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1.
Int J Mol Sci ; 24(23)2023 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-38068899

RESUMO

Circulating tumor DNA (ctDNA) has recently emerged as a real-time prognostic and predictive biomarker for monitoring cancer patients. Here, we aimed to ascertain whether tumor-agnostic ctDNA testing would be a feasible strategy to monitor disease progression and therapeutic response in muscle-invasive bladder cancer (MIBC) patients after radical cystectomy (RC). Forty-two MIBC patients who underwent RC were prospectively included. Blood samples from these patients were collected at different follow-up time points. Two specific mutations (TERT c.1-124C>T and ATM c.1236-2A>T) were analyzed in the patients' plasma samples by droplet digital PCR to determine their ctDNA status. During a median follow-up of 21 months, 24% of patients progressed in a median of six months. ctDNA status was identified as a prognostic biomarker of tumor progression before RC and 4 and 12 months later (HR 6.774, HR 3.673, and HR 30.865, respectively; p < 0.05). Lastly, dynamic changes in ctDNA status between baseline and four months later were significantly associated with patient outcomes (p = 0.045). In conclusion, longitudinal ctDNA analysis using a tumor-agnostic approach is a potential tool for monitoring MIBC patients after RC. The implementation of this testing in a clinical setting could improve disease management and patients' outcomes.


Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias da Bexiga Urinária , Humanos , DNA Tumoral Circulante/genética , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , DNA de Neoplasias , Biomarcadores , Músculos/patologia , Biomarcadores Tumorais/genética , Mutação
2.
Neurobiol Dis ; 174: 105885, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36208866

RESUMO

Mitochondrial dysfunction happens in both idiopathic (iPD) and LRRK2-related Parkinson's disease (LRRK2-PD). Nonetheless, previous studies suggested that a different type of mitochondrial pathology underlies the neurodegeneration in these two disorders. To further explore this hypothesis, we developed a novel multiplex digital PCR assay that allows the absolute quantification of cell-free mitochondrial DNA (cf-mtDNA) copy number and deletion ratio directly in cerebrospinal fluid (CSF) by simultaneously measuring two opposed regions of the mtDNA circular molecule, one of them in the commonly deleted major arc. The results confirmed that the content of cf-mtDNA in CSF was statistically significantly different between iPD and LRRK2-PD patients. Moreover, we found high cf-mtDNA deletion levels in CSF from patients with iPD, but not LRRK2-PD. The high cf-mtDNA deletion frequency in iPD was validated in an independent cohort. These results indicated that the content and deletion ratio of cf-mtDNA may differentiate iPD from LRRK2-PD, and provides further evidence of the different mitochondrial pathophysiology between these two forms of the disease.


Assuntos
Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/líquido cefalorraquidiano , Doença de Parkinson/genética , Doença de Parkinson/líquido cefalorraquidiano , DNA Mitocondrial/genética , Mitocôndrias/genética , Estudos de Coortes , Mutação
3.
Int J Mol Sci ; 23(19)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36233035

RESUMO

Cell-free DNA (cfDNA) has recently emerged as a real-time biomarker for diagnosis, monitoring and prediction of therapy response in tumoral disease. Here, we evaluated cfDNA as a prognostic biomarker for monitoring muscle-invasive bladder cancer (MIBC) patients at different follow-up time points. Blood samples from 37 MIBC patients who underwent radical cystectomy (RC) were collected at cystectomy and 1, 4, 12 and 24 months later. Plasma cfDNA amount and fragmentation patterns were determined. Four mutations were analyzed in cfDNA to detect circulating tumor DNA (ctDNA) during patient follow-up. During a median follow-up of 36 months, 46% of patients progressed; median time to progression was 10 months. cfDNA levels and ctDNA status four months after RC were identified as independent prognostic biomarkers of tumor progression (HR 5.290; p = 0.033) and cancer-specific survival (HR 4.199; p = 0.038), respectively. Furthermore, ctDNA clearance four months after RC was significantly associated with patients' clinical outcomes. In conclusion, cfDNA levels and ctDNA status four months after RC have prognostic implications in MIBC patients. In addition, cfDNA monitoring is useful to predict patient outcomes after RC. cfDNA analysis in the clinical setting could greatly improve MIBC patient management.


Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias da Bexiga Urinária , Biomarcadores , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , Humanos , Músculos/patologia , Prognóstico , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
4.
Cereb Cortex ; 30(6): 3781-3799, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32043120

RESUMO

Neural cell adhesion molecule 2 (NCAM2) is involved in the development and plasticity of the olfactory system. Genetic data have implicated the NCAM2 gene in neurodevelopmental disorders including Down syndrome and autism, although its role in cortical development is unknown. Here, we show that while overexpression of NCAM2 in hippocampal neurons leads to minor alterations, its downregulation severely compromises dendritic architecture, leading to an aberrant phenotype including shorter dendritic trees, retraction of dendrites, and emergence of numerous somatic neurites. Further, our data reveal alterations in the axonal tree and deficits in neuronal polarization. In vivo studies confirm the phenotype and reveal an unexpected role for NCAM2 in cortical migration. Proteomic and cell biology experiments show that NCAM2 molecules exert their functions through a protein complex with the cytoskeletal-associated proteins MAP2 and 14-3-3γ and ζ. We provide evidence that NCAM2 depletion results in destabilization of the microtubular network and reduced MAP2 signal. Our results demonstrate a role for NCAM2 in dendritic formation and maintenance, and in neural polarization and migration, through interaction of NCAM2 with microtubule-associated proteins.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Plasticidade Neuronal/genética , Animais , Movimento Celular/genética , Polaridade Celular/genética , Células HEK293 , Hipocampo , Humanos , Camundongos , Microtúbulos , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios
5.
Addict Biol ; 26(1): e12840, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-31833146

RESUMO

Repeated cocaine exposure causes long-lasting neuroadaptations that involve alterations in cellular signaling and gene expression mediated by dopamine in different brain regions, such as the striatum. Previous studies have pointed out to the dopamine D1 receptor as one major player in psychostimulants-induced behavioral, cellular, and molecular changes. However, the role of other dopamine receptors has not been fully characterized. Here we used dopamine D2 receptor knockout (D2-/- ) mice to explore the role of D2 receptor (D2R) in behavioral sensitization and its associated gene expression after acute and chronic cocaine and amphetamine administration. We also studied the impact of D2R elimination in D1R-mediated responses. We found that cocaine- and amphetamine-induced behavioral sensitization is deficient in D2-/- mice. The expression of dynorphin, primarily regulated by D1R and a marker of direct-pathway striatal neurons, is attenuated in naïve- and in cocaine- or amphetamine-treated D2-/- mice. Moreover, c-Fos expression observed in D2-/- mice was reduced in acutely but not in chronically treated animals. Interestingly, inactivation of D2R increased c-Fos expression in neurons of the striatopallidal pathway. Finally, elimination of D2R blunted the locomotor and striatal c-Fos response to the full D1 agonist SKF81297. In conclusion, D2R is critical for the development of behavioral sensitization and the associated gene expression, after cocaine administration, and it is required for the locomotor responses promoted by D1R activation.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Receptores de Dopamina D2/metabolismo , Anfetaminas/farmacologia , Animais , Benzazepinas , Corpo Estriado/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Receptores de Dopamina D1/metabolismo
6.
Int J Mol Sci ; 21(17)2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32878083

