RESUMEN
Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.
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Técnicas de Cultivo de Célula/métodos , Glioblastoma/metabolismo , Organoides/crecimiento & desarrollo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bancos de Muestras Biológicas , Femenino , Glioblastoma/genética , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Organoides/metabolismo , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
α-synuclein (αS) is an abundant, neuronal protein that assembles into fibrillar pathological inclusions in a spectrum of neurodegenerative diseases that include Lewy body diseases (LBD) and Multiple System Atrophy (MSA). The cellular and regional distributions of pathological inclusions vary widely between different synucleinopathies contributing to the spectrum of clinical presentations. Extensive cleavage within the carboxy (C)-terminal region of αS is associated with inclusion formation, although the events leading to these modifications and the implications for pathobiology are of ongoing study. αS preformed fibrils can induce prion-like spread of αS pathology in both in vitro and animal models of disease. Using C truncation-specific antibodies, we demonstrated here that prion-like cellular uptake and processing of αS preformed fibrils resulted in two major cleavages at residues 103 and 114. A third cleavage product (122 αS) accumulated upon application of lysosomal protease inhibitors. In vitro, both 1-103 and 1-114 αS polymerized rapidly and extensively in isolation and in the presence of full-length αS. 1-103 αS also demonstrated more extensive aggregation when expressed in cultured cells. Furthermore, we used novel antibodies to αS cleaved at residue Glu114, to assess x-114 αS pathology in postmortem brain tissue from patients with LBD and MSA, as well as three different transgenic αS mouse models of prion-like induction. The distribution of x-114 αS pathology was distinct from that of overall αS pathology. These studies reveal the cellular formation and behavior of αS C-truncated at residues 114 and 103 as well as the disease dependent distribution of x-114 αS pathology.
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Enfermedad por Cuerpos de Lewy , Atrofia de Múltiples Sistemas , alfa-Sinucleína , Animales , Ratones , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Ratones Transgénicos , Atrofia de Múltiples Sistemas/metabolismo , Atrofia de Múltiples Sistemas/patología , Priones/química , Priones/metabolismo , Humanos , Lisosomas/enzimología , Inhibidores de Proteasas , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Autopsia , Ácido Glutámico/metabolismoRESUMEN
Growing evidence has implicated systemic infection as a significant risk factor for the development and advancement of Alzheimer's disease (AD). With the emergence of SARS-CoV-2 (COVID-19) and the resultant pandemic, many individuals from the same aging population vulnerable to AD suffered a severe systemic infection with potentially unidentified long-term consequences for survivors. To study the impact of COVID-19 survival on the brain's intrinsic immune system in a population also suffering from AD, we profiled post-mortem brain tissue from patients in the UF Neuromedicine Human Brain and Tissue Bank with a diagnosis of AD who survived a COVID-19 infection (COVID-AD) and contrasted our findings with AD patients who did not experience a COVID-19 infection, including a group of brain donors who passed away before arrival of SARS-CoV-2 in the United States. We assessed disease-relevant protein pathology and microglial and astrocytic markers by quantitative immunohistochemistry and supplemented these data with whole tissue gene expression analysis performed on the NanoString nCounter® platform. COVID-AD patients showed slightly elevated Aß burden in the entorhinal, fusiform, and inferior temporal cortices compared to non-COVID-AD patients, while tau pathology burden did not differ between groups. Analysis of microglia revealed a significant loss of microglial homeostasis as well as exacerbated microgliosis in COVID-AD patients compared to non-COVID-AD patients in a brain region-dependent manner. Furthermore, COVID-AD patients showed reduced cortical astrocyte numbers, independent of functional subtype. Transcriptomic analysis supported these histological findings and, in addition, identified a dysregulation of oligodendrocyte and myelination pathways in the hippocampus of COVID-AD patients. In summary, our data demonstrate a profound impact of COVID-19 infection on neuroimmune and glial pathways in AD patients persisting for months post-infection, highlighting the importance of peripheral to central neuroimmune crosstalk in neurodegenerative diseases.
