RESUMEN
Alpha-synucleinopathies (α-synucleinopathies) such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are all characterized by aggregates of alpha-synuclein (α-syn), but display heterogeneous clinical and pathological phenotypes. The mechanism underlying this heterogeneity is thought to be due to diversity in the α-syn strains present across the diseases. α-syn obtained from the post-mortem brain of patients who lived with these conditions is heterogenous, and displays a different protease sensitivity, ultrastructure, cytotoxicity, and seeding potential. The primary aim of this review is to summarize previous studies investigating these concepts, which not only reflect the idea of different syn strains being present, but demonstrate that each property explains a small part of a much larger puzzle. Strains of α-syn appear at the center of the correlation between α-syn properties and the disease phenotype, likely influenced by external factors. There are considerable similarities in the properties of disease-specific α-syn strains, but MSA seems to consistently display more aggressive traits. Elucidating the molecular underpinnings of heterogeneity amongst α-synucleinopathies holds promise for future clinical translation, allowing for the development of personalized medicine approaches tackling the root cause of each α-synucleinopathy.
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Demencia , Atrofia de Múltiples Sistemas , Enfermedad de Parkinson , Sinucleinopatías , Humanos , alfa-Sinucleína/genética , Enfermedad de Parkinson/genética , Atrofia de Múltiples Sistemas/genéticaRESUMEN
Giant cell arteritis (GCA) usually presents with headache, scalp tenderness and raised inflammatory markers. GCA presenting with a clinically evident cranial nerve palsy is rare and may result in a delayed or missed diagnosis if not suspected. We present the rare case of a woman in her 70s with histologically confirmed GCA presenting with a unilateral sixth nerve palsy, which responded to treatment with high-dose oral prednisolone.
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Enfermedades del Nervio Abducens , Arteritis de Células Gigantes , Femenino , Humanos , Cefalea , Diagnóstico Erróneo , DolorRESUMEN
SARS-CoV-2, the causative agent of COVID-19, typically manifests as a respiratory illness, although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, as well as frequent thrombotic events, are increasingly recognised. How this maps onto SARS-CoV-2 organ tropism at the histological level, however, remains unclear. Here, we perform a comprehensive validation of a monoclonal antibody against the SARS-CoV-2 nucleocapsid protein (NP) followed by systematic multisystem organ immunohistochemistry analysis of the viral cellular tropism in tissue from 36 patients, 16 postmortem cases and 16 biopsies with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 status from the peaks of the pandemic in 2020 and four pre-COVID postmortem controls. SARS-CoV-2 anti-NP staining in the postmortem cases revealed broad multiorgan involvement of the respiratory, digestive, haematopoietic, genitourinary and nervous systems, with a typical pattern of staining characterised by punctate paranuclear and apical cytoplasmic labelling. The average time from symptom onset to time of death was shorter in positively versus negatively stained postmortem cases (mean = 10.3 days versus mean = 20.3 days, p = 0.0416, with no cases showing definitive staining if the interval exceeded 15 days). One striking finding was the widespread presence of SARS-CoV-2 NP in neurons of the myenteric plexus, a site of high ACE2 expression, the entry receptor for SARS-CoV-2, and one of the earliest affected cells in Parkinson's disease. In the bone marrow, we observed viral SARS-CoV-2 NP within megakaryocytes, key cells in platelet production and thrombus formation. In 15 tracheal biopsies performed in patients requiring ventilation, there was a near complete concordance between immunohistochemistry and PCR swab results. Going forward, our findings have relevance to correlating clinical symptoms with the organ tropism of SARS-CoV-2 in contemporary cases as well as providing insights into potential long-term complications of COVID-19. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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COVID-19 , SARS-CoV-2 , Humanos , Megacariocitos , Plexo Mientérico , NeuronasRESUMEN
BACKGROUND: Multimerization is a key process in prion-like disorders such as Alzheimer's disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau-tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers. METHODS: Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold. RESULTS: Our novel tau-proximity ligation assay (tau-PLA) directly visualized tau-tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions. CONCLUSIONS: Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Encéfalo/patología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Animales , Enfermedades Asintomáticas , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Ovillos Neurofibrilares/patología , Multimerización de Proteína , Proteínas tau/genéticaRESUMEN
We describe a human single-nuclei transcriptomic atlas for the substantia nigra (SN), generated by sequencing approximately 17,000 nuclei from matched cortical and SN samples. We show that the common genetic risk for Parkinson's disease (PD) is associated with dopaminergic neuron (DaN)-specific gene expression, including mitochondrial functioning, protein folding and ubiquitination pathways. We identify a distinct cell type association between PD risk and oligodendrocyte-specific gene expression. Unlike Alzheimer's disease (AD), we find no association between PD risk and microglia or astrocytes, suggesting that neuroinflammation plays a less causal role in PD than AD. Beyond PD, we find associations between SN DaNs and GABAergic neuron gene expression and multiple neuropsychiatric disorders. Conditional analysis reveals that distinct neuropsychiatric disorders associate with distinct sets of neuron-specific genes but converge onto shared loci within oligodendrocytes and oligodendrocyte precursors. This atlas guides our aetiological understanding by associating SN cell type expression profiles with specific disease risk.