RESUMO

Alzheimer's type dementia (AD) exhibits clinical heterogeneity, as well as differences in disease progression, as a subset of patients with a clinical diagnosis of AD progresses more rapidly (rpAD) than the typical AD of slow progression (spAD). Previous findings indicate that low cerebrospinal fluid (CSF) content of cell-free mitochondrial DNA (cf-mtDNA) precedes clinical signs of AD. We have now investigated the relationship between cf-mtDNA and other biomarkers of AD to determine whether a particular biomarker profile underlies the different rates of AD progression. We measured the content of cf-mtDNA, beta-amyloid peptide 1-42 (Aß), total tau protein (t-tau) and phosphorylated tau (p-tau) in the CSF from a cohort of 95 subjects consisting of 49 controls with a neurologic disorder without dementia, 30 patients with a clinical diagnosis of spAD and 16 patients with rpAD. We found that 37% of controls met at least one AD biomarker criteria, while 53% and 44% of subjects with spAD and rpAD, respectively, did not fulfill the two core AD biomarker criteria: high t-tau and low Aß in CSF. In the whole cohort, patients with spAD, but not with rpAD, showed a statistically significant 44% decrease of cf-mtDNA in CSF compared to control. When the cohort included only subjects selected by Aß and t-tau biomarker criteria, the spAD group showed a larger decrease of cf-mtDNA (69%), whereas in the rpAD group cf-mtDNA levels remained unaltered. In the whole cohort, the CSF levels of cf-mtDNA correlated positively with Aß and negatively with p-tau. Moreover, the ratio between cf-mtDNA and p-tau increased the sensitivity and specificity of spAD diagnosis up to 93% and 94%, respectively, in the biomarker-selected cohort. These results show that the content of cf-mtDNA in CSF correlates with the earliest pathological markers of the disease, Aß and p-tau, but not with the marker of neuronal damage t-tau. Moreover, these findings confirm that low CSF content of cf-mtDNA is a biomarker for the early detection of AD and support the hypothesis that low cf-mtDNA, together with low Aß and high p-tau, constitute a distinctive CSF biomarker profile that differentiates spAD from other neurological disorders.


Assuntos
Doença de Alzheimer/patologia , Biomarcadores/líquido cefalorraquidiano , DNA Mitocondrial/líquido cefalorraquidiano , DNA Mitocondrial/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/classificação , Estudos de Casos e Controles , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
7.
Mult Scler ; 24(4): 472-480, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28294696

RESUMO

BACKGROUND: Mitochondrial dysfunction is increasingly recognized as an important feature of multiple sclerosis (MS) pathology and may be relevant for clinical disease progression. However, it is unknown whether mitochondrial DNA (mtDNA) levels in the cerebrospinal fluid (CSF) associate with disease progression and therapeutic response. OBJECTIVES: To evaluate whether CSF concentrations of mtDNA in MS patients can serve as a marker of ongoing neuropathology and may be helpful to differentiate between MS disease subtypes. To explore the effect of disease-modifying therapies on mtDNA levels in the CSF. METHODS: CSF mtDNA was measured using a digital polymerase chain reaction (PCR) CSF mtDNA in two independent MS cohorts. The cohorts included 92 relapsing-remitting multiple sclerosis (RRMS) patients, 40 progressive multiple sclerosis (PMS) patients (27 secondary progressive and 13 primary progressive), 50 various neurologic disease controls, and 5 healthy controls. RESULTS: Patients with PMS showed a significant increase in CSF mtDNA compared to non-inflammatory neurologic disease controls. Patients with higher T2 lesion volumes and lower normalized brain volumes showed increased concentration of mtDNA. Patients treated with fingolimod had significantly lower mtDNA copy levels at follow-up compared to baseline. CONCLUSION: Our results showed a non-specific elevation of concentration of mtDNA in PMS patients. mtDNA concentrations respond to fingolimod and may be used to monitor biological effect of this treatment.


Assuntos
Biomarcadores/líquido cefalorraquidiano , DNA Mitocondrial/líquido cefalorraquidiano , Mitocôndrias/metabolismo , Esclerose Múltipla/tratamento farmacológico , Adulto , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/patologia , Esclerose Múltipla Crônica Progressiva/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano
8.
J Neurosci ; 35(14): 5504-21, 2015 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-25855168

RESUMO

In mature neurons, the number of synapses is determined by a neuronal activity-dependent dynamic equilibrium between positive and negative regulatory factors. We hypothesized that neuronal pentraxin (NP1), a proapoptotic protein induced by low neuronal activity, could be a negative regulator of synapse density because it is found in dystrophic neurites in Alzheimer's disease-affected brains. Here, we report that knockdown of NP1 increases the number of excitatory synapses and neuronal excitability in cultured rat cortical neurons and enhances excitatory drive and long-term potentiation in the hippocampus of behaving mice. Moreover, we found that NP1 regulates the surface expression of the Kv7.2 subunit of the Kv7 family of potassium channels that control neuronal excitability. Furthermore, pharmacological activation of Kv7 channels prevents, whereas inhibition mimics, the increase in synaptic proteins evoked by the knockdown of NP1. These results indicate that NP1 negatively regulates excitatory synapse number by modulating neuronal excitability and show that NP1 restricts excitatory synaptic plasticity.