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Enfermedad de Alzheimer , COVID-19 , Homeostasis , Humanos , COVID-19/inmunología , COVID-19/complicaciones , COVID-19/patología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Masculino , Femenino , Anciano , Anciano de 80 o más Años , Homeostasis/fisiología , Encéfalo/patología , Encéfalo/inmunología , Encéfalo/metabolismo , Neuroinmunomodulación/fisiología , Microglía/inmunología , Microglía/metabolismo , Microglía/patología , Persona de Mediana Edad , SARS-CoV-2 , Astrocitos/metabolismo , Astrocitos/inmunología , Astrocitos/patologíaRESUMEN
Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.
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Distrofia Miotónica , Humanos , Femenino , Ratones , Animales , Distrofia Miotónica/genética , Plexo Coroideo/metabolismo , Plexo Coroideo/patología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , ARN/genética , Ratones Noqueados , Expansión de Repetición de TrinucleótidoRESUMEN
Tau proteins within the adult central nervous system (CNS) are found to be abnormally aggregated into heterogeneous filaments in neurodegenerative diseases, termed tauopathies. These tau inclusions are pathological hallmarks of Alzheimer's disease (AD), Pick's disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). The neuropathological hallmarks of these diseases burden several cell types within the CNS, and have also been shown to be abundantly phosphorylated. The mechanism(s) by which tau aggregates in the CNS is not fully known, but it is hypothesized that hyperphosphorylated tau may precede and further promote filament formation, leading to the production of these pathological inclusions. In the studies herein, we generated and thoroughly characterized two novel conformation-dependent tau monoclonal antibodies that bind to residues Pro218-Glu222, but are sensitive to denaturing conditions and highly modulated by adjacent downstream phosphorylation sites. These epitopes are present in the neuropathological hallmarks of several tauopathies, including AD, PiD, CBD, and PSP. These novel antibodies will further enable investigation of tau-dependent pathological inclusion formation and enhance our understanding of the phosphorylation signatures within tauopathies with the possibility of new biomarker developments.
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Enfermedad de Alzheimer , Enfermedad de Pick , Tauopatías , Adulto , Humanos , Fosforilación , Anticuerpos Monoclonales , Sistema Nervioso CentralRESUMEN
AIMS: To illuminate the pathological synergy between Aß and tau leading to emergence of neurofibrillary tangles (NFT) in Alzheimer's disease (AD), here, we have performed a comparative neuropathological study utilising three distinctive variants of human tau (WT tau, P301L mutant tau and S320F mutant tau). Previously, in non-transgenic mice, we showed that WT tau or P301L tau does not form NFT while S320F tau can spontaneously aggregate into NFT, allowing us to test the selective vulnerability of these different tau conformations to the presence of Aß plaques. METHODS: We injected recombinant AAV-tau constructs into neonatal APP transgenic TgCRND8 mice or into 3-month-old TgCRND8 mice; both cohorts were aged 3 months post injection. This allowed us to test how different tau variants synergise with soluble forms of Aß (pre-deposit cohort) or with frank Aß deposits (post-deposit cohort). RESULTS: Expression of WT tau did not produce NFT or altered Aß in either cohort. In the pre-deposit cohort, S320F tau induced Aß plaque deposition, neuroinflammation and synaptic abnormalities, suggesting that early tau tangles affect the amyloid cascade. In the post-deposit cohort, contemporaneous expression of S320F tau did not exacerbate amyloid pathology, showing a dichotomy in Aß-tau synergy based on the nature of Aß. P301L tau produced NFT-type inclusions in the post-deposit cohort, but not in the pre-deposit cohort, indicating pathological synergy with pre-existing Aß deposits. CONCLUSIONS: Our data show that different tau mutations representing specific folding variants of tau synergise with Aß to different extents, depending on the presence of cerebral deposits.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Ovillos Neurofibrilares/patología , Placa Amiloide/patología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Ratones , Ratones Transgénicos , Ovillos Neurofibrilares/metabolismo , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/metabolismoRESUMEN
