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1.
Cell Calcium ; 100: 102480, 2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34607180

RESUMO

Calcineurin (CaN), acting downstream of intracellular calcium signals, orchestrates cellular remodeling in many cellular types. In astrocytes, major homeostatic players in the central nervous system (CNS), CaN is involved in neuroinflammation and gliosis, while its role in healthy CNS or in early neuro-pathogenesis is poorly understood. Here we report that in mice with conditional deletion of CaN in GFAP-expressing astrocytes (astroglial calcineurin KO, ACN-KO), at 1 month of age, transcription was largely unchanged, while the proteome was deranged in the hippocampus and cerebellum. Gene ontology analysis revealed overrepresentation of annotations related to myelin sheath, mitochondria, ribosome and cytoskeleton. Over-represented pathways were related to protein synthesis, oxidative phosphorylation, mTOR and neurological disorders, including Alzheimer's disease (AD) and seizure disorder. Comparison with published proteomic datasets showed significant overlap with the proteome of a familial AD mouse model and of human subjects with drug-resistant seizures. ACN-KO mice showed no alterations of motor activity, equilibrium, anxiety or depressive state. However, in Barnes maze ACN-KO mice learned the task but adopted serial search strategy. Strikingly, beginning from about 5 months of age ACN-KO mice developed spontaneous tonic-clonic seizures with an inflammatory signature of epileptic brains. Altogether, our data suggest that the deletion of astroglial CaN produces features of neurological disorders and predisposes mice to seizures. We suggest that calcineurin in astrocytes may serve as a novel Ca2+-sensitive switch which regulates protein expression and homeostasis in the central nervous system.

2.
Viruses ; 13(9)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34578326

RESUMO

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.


Assuntos
COVID-19/virologia , Interações Hospedeiro-Patógeno , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Fenômenos Fisiológicos Virais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/metabolismo , Ciclo Celular , Chlorocebus aethiops , Doxiciclina/farmacologia , Células HEK293 , Humanos , Ligação Proteica , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus , Transdução Genética , Células Vero
3.
Mol Neurodegener ; 16(1): 52, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376243

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease's complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell's physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification. METHODS: We analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis. RESULTS: Our procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs. CONCLUSIONS: Our analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

4.
Clin Chem Lab Med ; 59(11): 1832-1843, 2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34225393

RESUMO

OBJECTIVES: The search in the urinary sediment (U-sed) of fat particles with peculiar morphology is a simple and inexpensive tool for the diagnosis of Fabry disease (FD) nephropathy. In this study we investigated the morphology of a high number of such fat particles with the aim to obtain a morphological classification to be used for their identification. METHODS: Study of the morphology of fat particles in the U-sed of a cohort of FD patients using: bright field plus phase contrast microscopy (BF + PC), polarized light microscopy (POL), and transmission electron microscopy (TEM). Comparison of these results with those obtained for the fat particles seen in the U-sed of a control group (CG) of patients with non-FD glomerulopathies. RESULTS: FD: 18 U-sed from six patients (three samples/patient) were prospectively investigated and 506 fat particles identified. With BF + PC, these were classified in eight morphological categories (seven of which were confirmed by TEM), and with POL in 10 others. CG: eight U-sed from eight patients were investigated and 281 fat particles identified. These fell into four BF + PC morphological categories and into eight POL categories. While some categories were significantly more frequent in FD others were more frequent in the CG. CONCLUSIONS: Our study demonstrates that 1. The morphology of fat particles found in the U-sed of FD patients is much wider and complex than that described so far 2. Several significant differences exist in the morphology of such fat particles between FD and CG patients.