Assuntos
Proteína C-Reativa/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Proteína C-Reativa/genética , Células Cultivadas , Córtex Cerebral/citologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Antagonistas de Receptores de GABA-A/farmacologia , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/genética , Vigília/efeitos dos fármacos
9.
Neurobiol Dis ; 94: 10-7, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27260835

RESUMO

Mitochondrial DNA regulates mitochondrial function which is altered in both idiopathic and familial forms of Parkinson's disease. To investigate whether these two disease forms exhibit an altered regulation of mitochondrial DNA we measured cell free mitochondrial DNA content in cerebrospinal fluid (CSF) from idiopathic and LRRK2-related Parkinson's disease patients. The concentration of mitochondrial DNA was measured using a digital droplet polymerase chain reaction technique in a total of 98 CSF samples from a cohort of subjects including: 20 LRRK2(G2019S) mutation carriers with Parkinson's disease, 26 asymptomatic LRRK2(G2019S) mutation carriers, 31 patients with idiopathic Parkinson's disease and 21 first-degree relatives of LRRK2 Parkinson's disease patients without the mutation. Here we report that LRRK2(G2019S) mutation carriers with Parkinson's disease exhibit a high concentration of mitochondrial DNA in CSF compared with asymptomatic LRRK2(G2019S) mutation carriers and with idiopathic Parkinson's disease patients. In addition, idiopathic, but not LRRK2 Parkinson's disease is associated with low CSF concentration of α-synuclein. These results show that high mitochondrial DNA content in CSF distinguishes idiopathic from LRRK2-related Parkinson's disease suggesting that different biochemical pathways underlie neurodegeneration in these two disorders.


Assuntos
DNA Mitocondrial/líquido cefalorraquidiano , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/líquido cefalorraquidiano , Mitocôndrias/metabolismo , Doença de Parkinson/líquido cefalorraquidiano , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , alfa-Sinucleína/líquido cefalorraquidiano , alfa-Sinucleína/genética
10.
Alzheimers Dement ; 12(5): 546-55, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26806388

RESUMO

INTRODUCTION: Low content of cell-free mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) is a biomarker of early stage Alzheimer's disease (AD), but whether mtDNA is altered in a rapid neurodegenerative dementia such as Creutzfeldt-Jakob disease is unknown. METHODS: CSF mtDNA was measured using digital polymerase chain reaction (dPCR) in two independent cohorts comprising a total of 112 patients diagnosed with sporadic Creutzfeldt-Jakob disease (sCJD), probable AD, or non-Alzheimer's type dementia. RESULTS: Patients with AD exhibit low mtDNA content in CSF compared with patients diagnosed with sCJD or with non-Alzheimer's type dementias. The CSF concentration of mtDNA does not correlate with Aß, t-tau, p-tau, and 14-3-3 protein levels in CSF. DISCUSSION: Low-CSF mtDNA is not a consequence of brain damage and allows the differential diagnosis of AD from sCJD and other dementias. These results support the hypothesis that mtDNA in CSF is a pathophysiological biomarker of AD.


Assuntos
Doença de Alzheimer/diagnóstico , Síndrome de Creutzfeldt-Jakob/diagnóstico , DNA Mitocondrial/líquido cefalorraquidiano , Diagnóstico Diferencial , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Humanos , Proteínas tau/líquido cefalorraquidiano
11.
Ann Neurol ; 74(5): 655-68, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23794434