α-synuclein (αSyn) is an intrinsically disordered protein which can undergo structural transformations, resulting in the formation of stable, insoluble fibrils. αSyn amyloid-type nucleation can be induced by misfolded 'seeds' serving as a conformational template, tantamount to the prion-like mechanism. Accumulation of αSyn inclusions is a key feature of dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), and are found as additional pathology in Alzheimer's disease (AD) such as AD with amygdala predominant Lewy bodies (AD/ALB). While these disorders accumulate the same pathological protein, they exhibit heterogeneity in clinical and histological features; however, the mechanism(s) underlying this variability remains elusive. Accruing data from human autopsy studies, animal inoculation modeling, and in vitro characterization experiments, have lent credence to the hypothesis that conformational polymorphism of the αSyn amyloid-type fibril structure results in distinct "strains" with categorical infectivity traits. Herein, we directly compare the seeding abilities and outcome of human brain lysates from these diseases, as well as recombinant preformed human αSyn fibrils by the intracerebral inoculation of transgenic mice overexpressing either human wild-type αSyn or human αSyn with the familial A53T mutation. Our study has revealed that the initiating inoculum heavily dictates the phenotypic and pathological course of disease. Interestingly, we have also established relevant host-dependent distinctions between propagation profiles, including burden and spread of inclusion pathology throughout the neuroaxis, as well as severity of neurological symptoms. These findings provide compelling evidence supporting the hypothesis that diverse prion-type conformers may explain the variability seen in synucleinopathies.
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Enfermedad de Alzheimer , Atrofia de Múltiples Sistemas , Priones , Sinucleinopatías , Enfermedad de Alzheimer/patología , Amiloide , Animales , Humanos , Ratones , Ratones Transgénicos , Atrofia de Múltiples Sistemas/patología , Priones/genética , Priones/metabolismo , Sinucleinopatías/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Alzheimer's disease (AD) has multiple clinically and pathologically defined subtypes where the underlying causes of such heterogeneity are not well established. Rare TREM2 variants confer significantly increased risk for clinical AD in addition to other neurodegenerative disease clinical phenotypes. Whether TREM2 variants are associated with atypical clinical or pathologically defined subtypes of AD is not known. We studied here the clinical and pathological features associated with TREM2 risk variants in an autopsy-confirmed cohort. TREM2 variant cases were more frequently associated with non-amnestic clinical syndromes. Pathologically, TREM2 variant cases were associated with an atypical distribution of neurofibrillary tangle density with significantly lower hippocampal NFT burden relative to neocortical NFT accumulation. In addition, NFT density but not amyloid burden was associated with an increase of dystrophic microglia. TREM2 variant cases were not associated with an increased prevalence, extent, or severity of co-pathologies. These clinicopathological features suggest that TREM2 variants contribute to clinical and pathologic AD heterogeneity by altering the distribution of neurofibrillary degeneration and tau-dependent microglial dystrophy, resulting in hippocampal-sparing and non-amnestic AD phenotypes.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedades Neurodegenerativas/patología , Ovillos Neurofibrilares/patología , Hipocampo/patología , Microglía/patología , Glicoproteínas de Membrana/genética , Receptores Inmunológicos/genéticaRESUMEN
Pathological aggregation of amyloid-ß (Aß) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender-specific pathologies in the APP23 AD-like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender-specific effect of lack of IL12p40, the shared subunit of interleukin (IL)-12 and IL-23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aß plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aß1-40 without changes in Aß plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL-12/IL-23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD.