5.
Part Fibre Toxicol ; 18(1): 23, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34134756

RESUMO

BACKGROUND: Widespread use of silver in its different forms raises concerns about potential adverse effects after ingestion, the main exposure route for humans. The aim of this study was to investigate in CD-1 (ICR) male mice the tissue distribution and in vivo effects of 4-week oral exposure to 0.25 and 1 mg Ag/kg bw 10 nm citrate coated silver nanoparticles (AgNPs) and 1 mg Ag/kg bw silver acetate (AgAc) at the end of treatment (EoT) and after 4 weeks of recovery. RESULTS: There were no treatment-related clinical signs and mortality, and no significant effects on body and organ weights at the EoT and after recovery. Treatment-related changes in hematology and clinical chemistry were found after recovery, the most relevant being a dose-dependent lymphopenia and increased triglycerides in AgNP-treated mice, and increased levels of urea in all treated groups, associated with decreased albumin only in AgAc-treated mice. At the EoT the highest silver concentration determined by Triple Quadrupole ICP-MS analysis was found in the brain, followed by testis, liver, and spleen; much lower concentrations were present in the small intestine and kidney. Tissue silver concentrations were slightly higher after exposure to AgAc than AgNPs and dose dependent for AgNPs. After recovery silver was still present in the brain and testis, highlighting slow elimination. No histopathological changes and absence of silver staining by autometallography were observed in the organs of treated mice. At the EoT GFAP (astrocytes) immunoreactivity was significantly increased in the hippocampus of AgNP-treated mice in a dose-dependent manner and Iba1 (microglial cells) immunoreactivity was significantly increased in the cortex of 1 mg/kg bw AgNP-treated mice. After recovery, a significant reduction of Iba1 was observed in the cortex of all treated groups. TEM analysis of the hippocampus revealed splitting of basement membrane of the capillaries and swelling of astrocytic perivascular end-feet in 1 mg/kg bw AgNP- and AgAc-treated mice at the EoT. CONCLUSIONS: Our study revealed accumulation and slow clearance of silver in the brain after oral administration of 10 nm AgNPs and AgAc at low doses in mice, associated with effects on glial cells and ultrastructural alterations of the Blood-Brain Barrier.


Assuntos
Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Administração Oral , Animais , Encéfalo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Distribuição Tecidual
6.
Am J Cancer Res ; 11(5): 2303-2311, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34094686

RESUMO

Mitochondria have attracted attention in cancer research as organelles associated with tumor development and response to therapy. We recently reported acquisition of resistance to cisplatin (DDP) associated with a metabolic rewiring in ovarian cancer patient-derived xenografts (PDXs) models. DDP-resistant PDXs models were obtained mimicking the clinical setting, treating mice bearing sensitive-DDP tumors with multiple cycles of DDP until the development of resistance. To further characterize the metabolic rewiring, the present study focused on tumor mitochondria. We analysed by transmission electron microscopy the mitochondria structure in two models of DDP-resistant and the corresponding DDP-sensitive PDXs and evaluated tumor mDNA content, the expression of genes and proteins involved in mitochondria functionality, and mitochondria fitness-related processes, such as autophagy. We observed a decrease in the number of mitochondria paralleled by an increased volume in DDP-resistant versus DDP-sensitive PDXs. DDP-resistant PDXs presented a higher percentage of damaged mitochondria, in particular of type 2 (concave-shape), and type 3 (cristolysis) damage. We found no difference in the mDNA content, and the expression of genes involved in mitochondrial biogenesis was similar between the sensitive and resistant PDXs. An upregulation of some genes involved in mitochondrial fitness in DDP-R versus DDP-S PDXs was observed. At protein level, no difference in the expression of proteins involved in mitochondrial function and biogenesis, and in autophagy/mitophagy was found. We here reported that the acquisition of DDP resistance is associated with morphological alterations in mitochondria, even if we couldn't find any dysregulation in the studied genes/proteins that could explain the observed differences.

7.
J Biol Chem ; 296: 100664, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33865852

RESUMO

The formation of neurofibrillary tangles and amyloid plaques accompanies the progression of Alzheimer's disease. Tangles are made of fibrillar aggregates formed by the microtubule-associated protein tau, whereas plaques comprise fibrillar forms of amyloid-beta (Aß). Both form toxic oligomers during aggregation and are thought to interact synergistically to each promote the accumulation of the other. Recent in vitro studies have suggested that the monomeric nonphosphorylated full-length tau protein hinders the aggregation of Aß1-40 peptide, but whether the same is true for the more aggregation-prone Aß1-42 was not determined. We used in vitro and in vivo techniques to explore this question. We have monitored the aggregation kinetics of Aß1-42 by thioflavine T fluorescence in the presence or the absence of different concentrations of nonphosphorylated tau. We observed that elongation of Aß1-42 fibrils was inhibited by tau in a dose-dependent manner. Interestingly, the fibrils were structurally different in the presence of tau but did not incorporate tau. Surface plasmon resonance indicated that tau monomers bound to Aß1-42 oligomers (but not monomers) and hindered their interaction with the anti-Aß antibody 4G8, suggesting that tau binds to the hydrophobic central core of Aß recognized by 4G8. Tau monomers also antagonized the toxic effects of Aß oligomers in Caenorhabditis elegans. This suggests that nonphosphorylated tau might have a neuroprotective effect by binding Aß1-42 oligomers formed during the aggregation and shielding their hydrophobic patches.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Amiloide/antagonistas & inibidores , Caenorhabditis elegans/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Proteínas tau/farmacologia , Peptídeos beta-Amiloides/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Humanos , Cinética , Larva/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade
8.
J Biol Chem ; 296: 100490, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33662396

RESUMO

Fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and Gerstmann-Sträussler-Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.