RESUMO

OBJECTIVE: To identify a novel biochemical marker that precedes clinical symptoms in Alzheimer disease (AD). METHODS: Using quantitative polymerase chain reaction techniques, we measured circulating cell-free mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) from study participants, selected from a cohort of 282 subjects, who were classified according to their concentrations of amyloid ß1-42, total tau, and phosphorylated tau and by the presence or absence of dementia, into asymptomatic subjects at risk of AD, symptomatic patients diagnosed with sporadic AD, presymptomatic subjects carrying pathogenic PSEN1 mutations, and patients diagnosed with frontotemporal lobar degeneration (FTLD). We performed equivalent studies in a separate validation cohort of sporadic AD and FTLD patients. In addition, we measured mtDNA copy number in cultured cortical neurons from mutant amyloid precursor protein/presenilin1 (APP/PS1) transgenic mice. RESULTS: Asymptomatic patients at risk of AD and symptomatic AD patients, but not FTLD patients, exhibit a significant decrease in circulating cell-free mtDNA in the CSF. These observations were confirmed in the validation cohort. In addition, presymptomatic subjects carrying pathogenic PSEN1 gene mutations show low mtDNA content in CSF before the appearance of AD-related biomarkers in CSF. Moreover, mtDNA content in CSF discriminates with high sensitivity and specificity AD patients from either controls or patients with FTLD. Furthermore, cultured cortical neurons from APP/PS1 transgenic mice have fewer mtDNA copies before the appearance of altered synaptic markers. INTERPRETATION: Low content of mtDNA in CSF may be a novel biomarker for the early detection of preclinical AD. These findings support the hypothesis that mtDNA depletion is a characteristic pathophysiological factor of neurodegeneration in AD.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Disfunção Cognitiva/líquido cefalorraquidiano , DNA Mitocondrial/líquido cefalorraquidiano , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/líquido cefalorraquidiano , Animais , Biomarcadores/líquido cefalorraquidiano , Disfunção Cognitiva/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fosforilação , Presenilina-1/genética , Sintomas Prodrômicos , Proteínas tau/líquido cefalorraquidiano
12.
Genes (Basel) ; 15(3)2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38540444

RESUMO

Epithelial cells comprising the choroid plexus (CP) form a crucial barrier between the blood and the cerebrospinal fluid, thereby assuming a central position in brain homeostasis and signaling. Mounting evidence suggests that the impairment of CP function may be a significant contributor to Alzheimer's disease (AD) pathogenesis. CP function relies on the expression of specific receptors, and the potential involvement of olfactory receptors (ORs) and taste receptors (TASRs) in chemical surveillance within the CP is being investigated. Previous studies have implicated ORs and TASRs in neurodegenerative disorders like AD, although the direct evidence of their expression in the human CP remains to be established. In this study, we conducted a transcriptomic analysis encompassing eleven ORs and TASRs in the CP, comparing samples from healthy age-matched controls to those from patients with AD spanning Braak stages I to VI. Among these receptors, a striking finding emerged-OR2K2 exhibited robust expression, with a statistically significant upregulation noted at Braak stage I. Surprisingly, at the protein level, OR2K2 showed a significant decrease in both Braak stage I and VI. Additionally, we identified CP epithelial cells as the source of OR2K2 expression, where it colocalized with autophagy markers LC3 and p62. We postulate that OR2K2 could be subjected to degradation by autophagy in the early stages of AD, triggering a compensatory mechanism that leads to increased OR2K2 mRNA transcription. This study uncovers a potential role for OR2K2 in AD pathogenesis, offering a novel perspective on the intricate dynamics at play in this neurodegenerative disorder.


Assuntos
Doença de Alzheimer , Receptores Odorantes , Humanos , Doença de Alzheimer/patologia , Plexo Corióideo/metabolismo , Plexo Corióideo/patologia , Expressão Gênica , Encéfalo/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo
13.
EBioMedicine ; 102: 105065, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38502973