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Enfermedad de Alzheimer , Interleucina-12/deficiencia , Subunidad p19 de la Interleucina-23/deficiencia , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Interleucina-12/genética , Subunidad p40 de la Interleucina-12/deficiencia , Subunidad p40 de la Interleucina-12/genética , Subunidad p19 de la Interleucina-23/genética , Masculino , Ratones , Ratones Transgénicos , Placa AmiloideRESUMEN
Identification of multiple immune-related genetic risk factors for sporadic AD (sAD) have put the immune system center stage in mechanisms underlying this disorder. Comprehensive analysis of microglia in different stages of AD in human brains revealed microglia activation to follow the progression of AD neuropathological changes and requiring the co-occurrence of beta-Amyloid (Aß) and tau pathology. Carriers of AD-associated risk variants in TREM2 (Triggering receptor expressed on myeloid cells 2) showed a reduction of plaque-associated microglia and a substantial increase in dystrophic neurites and overall pathological tau compared with age and disease stage matched AD patients without TREM2 risk variants. These findings were substantiated by digital spatial profiling of the plaque microenvironment and targeted gene expression profiling on the NanoString nCounter system, which revealed striking brain region dependent differences in immune response patterns within individual cases. The demonstration of profound brain region and risk-variant specific differences in immune activation in human AD brains impacts the applicability of immune-therapeutic approaches for sAD and related neurodegenerative diseases.
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Enfermedad de Alzheimer/genética , Encéfalo/patología , Glicoproteínas de Membrana/genética , Microglía/patología , Placa Amiloide/patología , Receptores Inmunológicos/genética , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/inmunología , Progresión de la Enfermedad , Humanos , Masculino , Microglía/inmunología , Neuritas/inmunología , Neuritas/patología , Placa Amiloide/inmunología , Proteínas tau/metabolismoRESUMEN
Diets high in fat (HFD) are known to cause an immune response in the periphery as well as the central nervous system. In peripheral adipose tissue, this immune response is primarily mediated by macrophages that are recruited to the tissue. Similarly, reactivity of microglia, the innate immune cells of the brain, has been shown to occur in the hypothalamus of mice fed a high-fat diet. To characterize the nature of the microglial response to diets high in fat in a temporal fashion, we studied the phenotypic spectrum of hypothalamic microglia of mice fed high-fat diet for 3 days and 8 weeks by assessing their tissue reaction and inflammatory signature. While we observed a significant increase in Iba1+ myeloid cells and a reaction of GFAP+ astrocytes in the hypothalamus after 8 weeks of HFD feeding, we found the hypothalamic myeloid cell reaction to be limited to endogenous microglia and not mediated by infiltrating myeloid cells. Moreover, obese humans were found to present with signs of hypothalamic gliosis and exacerbated microglia dystrophy, suggesting a targeted microglia response to diet in humans as well. Notably, the glial reaction occurring in the mouse hypothalamus was not accompanied by an increase in pro-inflammatory cytokines, but rather by an anti-inflammatory reaction. Gene expression analyses of isolated microglia not only confirmed this observation, but also revealed a downregulation of microglia genes important for sensing signals in the microenvironment. Finally, we demonstrate that long-term exposure of microglia to HFD in vivo does not impair the cell's ability to respond to additional stimuli, like lipopolysaccharide. Taken together, our findings support the notion that microglia react to diets high in fat in a region-specific manner in rodents as well as in humans; however, this response changes over time as it is not exclusively pro-inflammatory nor does exposure to HFD prime microglia in the hypothalamus.
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Astrocitos/metabolismo , Sistema Nervioso Central/metabolismo , Dieta Alta en Grasa , Microglía/metabolismo , Obesidad/complicaciones , Alimentación Animal , Animales , Gliosis/metabolismo , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
Although soluble species of the amyloid-ß peptide Aß42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-ß (Aß). Here, we show that Aß peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aß42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aß in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aß in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aß42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aß42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aß42 wild-type peptide. Shorter peptides (Aß38 or Aß40) and other longer peptides (nontoxic Aß42 G33A substitution or Aß43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aß42 impacts gene regulation, and deleterious effects of Aß42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.