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Expresión Génica , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Encéfalo , Neuronas Dopaminérgicas/metabolismo , Humanos , Microglía/metabolismo , Mitocondrias/metabolismo , Enfermedades del Sistema Nervioso/patología , Sustancia Negra/patología , TranscriptomaRESUMEN
Parkinson's disease (PD) is characterized by the death of dopamine neurons in the substantia nigra pars compacta (SNc) and accumulation of α-synuclein. Impaired autophagy has been implicated and activation of autophagy proposed as a treatment strategy. We generate a human α-synuclein-expressing mouse model of PD with macroautophagic failure in dopamine neurons to understand the interaction between impaired macroautophagy and α-synuclein. We find that impaired macroautophagy generates p62-positive inclusions and progressive neuron loss in the SNc. Despite this parkinsonian pathology, motor phenotypes accompanying human α-synuclein overexpression actually improve with impaired macroautophagy. Real-time fast-scan cyclic voltammetry reveals that macroautophagy impairment in dopamine neurons increases evoked extracellular concentrations of dopamine, reduces dopamine uptake, and relieves paired-stimulus depression. Our findings show that impaired macroautophagy paradoxically enhances dopamine neurotransmission, improving movement while worsening pathology, suggesting that changes to dopamine synapse function compensate for and conceal the underlying PD pathogenesis, with implications for therapies that target autophagy.
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Autofagia , Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Dopamina/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sustancia Negra/fisiopatología , Transmisión Sináptica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are neurodegenerative disorders resulting in progressive motor/cognitive deficits among other symptoms. They are characterised by stereotypical brain cell loss accompanied by the formation of proteinaceous aggregations of the protein α-synuclein (α-syn), being, therefore, termed α-synucleinopathies. Although the presence of α-syn inclusions is a common hallmark of these disorders, the exact nature of the deposited protein is specific to each disease. Different neuroanatomical regions and cellular populations manifest a differential vulnerability to the appearance of protein deposits, cell dysfunction, and cell death, leading to phenotypic diversity. The present review describes the multiple factors that contribute to the selective vulnerability in α-synucleinopathies. We explore the intrinsic cellular properties in the affected regions, including the physiological and pathophysiological roles of endogenous α-syn, the metabolic and genetic build-up of the cells and their connectivity. These factors converge with the variability of the α-syn conformational strains and their spreading capacity to dictate the phenotypic diversity and regional vulnerability of each disease. Finally, we describe the exogenous and environmental factors that potentially contribute by igniting and modulating the differential pathology in α-synucleinopathies. In conclusion, we think that it is the confluence of this disruption of the cellular metabolic state and α-syn structural equilibrium through the anatomical connectivity which appears to initiate cascades of pathological processes triggered by genetic, environmental, or stochastic events that result in the "death by a thousand cuts" profile of α-synucleinopathies.
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Encéfalo/patología , Atrofia de Múltiples Sistemas/patología , Enfermedad de Parkinson/patología , Sinucleinopatías/patología , Animales , Humanos , Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/patologíaRESUMEN
Alpha-synuclein oligomers are thought to be toxic mediators of Parkinson's disease and other alpha-synucleinopathies, but their histological detection in situ in diseased brain has been a challenge in the field for some time. Here we describe a method, the alpha-synuclein proximity ligation assay (AS-PLA), to detect alpha-synuclein oligomers in paraffin-embedded brain sections. Using AS-PLA previously unobserved alpha-synuclein oligomeric pathology is revealed.
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Encéfalo/metabolismo , Multimerización de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismoRESUMEN
Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of 'sporadic' cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80â¯kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3â¯months of age, TDP-43M337V mice are phenotypically normal but by around 6â¯months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6â¯months of age can facilitate the study of early pathways underlying ALS.