RESUMO

BACKGROUND: Idiopathic rapid eye movement (REM) sleep behaviour disorder (IRBD) represents the prodromal stage of Lewy body disorders (Parkinson's disease (PD) and dementia with Lewy bodies (DLB)) which are linked to variations in circulating cell-free mitochondrial DNA (cf-mtDNA). Here, we assessed whether altered cf-mtDNA release and integrity are already present in IRBD. METHODS: We used multiplex digital PCR (dPCR) to quantify cf-mtDNA copies and deletion ratio in cerebrospinal fluid (CSF) and serum in a cohort of 71 participants, including 1) 17 patients with IRBD who remained disease-free (non-converters), 2) 34 patients initially diagnosed with IRBD who later developed either PD or DLB (converters), and 3) 20 age-matched controls without IRBD or Parkinsonism. In addition, we investigated whether CD9-positive extracellular vesicles (CD9-EVs) from CSF and serum samples contained cf-mtDNA. FINDINGS: Patients with IRBD, both converters and non-converters, exhibited more cf-mtDNA with deletions in the CSF than controls. This finding was confirmed in CD9-EVs. The high levels of deleted cf-mtDNA in CSF corresponded to a significant decrease in cf-mtDNA copies in CD9-EVs in both IRBD non-converters and converters. Conversely, a significant increase in cf-mtDNA copies was found in serum and CD9-EVs from the serum of patients with IRBD who later converted to a Lewy body disorder. INTERPRETATION: Alterations in cf-mtDNA copy number and deletion ratio known to occur in Lewy body disorders are already present in IRBD and are not a consequence of Lewy body disease conversion. This suggests that mtDNA dysfunction is a primary molecular mechanism of the pathophysiological cascade that precedes the full clinical motor and cognitive manifestation of Lewy body disorders. FUNDING: Funded by Michael J. Fox Foundation research grant MJFF-001111. Funded by MICIU/AEI/10.13039/501100011033 "ERDF A way of making Europe", grants PID2020-115091RB-I00 (RT) and PID2022-143279OB-I00 (ACo). Funded by Instituto de Salud Carlos III and European Union NextGenerationEU/PRTR, grant PMP22/00100 (RT and ACo). Funded by AGAUR/Generalitat de Catalunya, grant SGR00490 (RT and ACo). MP has an FPI fellowship, PRE2018-083297, funded by MICIU/AEI/10.13039/501100011033 "ESF Investing in your future".


Assuntos
Doença de Parkinson , Transtornos Parkinsonianos , Transtorno do Comportamento do Sono REM , Humanos , Transtorno do Comportamento do Sono REM/diagnóstico , Transtorno do Comportamento do Sono REM/genética , Doença de Parkinson/genética , Previsões , DNA Mitocondrial/genética
14.
J Neurosci ; 32(4): 1453-66, 2012 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-22279230

RESUMO

In cultured cerebellar granule neurons, low neuronal activity triggers the intrinsic program of apoptosis, which requires protein synthesis-dependent BAX translocation to mitochondria, a process that may underlie neuronal damage in neurodegeneration. However, the mechanisms that link neuronal activity with the induction of the mitochondrial program of apoptosis remain unclear. Neuronal pentraxin 1 (NP1) is a pro-apoptotic protein induced by low neuronal activity that is increased in damaged neurites in Alzheimer's disease-affected brains. Here we report that NP1 facilitates the accumulation of BAX in mitochondria and regulates mitochondrial dynamics during apoptosis in rat and mouse cerebellar granule neurons in culture. Reduction of neuronal activity increases NP1 protein levels in mitochondria and contributes to mitochondrial fragmentation in a Bax-dependent manner. In addition, NP1 is involved in mitochondrial transport in healthy neurons. These results show that NP1 is targeted to mitochondria acting upstream of BAX and uncover a novel function for NP1 in the regulation of mitochondrial dynamics and trafficking during apoptotic neurodegeneration.


Assuntos
Proteína C-Reativa/fisiologia , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Simulação de Dinâmica Molecular , Ratos , Ratos Sprague-Dawley
15.
J Neurosci ; 32(36): 12431-6, 2012 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-22956834

RESUMO

Peripheral nerve injury causes spontaneous and long-lasting pain, hyperalgesia, and allodynia. Excitatory amino acid receptor-dependent increases in descending facilitatory drive from the brainstem rostral ventromedial medulla (RVM) contribute to injury-evoked hypersensitivity. Although increased excitability likely reflects changes in synaptic efficacy, the cellular mechanisms underlying injury-induced synaptic plasticity are poorly understood. Neuronal pentraxin 1 (NP1), a protein with exclusive CNS expression, is implicated in synaptogenesis and AMPA receptor recruitment to immature synapses. Its role in the adult brain and in descending pain facilitation is unknown. Here, we use the spared nerve injury (SNI) model in rodents to examine this issue. We show that SNI increases RVM NP1 expression and constitutive deletion or silencing NP1 in the RVM, before or after SNI, attenuates allodynia and hyperalgesia in rats. Selective rescue of RVM NP1 expression restores behavioral hypersensitivity of knock-out mice, demonstrating a key role of RVM NP1 in the pathogenesis of neuropathic pain.