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Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Transporte Activo de Núcleo Celular , Enfermedad de Alzheimer/metabolismo , Sustitución de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/deficiencia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Neuronas/metabolismo , Neuronas/ultraestructura , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Presenilina-1/deficiencia , Presenilina-1/genética , Presenilina-1/metabolismo , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad EstáticaRESUMEN
In bacterial meningitis, excessive immune responses carry significant potential for damage to brain tissue even after successful antibiotic therapy. Bacterial meningitis is regarded primarily as the domain of innate immunity, and the role of lymphocytes remains unclear. We studied the contribution of lymphocytes to acute inflammation and neurodegeneration in experimental Toll-like receptor 2-driven meningitis, comparing wild-type mice with RAG-1-deficient mice that have no mature T and B lymphocytes. At 24 h after intrathecal challenge with the synthetic bacterial lipopeptide Pam(3)CysSK(4), RAG-1-deficient mice displayed more pronounced clinical impairment and an increased concentration of neutrophils, reduced expression of interleukin-10 (IL-10) mRNA, and increased expression of CXCL1 mRNA in the cerebrospinal fluid. Conversely, neuronal loss in the dentate gyrus was reduced in RAG-1-deficient mice, and expression of IL-10, transforming growth factor ß and CCL2 mRNA by microglia was increased compared to wild-type mice. Adoptive transfer of wild-type lymphocytes reversed the enhanced meningeal inflammation and functional impairment observed in RAG-1-deficient mice. Our findings suggest compartment-specific effects of lymphocytes during acute bacterial meningitis, including attenuation of meningeal inflammation and shifting of microglial activation toward a more neurotoxic phenotype.
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Inmunidad Innata , Linfocitos/inmunología , Meningitis Bacterianas/inmunología , Meningitis Bacterianas/patología , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Tauopathies are neurodegenerative disorders in which the pathological intracellular aggregation of the protein tau causes cognitive deficits. Additionally, clinical studies report muscle weakness in populations with tauopathy. However, whether neuronal pathological tau species confer muscle weakness, and whether skeletal muscle maintains contractile capacity in primary tauopathy remains unknown. Here, we identified skeletal muscle abnormalities in a mouse model of primary tauopathy, expressing human mutant P301L-tau using adeno-associated virus serotype 8 (AAV8). AAV8-P301L mice showed grip strength deficits, hyperactivity, and abnormal histological features of skeletal muscle. Additionally, spatially resolved gene expression of muscle cross sections were altered in AAV8-P301L myofibers. Transcriptional changes showed alterations of genes encoding sarcomeric proteins, proposing a weakness phenotype. Strikingly, specific force of the soleus muscle was blunted in AAV8-P301L tau male mice. Our findings suggest tauopathy has peripheral consequences in skeletal muscle that contribute to weakness in tauopathy.
RESUMEN
Acetylation of key Lysine residues characterizes aggregates of the microtubule-associated protein tau constituting the neuropathological hallmark of many neurodegenerative diseases, such as Alzheimer's disease (AD) and Progressive Supranuclear Palsy (PSP). This has led to the idea that acetylation influences tau aggregation. Using a HEK293 cell-based aggregation assay, we tested whether acetylation-mimicking substitutions (KâQ) on five AD-associated acetyl-modified sites (AcK-311, 353, 369, 370, 375) influenced its propensity to aggregate when exposed to tau seeds derived from two clinically distinctive diseases - AD and PSP. In combination, the presence of 5KâQ sites ablated tau aggregation induced by seeds from both AD and PSP patients, indicating that acetylation within the filament core domain of tau could have an inhibitory effect on seed-mediated aggregation. We had previously identified that a phosphorylation-mimetic on Ser305 (SâE) abrogated tau aggregation by seeds from AD patients, without affecting seeding by PSP patients. Combining the S305âE to the 5KâQ acetyl-modified sites, we found that this tau could now be seeded only by PSP patients, but not by AD patients, confirming Ser305 as a critical determinant of strain-specific tau seeding. On the other hand, acetylation-nullifying substitutions (KâR or KâA) on these same Lys sites did not alter tau seeding abilities compared to the parental tau construct. Notably, the combined acetylation-nullifying Alanine substitutions on these 5 Lys sites resulted in spontaneous self-aggregation, with the filaments resembling amorphous deposits. All together, we demonstrate that cooperative acetyl-occupancy in the tau filament core influences seeded propagation of misfolded tau as well as drives self-aggregation.