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Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/genética , Neuronas Motoras/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Fuerza de la Mano , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Mutación , Unión Neuromuscular/patología , Proteínas de Unión al ARN/metabolismo , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
BACKGROUND: Although primarily a neurodegenerative process, there is increasing awareness of peripheral disease mechanisms in Parkinson's disease. To investigate disease processes in accessible patient cells, we studied peripheral blood mononuclear cells in recently diagnosed PD patients and rapid eye movement-sleep behavior disorder patients who have a greatly increased risk of developing PD. We hypothesized that peripheral blood mononuclear cells may recapitulate cellular pathology found in the PD brain and investigated these cells for mitochondrial dysfunction and oxidative stress. METHODS: Peripheral blood mononuclear cells were isolated and studied from PD patients, rapid eye movement-sleep behavior disorder patients and age- and sex-matched control individuals from the well-characterized Oxford Discovery cohort. All participants underwent thorough clinical assessment. RESULTS: Initial characterization showed that PD patients had elevated levels of CD14 + monocytes and monocytes expressing C-C motif chemokine receptor 2. Mitochondrial dysfunction and oxidative stress were increased in PD patient peripheral blood mononuclear cells, with elevated levels of mitochondrial reactive oxygen species specifically in patient monocytes. This was combined with reduced levels of the antioxidant superoxide dismutase in blood cells from PD patients and, importantly, also in rapid eye movement-sleep behavior disorder patients. This mitochondrial dysfunction was associated with a concomitant increase in glycolysis in both PD and rapid eye movement-sleep behavior disorder patient blood cells independent of glucose uptake or monocyte activation. CONCLUSIONS: This work demonstrates functional bioenergetic deficits in PD and rapid eye movement-sleep behavior disorder patient blood cells during the early stages of human disease. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.
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Glucólisis/fisiología , Leucocitos Mononucleares/ultraestructura , Enfermedades Mitocondriales/etiología , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/complicaciones , Estudios de Casos y Controles , Citocinas/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Masculino , Mitocondrias/metabolismo , Mitocondrias/patología , Consumo de Oxígeno/fisiología , Enfermedad de Parkinson/patología , Síntomas Prodrómicos , Trastorno de la Conducta del Sueño REM/sangre , Trastorno de la Conducta del Sueño REM/complicaciones , Trastorno de la Conducta del Sueño REM/patología , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores CCR2/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Alpha-synuclein is a protein implicated in Parkinson's disease and thought to be one of the main pathological drivers in the disease, although it remains unclear how this protein elicits its neurotoxic effects. Recent findings indicate that the assembly of toxic oligomeric species of alpha-synuclein may be one of the key processes for the pathology and spread of the disease. The absence of a sensitive in situ detection method has hindered the study of these oligomeric species and the role they play in the human brain until recently. In this review, we assess the evidence for the toxicity and prion-like activity of oligomeric forms of alpha-synuclein and discuss the advances in our understanding of the role of alpha-synuclein in Parkinson's disease that may be brought about by the specific and sensitive detection of distinct oligomeric species in post-mortem patient brain. Finally, we discuss current approaches being taken to therapeutically target alpha-synuclein oligomers and their implications.
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Antiparkinsonianos/uso terapéutico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/terapia , alfa-Sinucleína/metabolismo , Animales , Antiparkinsonianos/farmacología , Biomarcadores/metabolismo , Humanos , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/terapia , alfa-Sinucleína/toxicidadRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.