Assuntos
Mutação , Proteínas Priônicas/metabolismo , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Doença de Gerstmann-Straussler-Scheinker/genética , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Doença de Gerstmann-Straussler-Scheinker/patologia , Complexo de Golgi/metabolismo , Humanos , Insônia Familiar Fatal/genética , Insônia Familiar Fatal/metabolismo , Insônia Familiar Fatal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Proteínas Priônicas/genética , Dobramento de Proteína , Via Secretória , Quinases da Família src/química
9.
J Am Soc Nephrol ; 32(5): 1114-1130, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33722931

RESUMO

BACKGROUND: Podocyte dysfunction and loss are major determinants in the development of proteinuria. FSGS is one of the most common causes of proteinuria, but the mechanisms leading to podocyte injury or conferring protection against FSGS remain poorly understood. The cytosolic protein M-Sec has been involved in the formation of tunneling nanotubes (TNTs), membrane channels that transiently connect cells and allow intercellular organelle transfer. Whether podocytes express M-Sec is unknown and the potential relevance of the M-Sec-TNT system in FSGS has not been explored. METHODS: We studied the role of the M-Sec-TNT system in cultured podocytes exposed to Adriamycin and in BALB/c M-Sec knockout mice. We also assessed M-Sec expression in both kidney biopsies from patients with FSGS and in experimental FSGS (Adriamycin-induced nephropathy). RESULTS: Podocytes can form TNTs in a M-Sec-dependent manner. Consistent with the notion that the M-Sec-TNT system is cytoprotective, podocytes overexpressed M-Sec in both human and experimental FSGS. Moreover, M-Sec deletion resulted in podocyte injury, with mitochondrial abnormalities and development of progressive FSGS. In vitro, M-Sec deletion abolished TNT-mediated mitochondria transfer between podocytes and altered mitochondrial bioenergetics. Re-expression of M-Sec reestablishes TNT formation and mitochondria exchange, rescued mitochondrial function, and partially reverted podocyte injury. CONCLUSIONS: These findings indicate that the M-Sec-TNT system plays an important protective role in the glomeruli by rescuing podocytes via mitochondrial horizontal transfer. M-Sec may represent a promising therapeutic target in FSGS, and evidence that podocytes can be rescued via TNT-mediated horizontal transfer may open new avenues of research.


Assuntos
Glomerulosclerose Segmentar e Focal/metabolismo , Podócitos/metabolismo , Fatores de Necrose Tumoral/metabolismo , Idoso , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Doxorrubicina , Feminino , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Nanotubos , Podócitos/patologia
10.
J Exp Clin Cancer Res ; 38(1): 496, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31847869

RESUMO

In the original publication of this article [1], the images of Figs. 4 and 5 were exchanged and the legends of the two figures did not correspond due to a typesetting error.

11.
J Exp Clin Cancer Res ; 38(1): 436, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31665044

RESUMO

BACKGROUND: All-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied. METHODS: We used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and -resistant cell-lines. Bio-computing approaches were used to analyse the high-throughput lipidomic and transcriptomic data. RESULTS: ATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative-phosphorylation. ATRA reduces the amounts of cardiolipins and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells. CONCLUSIONS: The observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria.