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Amyloid-ß (Aß) deposition throughout the neuroaxis is a classical hallmark of several neurodegenerative diseases, most notably Alzheimer's disease (AD). Aß peptides of varied length and diverse structural conformations are deposited within the parenchyma and vasculature in the brains of individuals with AD. Neuropathologically, Aß pathology can be assessed using antibodies to label and characterize their features, which in turn leads to a more extensive understanding of the pathological process. In the present study, we generated a novel monoclonal antibody, which we found to be specific for the N-terminal region of Aß. This antibody reacted to amyloid precursor protein expressed in cultured cells and labels Aß plaques and cerebral amyloid angiopathy in brain tissue from a mouse model of amyloidosis as well as post-mortem brain tissue from patients diagnosed with AD. This highly specific novel antibody will serve as a unique tool for future studies investigating Aß deposition in novel mouse models and cross-sectional studies using post-mortem human tissue.
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The disease-specific accumulation of pathological proteins has long been the major focus of research in neurodegenerative diseases (ND), including Alzheimer's disease (AD) and related dementias (RD), but the recent identification of a multitude of genetic risk factors for ND in immune-associated genes highlights the importance of immune processes in disease pathogenesis and progression. Studies in animal models have characterized the local immune response to disease-specific proteins in AD and ADRD, but due to the complexity of disease processes and the co-existence of multiple protein pathologies in human donor brains, the precise role of immune processes in ND is far from understood. To better characterize the interplay between different extracellular and intracellular protein pathologies and the brain's intrinsic immune system in ND, we set out to comprehensively profile the local immune response in postmortem brain samples of individuals with "pure" beta-Amyloid and tau pathology (AD), "pure" α-Synuclein pathology in Lewy body diseases (LBD), as well as cases with Alzheimer's disease neuropathological changes (ADNC) and Lewy body pathology (MIX). Combining immunohistochemical profiling of microglia and digital image analysis, along with deep immunophenotyping using gene expression profiling on the NanoString nCounter® platform and digital spatial profiling on the NanoString GeoMx® platform we identified a robust immune activation signature in AD brain samples. This signature is maintained in persons with mixed pathologies, irrespective of co-existence of AD pathology and Lewy body (LB) pathology, while LBD brain samples with "pure" LB pathology exhibit an attenuated and distinct immune signature. Our studies highlight disease- and brain region-specific immune response profiles to intracellular and extracellular protein pathologies and further underscore the complexity of neuroimmune interactions in ND.
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Enfermedad de Alzheimer , Enfermedad por Cuerpos de Lewy , Enfermedades Neurodegenerativas , Animales , Humanos , Enfermedad de Alzheimer/patología , Enfermedades Neurodegenerativas/patología , Proteínas tau/metabolismo , alfa-Sinucleína/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/patologíaRESUMEN
Background: White matter loss is a well-documented phenomenon in Alzheimer's disease (AD) patients that has been recognized for decades. However, the underlying reasons for the failure of oligodendrocyte progenitor cells (OPCs) to repair myelin deficits in these patients remain elusive. A single nucleotide polymorphism (SNP) in Clusterin has been identified as a risk factor for late-onset Alzheimer's disease and linked to a decrease in white matter integrity in healthy adults, but its specific role in oligodendrocyte function and myelin maintenance in Alzheimer's disease pathology remains unclear. Methods: To investigate the impact of Clusterin on OPCs in the context of Alzheimer's disease, we employed a combination of immunofluorescence and transmission electron microscopy techniques, primary culture of OPCs, and an animal model of Alzheimer's disease. Results: Our findings demonstrate that Clusterin, a risk factor for late-onset AD, is produced by OPCs and inhibits their differentiation into oligodendrocytes. Specifically, we observed upregulation of Clusterin in OPCs in the 5xFAD mouse model of AD. We also found that the phagocytosis of debris, including amyloid beta (Aß), myelin, and apoptotic cells leads to the upregulation of Clusterin in OPCs. In vivo experiments confirmed that Aß oligomers stimulate Clusterin upregulation and that OPCs are capable of phagocytosing Aß. Furthermore, we discovered that Clusterin significantly inhibits OPC differentiation and hinders the production of myelin proteins. Finally, we demonstrate that Clusterin inhibits OPC differentiation by reducing the production of IL-9 by OPCs. Conclusion: Our data suggest that Clusterin may play a key role in the impaired myelin repair observed in AD and could serve as a promising therapeutic target for addressing AD-associated cognitive decline.
