct increase in late stage AD. These disease-associated sites are located outside of the protofilament core sequence. We demonstrate that calpain 1 specifically cleaves at both the normal and diseased sites in vitro, and the site selection is conformation-dependent. Monomeric tau is predominantly cleaved at G323/G326 (normal sites), whereas oligomerization increases cleavages at the late-AD-associated sites. The fragmentation patterns specific to disease and healthy states suggest novel regulatory mechanisms of tau aggregation in the human brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Calpaína/metabolismo , Progresión de la Enfermedad , Proteínas tau/química , Proteínas tau/metabolismo , Anciano de 80 o más Años , Encéfalo/metabolismo , Femenino , Humanos , Masculino , ProteolisisRESUMEN
Common variant genome-wide association studies (GWASs) have, to date, identified >24 risk loci for Parkinson's disease (PD). To discover additional loci, we carried out a GWAS comparing 6,476 PD cases with 302,042 controls, followed by a meta-analysis with a recent study of over 13,000 PD cases and 95,000 controls at 9,830 overlapping variants. We then tested 35 loci (P < 1 × 10-6) in a replication cohort of 5,851 cases and 5,866 controls. We identified 17 novel risk loci (P < 5 × 10-8) in a joint analysis of 26,035 cases and 403,190 controls. We used a neurocentric strategy to assign candidate risk genes to the loci. We identified protein-altering or cis-expression quantitative trait locus (cis-eQTL) variants in linkage disequilibrium with the index variant in 29 of the 41 PD loci. These results indicate a key role for autophagy and lysosomal biology in PD risk, and suggest potential new drug targets for PD.
Asunto(s)
Estudio de Asociación del Genoma Completo , Enfermedad de Parkinson/genética , Antiparkinsonianos/farmacología , Autofagia/genética , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Humanos , Desequilibrio de Ligamiento , Lisosomas/fisiología , Terapia Molecular Dirigida , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/epidemiología , Riesgo , Factores de TranscripciónRESUMEN
The AMPA-type glutamate receptors mediate the majority of the fast excitatory synaptic transmission and critically contribute to synaptic plasticity in the brain, hence the existence of numerous trafficking proteins dedicated to regulation of their synaptic delivery and turnover. Stargazin (also termed gamma2) is a member of a recently identified protein family termed transmembrane AMPA receptor regulatory proteins (TARPs). TARPs physically associate with AMPA receptors and participate in their surface delivery and anchoring at the postsynaptic membrane. Here, we report that next to its trafficking roles, stargazin may also act as a positive allosteric modulator of AMPA receptor ion channel function. Coexpression of stargazin with AMPA receptor subunits, either in Xenopus oocytes or in human embryonic kidney 293 cells, significantly reduced receptor desensitization in response to glutamate. Receptor deactivation rates were also slowed, and the recovery from desensitization was accelerated. Structurally, based on the data showing a tight correlation between desensitization and the stability of the AMPA receptor intradimer interface, we propose that binding of stargazin may stabilize the receptor conformation. Functionally, our data suggest that AMPA receptors complexed with stargazin (and possibly also with other TARPs) at the postsynaptic membrane are significantly more responsive to synaptically released glutamate compared with AMPA receptors lacking stargazin/TARP interaction. The putative existence of such two states of synaptic AMPA receptors, with and without stargazin/TARP binding, may provide a novel mechanism for regulation of excitatory synaptic strength during development and/or in synaptic plasticity in the adult brain.
Asunto(s)
Canales de Calcio/fisiología , Receptores AMPA/metabolismo , Animales , Canales de Calcio/biosíntesis , Línea Celular , Relación Dosis-Respuesta a Droga , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Humanos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptores AMPA/agonistas , Xenopus laevisRESUMEN
The spread of tau pathology correlates with cognitive decline in Alzheimer's disease. In vitro, tau antibodies can block cell-to-cell tau spreading. Although mechanisms of anti-tau function in vivo are unknown, effector function might promote microglia-mediated clearance. In this study, we investigated whether antibody effector function is required for targeting tau. We compared efficacy in vivo and in vitro of two versions of the same tau antibody, with and without effector function, measuring tau pathology, neuron health, and microglial function. Both antibodies reduced accumulation of tau pathology in Tau-P301L transgenic mice and protected cultured neurons against extracellular tau-induced toxicity. Only the full-effector antibody enhanced tau uptake in cultured microglia, which promoted release of proinflammatory cytokines. In neuron-microglia co-cultures, only effectorless anti-tau protected neurons, suggesting full-effector tau antibodies can induce indirect toxicity via microglia. We conclude that effector function is not required for efficacy, and effectorless tau antibodies may represent a safer approach to targeting tau.
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Enfermedad de Alzheimer/metabolismo , Microglía/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/patología , Animales , Anticuerpos/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Técnicas de Cocultivo/métodos , Citocinas/metabolismo , Ratones Transgénicos , Neuronas/metabolismoRESUMEN
Excessive glutamate signaling is thought to underlie neurodegeneration in multiple contexts, yet the pro-degenerative signaling pathways downstream of glutamate receptor activation are not well defined. We show that dual leucine zipper kinase (DLK) is essential for excitotoxicity-induced degeneration of neurons in vivo. In mature neurons, DLK is present in the synapse and interacts with multiple known postsynaptic density proteins including the scaffolding protein PSD-95. To examine DLK function in the adult, DLK-inducible knockout mice were generated through Tamoxifen-induced activation of Cre-ERT in mice containing a floxed DLK allele, which circumvents the neonatal lethality associated with germline deletion. DLK-inducible knockouts displayed a modest increase in basal synaptic transmission but had an attenuation of the JNK/c-Jun stress response pathway activation and significantly reduced neuronal degeneration after kainic acid-induced seizures. Together, these data demonstrate that DLK is a critical upstream regulator of JNK-mediated neurodegeneration downstream of glutamate receptor hyper-activation and represents an attractive target for the treatment of indications where excitotoxicity is a primary driver of neuronal loss.
Asunto(s)
Quinasas Quinasa Quinasa PAM/fisiología , Degeneración Nerviosa/fisiopatología , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Homólogo 4 de la Proteína Discs Large , Ácido Glutámico/fisiología , Guanilato-Quinasas/fisiología , Ácido Kaínico/toxicidad , Quinasas Quinasa Quinasa PAM/deficiencia , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , N-Metilaspartato/fisiología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Proteínas del Tejido Nervioso/fisiología , Sinapsis/fisiologíaRESUMEN
The N-terminal domain (NTD) of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1-N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor delta2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.