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Envejecimiento/metabolismo , Antiparkinsonianos/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Levodopa/farmacología , Mutación , Enfermedad de Parkinson/genética , Potenciales de Acción , Envejecimiento/patología , Sustitución de Aminoácidos , Animales , Muerte Celular/genética , Cromosomas Artificiales Bacterianos/química , Cromosomas Artificiales Bacterianos/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Masculino , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Regiones Promotoras Genéticas , Dominios Proteicos , Ratas , Ratas Transgénicas , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
Oligomeric forms of alpha-synuclein are emerging as key mediators of pathogenesis in Parkinson's disease. Our understanding of the exact contribution of alpha-synuclein oligomers to disease is limited by the lack of a technique for their specific detection. We describe a novel method, the alpha-synuclein proximity ligation assay, which specifically recognizes alpha-synuclein oligomers. In a blinded study with post-mortem brain tissue from patients with Parkinson's disease (n = 8, age range 73-92 years, four males and four females) and age- and sex-matched controls (n = 8), we show that the alpha-synuclein proximity ligation assay reveals previously unrecognized pathology in the form of extensive diffuse deposition of alpha-synuclein oligomers. These oligomers are often localized, in the absence of Lewy bodies, to neuroanatomical regions mildly affected in Parkinson's disease. Diffuse alpha-synuclein proximity ligation assay signal is significantly more abundant in patients compared to controls in regions including the cingulate cortex (1.6-fold increase) and the reticular formation of the medulla (6.5-fold increase). In addition, the alpha-synuclein proximity ligation assay labels very early perikaryal aggregates in morphologically intact neurons that may precede the development of classical Parkinson's disease lesions, such as pale bodies or Lewy bodies. Furthermore, the alpha-synuclein proximity ligation assay preferentially detects early-stage, loosely compacted lesions such as pale bodies in patient tissue, whereas Lewy bodies, considered heavily compacted late lesions are only very exceptionally stained. The alpha-synuclein proximity ligation assay preferentially labels alpha-synuclein oligomers produced in vitro compared to monomers and fibrils, while stained oligomers in human brain display a distinct intermediate proteinase K resistance, suggesting the detection of a conformer that is different from both physiological, presynaptic alpha-synuclein (proteinase K-sensitive) and highly aggregated alpha-synuclein within Lewy bodies (proteinase K-resistant). These disease-associated conformers represent previously undetected Parkinson's disease pathology uncovered by the alpha-synuclein proximity ligation assay.
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Giro del Cíngulo/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Formación Reticular/metabolismo , alfa-Sinucleína/metabolismo , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Diagnóstico Precoz , Femenino , Técnica del Anticuerpo Fluorescente , Giro del Cíngulo/patología , Células HEK293 , Humanos , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Masculino , Neuronas/metabolismo , Enfermedad de Parkinson/diagnóstico , Polimerizacion , Formación Reticular/patología , alfa-Sinucleína/químicaRESUMEN
Atypical meningioma is a rare cause of perineural tumour spread. In this report, we present the case of a 46-year-old female with an atypical meningioma of the skull base demonstrating perineural tumour spread. We describe the imaging features of this condition and its distinguishing features from other tumours exhibiting perineural spread.
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Neoplasias Meníngeas/diagnóstico por imagen , Neoplasias Meníngeas/patología , Meningioma/diagnóstico por imagen , Meningioma/patología , Invasividad Neoplásica/diagnóstico por imagen , Invasividad Neoplásica/patología , Neoplasias de la Base del Cráneo/diagnóstico por imagen , Neoplasias de la Base del Cráneo/patología , Diagnóstico Diferencial , Femenino , Humanos , Biopsia Guiada por Imagen , Imagen por Resonancia Magnética , Neoplasias Meníngeas/terapia , Meningioma/terapia , Persona de Mediana Edad , Neoplasias de la Base del Cráneo/terapia , Tomografía Computarizada por Rayos XRESUMEN
Friedreich's ataxia (FRDA) is caused by large GAA expansions in intron 1 of the frataxin gene (FXN), which lead to reduced FXN expression through a mechanism not fully understood. Understanding such mechanism is essential for the identification of novel therapies for FRDA and this can be accelerated by the development of cell models which recapitulate the genomic context of the FXN locus and allow direct comparison of normal and expanded FXN loci with rapid detection of frataxin levels. Here we describe the development of the first GAA-expanded FXN genomic DNA reporter model of FRDA. We modified BAC vectors carrying the whole FXN genomic DNA locus by inserting the luciferase gene in exon 5a of the FXN gene (pBAC-FXN-Luc) and replacing the six GAA repeats present in the vector with an â¼310 GAA repeat expansion (pBAC-FXN-GAA-Luc). We generated human clonal cell lines carrying the two vectors using site-specific integration to allow direct comparison of normal and expanded FXN loci. We demonstrate that the presence of expanded GAA repeats recapitulates the epigenetic modifications and repression of gene expression seen in FRDA. We applied the GAA-expanded reporter model to the screening of a library of novel small molecules and identified one molecule which up-regulates FXN expression in FRDA patient primary cells and restores normal histone acetylation around the GAA repeats. These results suggest the potential use of genomic reporter cell models for the study of FRDA and the identification of novel therapies, combining physiologically relevant expression with the advantages of quantitative reporter gene expression.