RESUMEN
Alzheimer's disease (AD) is a complex neurodegenerative disorder that develops over decades. AD brain proteomics reveals vast alterations in protein levels and numerous altered biologic pathways. Here, we compare AD brain proteome and network changes with the brain proteomes of amyloid ß (Aß)-depositing mice to identify conserved and divergent protein networks with the conserved networks identifying an Aß amyloid responsome. Proteins in the most conserved network (M42) accumulate in plaques, cerebrovascular amyloid (CAA), and/or dystrophic neuronal processes, and overexpression of two M42 proteins, midkine (Mdk) and pleiotrophin (PTN), increases the accumulation of Aß in plaques and CAA. M42 proteins bind amyloid fibrils in vitro, and MDK and PTN co-accumulate with cardiac transthyretin amyloid. M42 proteins appear intimately linked to amyloid deposition and can regulate amyloid deposition, suggesting that they are pathology modifiers and thus putative therapeutic targets. We posit that amyloid-scaffolded accumulation of numerous M42+ proteins is a central mechanism mediating downstream pathophysiology in AD.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Encéfalo , Placa Amiloide , Proteómica , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Proteómica/métodos , Animales , Péptidos beta-Amiloides/metabolismo , Humanos , Placa Amiloide/metabolismo , Placa Amiloide/patología , Ratones , Encéfalo/metabolismo , Encéfalo/patología , Proteoma/metabolismo , Ratones Transgénicos , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Citocinas/metabolismo , MasculinoRESUMEN
Introduction: Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, yet our comprehension predominantly relies on studies within the non-Hispanic White (NHW) population. Here we aimed to provide comprehensive insights into the proteomic landscape of AD across diverse racial and ethnic groups. Methods: Dorsolateral prefrontal cortex (DLPFC) and superior temporal gyrus (STG) brain tissues were donated from multiple centers (Mayo Clinic, Emory University, Rush University, Mt. Sinai School of Medicine) and were harmonized through neuropathological evaluation, specifically adhering to the Braak staging and CERAD criteria. Among 1105 DLPFC tissue samples (998 unique individuals), 333 were from African American donors, 223 from Latino Americans, 529 from NHW donors, and the rest were from a mixed or unknown racial background. Among 280 STG tissue samples (244 unique individuals), 86 were African American, 76 Latino American, 116 NHW and the rest were mixed or unknown ethnicity. All tissues were uniformly homogenized and analyzed by tandem mass tag mass spectrometry (TMT-MS). Results: As a Quality control (QC) measure, proteins with more than 50% missing values were removed and iterative principal component analysis was conducted to remove outliers within brain regions. After QC, 9,180 and 9,734 proteins remained in the DLPC and STG proteome, respectively, of which approximately 9,000 proteins were shared between regions. Protein levels of microtubule-associated protein tau (MAPT) and amyloid-precursor protein (APP) demonstrated AD-related elevations in DLPFC tissues with a strong association with CERAD and Braak across racial groups. APOE4 protein levels in brain were highly concordant with APOE genotype of the individuals. Discussion: This comprehensive region resolved large-scale proteomic dataset provides a resource for the understanding of ethnoracial-specific protein differences in AD brain.