Assuntos
Proteína C-Reativa/antagonistas & inibidores , Proteína C-Reativa/fisiologia , Hiperalgesia/metabolismo , Hiperalgesia/prevenção & controle , Bulbo/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Neuralgia/metabolismo , Neuralgia/prevenção & controle , Animais , Proteína C-Reativa/genética , Inativação Gênica/fisiologia , Hiperalgesia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neuralgia/genética , Neurônios/metabolismo , Manejo da Dor/métodos , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley
16.
Transl Neurodegener ; 12(1): 10, 2023 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-36895045

RESUMO

BACKGROUND: Persistent inflammatory response in the brain can lead to tissue damage and neurodegeneration. In Alzheimer's disease (AD), there is an aberrant activation of inflammasomes, molecular platforms that drive inflammation through caspase-1-mediated proteolytic cleavage of proinflammatory cytokines and gasdermin D (GSDMD), the executor of pyroptosis. However, the mechanisms underlying the sustained activation of inflammasomes in AD are largely unknown. We have previously shown that high brain cholesterol levels promote amyloid-ß (Aß) accumulation and oxidative stress. Here, we investigate whether these cholesterol-mediated changes may regulate the inflammasome pathway. METHODS: SIM-A9 microglia and SH-SY5Y neuroblastoma cells were cholesterol-enriched using a water-soluble cholesterol complex. After exposure to lipopolysaccharide (LPS) plus muramyl dipeptide or Aß, activation of the inflammasome pathway was analyzed by immunofluorescence, ELISA and immunoblotting analysis. Fluorescently-labeled Aß was employed to monitor changes in microglia phagocytosis. Conditioned medium was used to study how microglia-neuron interrelationship modulates the inflammasome-mediated response. RESULTS: In activated microglia, cholesterol enrichment promoted the release of encapsulated IL-1ß accompanied by a switch to a more neuroprotective phenotype, with increased phagocytic capacity and release of neurotrophic factors. In contrast, in SH-SY5Y cells, high cholesterol levels stimulated inflammasome assembly triggered by both bacterial toxins and Aß peptides, resulting in GSDMD-mediated pyroptosis. Glutathione (GSH) ethyl ester treatment, which recovered the cholesterol-mediated depletion of mitochondrial GSH levels, significantly reduced the Aß-induced oxidative stress in the neuronal cells, resulting in lower inflammasome activation and cell death. Furthermore, using conditioned media, we showed that neuronal pyroptosis affects the function of the cholesterol-enriched microglia, lowering its phagocytic activity and, therefore, the ability to degrade extracellular Aß. CONCLUSIONS: Changes in intracellular cholesterol levels differentially regulate the inflammasome-mediated immune response in microglia and neuronal cells. Given the microglia-neuron cross-talk in the brain, cholesterol modulation should be considered a potential therapeutic target for AD treatment, which may help to block the aberrant and chronic inflammation observed during the disease progression.


Assuntos
Doença de Alzheimer , Hipercolesterolemia , Neuroblastoma , Humanos , Inflamassomos/metabolismo , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Hipercolesterolemia/metabolismo , Neuroblastoma/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Inflamação/metabolismo
17.
Nat Genet ; 55(10): 1632-1639, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37723262

RESUMO

Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization1,2. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy3,4. However, the causative mechanisms of paternal mtDNA elimination have not been defined5,6. We found that mitochondria in human spermatozoa are devoid of intact mtDNA and lack mitochondrial transcription factor A (TFAM)-the major nucleoid protein required to protect, maintain and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial presequence, ordinarily removed upon mitochondrial import. Phosphorylation of this presequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM relocalization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mtDNA, thereby explaining maternal inheritance in this species.


Assuntos
DNA Mitocondrial , Herança Materna , Humanos , Masculino , DNA Mitocondrial/genética , Herança Materna/genética , Sêmen/metabolismo , Mitocôndrias/genética , Espermatozoides/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
18.
Sci Rep ; 13(1): 18997, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37923774