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Ataxia de Friedreich/genética , Terapia Genética , Proteínas de Unión a Hierro/genética , Expansión de Repetición de Trinucleótido/genética , Línea Celular , Ataxia de Friedreich/patología , Ataxia de Friedreich/terapia , Expresión Génica , Genes Reporteros , Genómica , Heterocromatina/genética , Humanos , Intrones , FrataxinaRESUMEN
Amyotrophic lateral sclerosis and frontotemporal dementia form two poles of a genetically, pathologically and clinically-related disease continuum. Analysis of the genes and proteins at the heart of this continuum highlights dysfunction of RNA processing and aggrephagy as crucial disease-associated pathways. TAR DNA binding protein and fused in sarcoma (FUS) are both RNA processing proteins whose dysfunction impacts on global cellular RNA regulation. The recent discovery that expression of repeat expansions in the C9orf72 gene may induce RNA foci that could sequester RNA binding proteins such as TAR DNA binding protein and FUS highlights a further possibly important mechanism of RNA dysfunction in disease. Furthermore, sequestration of key RNA binding proteins may also play an important role in sporadic disease due to the association of TAR DNA binding protein and FUS with stress granules. In a further functional convergence, ubiquilin 2, p62, valosin-containing protein and optineurin are all linked to aggrephagy, a cargo-specific subtype of autophagy important for degrading ubiquitinated target proteins through the lysosome. Notably these two key pathways interact; TAR DNA binding protein and FUS bind and regulate key aggrephagy-related genes whereas dysfunction of aggrephagy leads to cytoplasmic relocalization and aggregation of TAR DNA binding protein. The convergence of amyotrophic lateral sclerosis and frontotemporal dementia linked genes into these two pathways highlights RNA dysfunction and aggrephagy as promising areas for drug discovery. In this review we discuss the importance of each of these pathways and suggest mechanisms by which they may cause both sporadic and familial disease.
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Esclerosis Amiotrófica Lateral/genética , Autofagia/genética , Demencia Frontotemporal/genética , Procesamiento Postranscripcional del ARN/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/fisiopatología , Humanos , Transducción de Señal/genéticaRESUMEN
Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene on chromosome 12 cause autosomal dominant PD (Parkinson's disease), which is indistinguishable from sporadic forms of the disease. Numerous attempts have therefore been made to model PD in rodents via the transgenic expression of LRRK2 and its mutant variants and to elucidate the function of LRRK2 by knocking out rodent Lrrk2. Although these models often only partially recapitulate PD pathology, they have helped to elucidate both the normal and pathological function of LRRK2. In particular, LRRK2 has been suggested to play roles in cytoskeletal dynamics, synaptic machinery, dopamine homoeostasis and autophagic processes. Our understanding of how these pathways are affected, their contribution towards PD development and their interaction with one another is still incomplete, however. The present review summarizes the findings from LRRK2 rodent models and draws potential connections between the apparently disparate cellular processes altered, in order to better understand the underlying mechanisms of LRRK2 dysfunction and illuminate future therapeutic interventions.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Eliminación de Gen , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Noqueados , Ratones Transgénicos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Asunto(s)
Autofagia , Bioensayo/métodos , Animales , Autofagia/genética , Humanos , Modelos BiológicosRESUMEN
Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.
Asunto(s)
Autofagia/inmunología , Bioensayo/métodos , Senescencia Celular/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Adulto , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Células Cultivadas , Células HEK293 , Histonas/metabolismo , Humanos , Ratones , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Neurons are quiescent cells that survive for several decades, many times the turnover time of most organelles and proteins, and so with advancing age neurons become affected by degenerative diseases. Autophagy is thought to be an important cellular mechanism preventing cell degeneration in such long-lived cells. We have recently found that the Parkinson disease (PD) gene leucine rich repeat kinase 2 (LRRK2) is directly involved in this process by acting as a negative regulator of autophagic activity. We created a novel genomic DNA reporter cellular model using a new recombineering strategy called Sequential insertion of Target with ovErlapping Primers (STEP) to express a genomic DNA locus YPet-LRRK2 fusion protein. Expression of the R1441C mutant form of LRRK2 induces a cellular phenotype of impaired autophagic balance at the convergent crossroads of the endocytic and autophagic avenues. Conversely, RNAi-induced knockdown of LRRK2 increases autophagic activity. Taken together, these data demonstrate the key role of LRRK2 in regulating autophagy and suggest modulation of LRRK2 function may represent a promising therapeutic target to help restore autophagic equilibrium in neurodegenerative diseases.