Assuntos
Neoplasias da Mama/metabolismo , Cardiolipinas/metabolismo , Perfilação da Expressão Gênica/métodos , Mitocôndrias/metabolismo , Tretinoína/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lipidômica/métodos , Espectrometria de Massas , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Análise de Célula Única , Sequenciamento Completo do Exoma
12.
J Neuroinflammation ; 16(1): 9, 2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651101

RESUMO

BACKGROUND: Phagocytosis is a key function of myeloid cells and is highly involved in brain ischemic injury. It has been scarcely studied in vivo, thus preventing a deep knowledge of the processes occurring in the ischemic environment. Structured illumination microscopy (SIM) is a superresolution technique which helps study phagocytosis, a process involving the recruitment of vesicles sized below the resolution limits of standard confocal microscopy. METHODS: Mice underwent permanent occlusion of the middle cerebral artery and were sacrificed at 48 h or 7 days after insult. Immunofluorescence for CD11b, myeloid cell membrane marker, and CD68, lysosomal marker was done in the ischemic area. Images were acquired using a SIM system and verified with SIM check. Lysosomal distribution was measured in the ischemic area by the gray level co-occurrence matrix (GLCM). SIM dataset was compared with transmission electron microscopy images of macrophages in the ischemic tissue at the same time points. Cultured microglia were stimulated with LPS to uptake 100 nm fluorescent beads and imaged by time-lapse SIM. GLCM was used to analyze bead distribution over the cytoplasm. RESULTS: SIM images reached a resolution of 130 nm and passed the quality control diagnose, ruling out possible artifacts. After ischemia, GLCM applied to the CD68 images showed that myeloid cells at 48 h had higher angular second moment (ASM), inverse difference moment (IDM), and lower entropy than myeloid cells at 7 days indicating higher lysosomal clustering at 48 h. At this time point, lysosomal clustering was proximal (< 700 nm) to the cell membrane indicating active target internalization, while at 7 days, it was perinuclear, consistent with final stages of phagocytosis or autophagy. Electron microscopy images indicated a similar pattern of lysosomal distribution thus validating the SIM dataset. GLCM on time-lapse SIM from phagocytic microglia cultures revealed a temporal decrease in ASM and IDM and increase in entropy, as beads were uptaken, indicating that GLCM informs on the progression of phagocytosis. CONCLUSIONS: GLCM analysis on SIM dataset quantitatively described different phases of macrophage phagocytic behavior revealing the dynamics of lysosomal movements in the ischemic brain indicating initial active internalization vs. final digestion/autophagy.


Assuntos
Encéfalo/diagnóstico por imagem , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/patologia , Células Mieloides/fisiologia , Imagem Óptica/métodos , Fagocitose/fisiologia , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Lipopolissacarídeos/farmacologia , Lisossomos/patologia , Lisossomos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/ultraestrutura , Microscopia Eletrônica de Transmissão , Células Mieloides/ultraestrutura , Medula Espinal/citologia , Fatores de Tempo
13.
Neuroimage ; 184: 490-495, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30240904

RESUMO

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aß) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aß plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases.


Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Imageamento Tridimensional/métodos , Neuroimagem/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Transgênicos
14.
Brain ; 141(9): 2685-2699, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30084913

RESUMO

Traumatic brain injury is a risk factor for subsequent neurodegenerative disease, including chronic traumatic encephalopathy, a tauopathy mostly associated with repetitive concussion and blast, but not well recognized as a consequence of severe traumatic brain injury. Here we show that a single severe brain trauma is associated with the emergence of widespread hyperphosphorylated tau pathology in a proportion of humans surviving late after injury. In parallel experimental studies, in a model of severe traumatic brain injury in wild-type mice, we found progressive and widespread tau pathology, replicating the findings in humans. Brain homogenates from these mice, when inoculated into the hippocampus and overlying cerebral cortex of naïve mice, induced widespread tau pathology, synaptic loss, and persistent memory deficits. These data provide evidence that experimental brain trauma induces a self-propagating tau pathology, which can be transmitted between mice, and call for future studies aimed at investigating the potential transmissibility of trauma associated tau pathology in humans.


Assuntos
Lesões Encefálicas Traumáticas/complicações , Tauopatias/etiologia , Tauopatias/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Concussão Encefálica/patologia , Lesões Encefálicas Traumáticas/fisiopatologia , Córtex Cerebral/patologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia , Fosforilação , Proteínas tau/metabolismo , Proteínas tau/fisiologia
15.
ACS Nano ; 12(7): 7292-7300, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29953205

RESUMO

Engineered nanoparticles offer the chance to improve drug transport and delivery through biological barriers, exploiting the possibility to leave the blood circulation and traverse the endothelial vascular bed, blood-brain barrier (BBB) included, to reach their target. It is known that nanoparticles gather molecules on their surface upon contact with biological fluids, forming the "protein corona", which can affect their fate and therapeutic/diagnostic performance, yet no information on the corona's evolution across the barrier has been gathered so far. Using a cellular model of the BBB and gold nanoparticles, we show that the composition of the corona undergoes dramatic quantitative and qualitative molecular modifications during passage from the "blood" to the "brain" side, while it is stable once beyond the BBB. Thus, we demonstrate that the nanoparticle corona dynamically and drastically evolves upon crossing the BBB and that its initial composition is not predictive of nanoparticle fate and performance once beyond the barrier at the target organ.