RESUMO

Somatic single-nucleotide variants (SNVs) occur every time a cell divides, appearing even in healthy tissues at low frequencies. These mutations may accumulate as neutral variants during aging, or eventually, promote the development of neoplasia. Here, we present the SP-ddPCR, a droplet digital PCR (ddPCR) based approach that utilizes customized SuperSelective primers aiming at quantifying the proportion of rare SNVs. For that purpose, we selected five potentially pathogenic variants identified by whole-exome sequencing (WES) occurring at low variant allele frequency (VAF) in at-risk colon healthy mucosa of patients diagnosed with colorectal cancer or advanced adenoma. Additionally, two APC SNVs detected in two cancer lesions were added to the study for WES-VAF validation. SuperSelective primers were designed to quantify SNVs at low VAFs both in silico and in clinical samples. In addition to the two APC SNVs in colonic lesions, SP-ddPCR confirmed the presence of three out of five selected SNVs in the normal colonic mucosa with allelic frequencies ≤ 5%. Moreover, SP-ddPCR showed the presence of two potentially pathogenic variants in the distal normal mucosa of patients with colorectal carcinoma. In summary, SP-ddPCR offers a rapid and feasible methodology to validate next-generation sequencing data and accurately quantify rare SNVs, thus providing a potential tool for diagnosis and stratification of at-risk patients based on their mutational profiling.


Assuntos
Neoplasias , Humanos , Mutação , Primers do DNA , Colo , Reação em Cadeia da Polimerase , Sequenciamento de Nucleotídeos em Larga Escala/métodos
19.
J Biol Chem ; 286(31): 27311-21, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21665950

RESUMO

ß-Amyloid (Aß), a peptide generated from the amyloid precursor protein, is widely believed to underlie the pathophysiology of Alzheimer disease (AD). Emerging evidences suggest that soluble Aß oligomers adversely affect synaptic function, leading to cognitive failure associated with AD. The Aß-induced synaptic dysfunction has been attributed to the synaptic removal of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs). However, the molecular mechanisms underlying the loss of AMPAR induced by Aß at synapses are largely unknown. In this study we have examined the effect of Aß oligomers on phosphorylated GluA1 at serine 845, a residue that plays an essential role in the trafficking of AMPARs toward extrasynaptic sites and the subsequent delivery to synapses during synaptic plasticity events. We found that Aß oligomers reduce basal levels of Ser-845 phosphorylation and surface expression of AMPARs affecting AMPAR subunit composition. Aß-induced GluA1 dephosphorylation and reduced receptor surface levels are mediated by an increase in calcium influx into neurons through ionotropic glutamate receptors and activation of the calcium-dependent phosphatase calcineurin. Moreover, Aß oligomers block the extrasynaptic delivery of AMPARs induced by chemical synaptic potentiation. In addition, reduced levels of total and phosphorylated GluA1 are associated with initial spatial memory deficits in a transgenic mouse model of AD. These findings indicate that Aß oligomers could act as a synaptic depressor affecting the mechanisms involved in the targeting of AMPARs to the synapses during early stages of the disease.


Assuntos
Peptídeos beta-Amiloides/química , Biopolímeros/farmacologia , Receptores de AMPA/metabolismo , Sinapses/fisiologia , Animais , Biopolímeros/química , Western Blotting , Membrana Celular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transporte Proteico
20.
Nat Commun ; 13(1): 5272, 2022 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-36071061

RESUMO

Astrocytes are key elements of brain circuits that are involved in different aspects of the neuronal physiology relevant to brain functions. Although much effort is being made to understand how the biology of astrocytes affects brain circuits, astrocytic network heterogeneity and plasticity is still poorly defined. Here, we have combined structural and functional imaging of astrocyte activity recorded in mice using the Ca2+-modulated photoactivatable ratiometric integrator and specific optostimulation of glutamatergic pathways to map the functional neuron-astrocyte circuitries in the nucleus accumbens (NAc). We showed pathway-specific astrocytic responses induced by selective optostimulation of main inputs from the prefrontal cortex, basolateral amygdala, and ventral hippocampus. Furthermore, co-stimulation of glutamatergic pathways induced non-linear Ca2+-signaling integration, revealing integrative properties of NAc astrocytes. All these results demonstrate the existence of specific neuron-astrocyte circuits in the NAc, providing an insight to the understanding of how the NAc integrates information.


Assuntos
Complexo Nuclear Basolateral da Amígdala , Núcleo Accumbens , Animais , Astrócitos/metabolismo , Hipocampo/fisiologia , Camundongos , Neurônios/metabolismo , Núcleo Accumbens/metabolismo
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