Assuntos
Barreira Hematoencefálica/metabolismo , Nanopartículas/metabolismo , Coroa de Proteína/metabolismo , Barreira Hematoencefálica/química , Células Endoteliais/química , Células Endoteliais/metabolismo , Humanos , Nanopartículas/química , Coroa de Proteína/química
16.
Hum Mol Genet ; 27(14): 2477-2489, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29718201

RESUMO

Marinesco-Sjögren syndrome (MSS) is a rare, early onset, autosomal recessive multisystem disorder characterized by cerebellar ataxia, cataracts and myopathy. Most MSS cases are caused by loss-of-function mutations in the gene encoding SIL1, a nucleotide exchange factor for the molecular chaperone BiP which is essential for correct protein folding in the endoplasmic reticulum. Woozy mice carrying a spontaneous Sil1 mutation recapitulate key pathological features of MSS, including cerebellar atrophy with degeneration of Purkinje cells and progressive myopathy. Because the PERK branch of the unfolded protein response is activated in degenerating neurons of woozy mice, and inhibiting PERK-mediated translational attenuation has shown protective effects in protein-misfolding neurodegenerative disease models, we tested the therapeutic efficacy of GSK2606414, a potent inhibitor of PERK. Mice were chronically treated with GSK2606414 starting from a presymptomatic stage, and the effects were evaluated on biochemical, histopathological and clinical readouts. GSK2606414 delayed Purkinje cell degeneration and the onset of motor deficits, prolonging the asymptomatic phase of the disease; it also reduced the skeletal muscle abnormalities and improved motor performance during the symptomatic phase. The protein but not the mRNA level of ORP150, a nucleotide exchange factor which can substitute for SIL1, was increased in the cerebellum of GSK2606414-treated woozy mice, suggesting that translational recovery promoted the synthesis of this alternative BiP co-factor. Targeting PERK signaling may have beneficial disease-modifying effects in carriers of SIL1 mutations.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Choque Térmico HSP70/genética , Degeneração Neural/genética , Degenerações Espinocerebelares/terapia , eIF-2 Quinase/genética , Adenina/administração & dosagem , Adenina/análogos & derivados , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/fisiopatologia , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Heterozigoto , Humanos , Indóis/administração & dosagem , Mutação com Perda de Função/genética , Camundongos , Atividade Motora/fisiologia , Degeneração Neural/fisiopatologia , Dobramento de Proteína , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/patologia , Degenerações Espinocerebelares/genética , Degenerações Espinocerebelares/patologia , Resposta a Proteínas não Dobradas/genética
17.
PLoS One ; 12(3): e0173512, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28282408

RESUMO

Excess of uric acid is mainly treated with xanthine oxidase (XO) inhibitors, also called uricostatics because they block the conversion of hypoxanthine and xanthine into urate. Normally, accumulation of upstream metabolites is prevented by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The recycling pathway, however, is impaired in the presence of HPRT deficiency, as observed in Lesch-Nyhan disease. To gain insights into the consequences of purine accumulation with HPRT deficiency, we investigated the effects of the XO inhibitor allopurinol in Hprt-lacking (HPRT-/-) mice. Allopurinol was administered in the drinking water of E12-E14 pregnant mothers at dosages of 150 or 75 µg/ml, and mice sacrificed after weaning. The drug was well tolerated by wild-type animals and heterozygous HPRT+/- mice. Instead, a profound alteration of the renal function was observed in the HPRT-/- model. Increased hypoxanthine and xanthine concentrations were found in the blood. The kidneys showed a yellowish appearance, diffuse interstitial nephritis, with dilated tubules, inflammatory and fibrotic changes of the interstitium. There were numerous xanthine tubular crystals, as determined by HPLC analysis. Oil red O staining demonstrated lipid accumulation in the same location of xanthine deposits. mRNA analysis showed increased expression of adipogenesis-related molecules as well as profibrotic and proinflammatory pathways. Immunostaining showed numerous monocyte-macrophages and overexpression of alpha-smooth muscle actin in the tubulointerstitium. In vitro, addition of xanthine to tubular cells caused diffuse oil red O positivity and modification of the cell phenotype, with loss of epithelial features and appearance of mesenchymal characteristics, similarly to what was observed in vivo. Our results indicate that in the absence of HPRT, blockade of XO by allopurinol causes rapidly developing renal failure due to xanthine deposition within the mouse kidney. Xanthine seems to be directly involved in promoting lipid accumulation and subsequent phenotype changes of tubular cells, with activation of inflammation and fibrosis.


Assuntos
Alopurinol/farmacologia , Síndrome de Lesch-Nyhan/tratamento farmacológico , Nefrite/tratamento farmacológico , Xantina Oxidase/antagonistas & inibidores , Xantina/metabolismo , Animais , Hipoxantina Fosforribosiltransferase/genética , Síndrome de Lesch-Nyhan/genética , Síndrome de Lesch-Nyhan/metabolismo , Síndrome de Lesch-Nyhan/patologia , Camundongos , Camundongos Knockout , Nefrite/genética , Nefrite/metabolismo , Nefrite/patologia , Xantina Oxidase/genética , Xantina Oxidase/metabolismo
18.
J Alzheimers Dis ; 57(3): 857-871, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28282805

RESUMO

The 24-residue peptide humanin (HN) has been proposed as a peptide-based inhibitor able to interact directly with amyloid-ß (Aß) oligomers and interfere with the formation and/or biological properties of toxic Aß species. When administered exogenously, HN, or its synthetic S14G-derivative (HNG), exerted multiple cytoprotective effects, counteracting the Aß-induced toxicity. Whether these peptides interact directly with Aß, particularly with the soluble oligomeric assemblies, remains largely unknown. We here investigated the ability of HN and HNG to interact directly with highly aggregating Aß42, and interfere with the formation and toxicity of its oligomers. Experiments were run in cell-free conditions and in vivo in a transgenic C. elegans strain in which the Aß toxicity was specifically due to oligomeric species. Thioflavin-T assay indicated that both HN and HNG delay the formation and reduce the final amount of Aß42 fibrils. In vitro surface plasmon resonance studies indicated that they interact with Aß42 oligomers favoring the formation of amorphous larger assemblies, observed with turbidity and electron microscopy. In vivo studies indicated that both HN and HNG decrease the relative abundance of A11-positive prefibrillar oligomers as well as OC-positive fibrillar oligomers and had similar protective effects. However, while HN possibly decreased the oligomers by promoting their assembly into larger aggregates, the reduction of oligomers caused by HNG can be ascribed to a marked decrease of the total Aß levels, likely the consequence of the HNG-induced overexpression of the Aß-degrading enzyme neprilysin. These findings provide information on the mechanisms underlying the anti-oligomeric effects of HN and HNG and illustrate the role of S14G substitution in regulating the in vivo mechanism of action.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/uso terapêutico , Paralisia/induzido quimicamente , Paralisia/tratamento farmacológico , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dicroísmo Circular/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Neprilisina/genética , Neprilisina/metabolismo , Paralisia/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/ultraestrutura , Ressonância de Plasmônio de Superfície
19.
Front Aging Neurosci ; 8: 146, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27445790

RESUMO

Frontotemporal lobar degeneration (FTLD) is a severe neurodegenerative disorder that is diagnosed with increasing frequency in clinical setting. Currently, no therapy is available and in addition the molecular basis of the disease are far from being elucidated. Consequently, it is of pivotal importance to develop reliable and cost-effective in vitro models for basic research purposes and drug screening. To this respect, recent results in the field of Alzheimer's disease have suggested that a tridimensional (3D) environment is an added value to better model key pathologic features of the disease. Here, we have tried to add complexity to the 3D cell culturing concept by using a microfluidic bioreactor, where cells are cultured under a continuous flow of medium, thus mimicking the interstitial fluid movement that actually perfuses the body tissues, including the brain. We have implemented this model using a neuronal-like cell line (SH-SY5Y), a widely exploited cell model for neurodegenerative disorders that shows some basic features relevant for FTLD modeling, such as the release of the FTLD-related protein progranulin (PRGN) in specific vesicles (exosomes). We have efficiently seeded the cells on 3D scaffolds, optimized a disease-relevant oxidative stress experiment (by targeting mitochondrial function that is one of the possible FTLD-involved pathological mechanisms) and evaluated cell metabolic activity in dynamic culture in comparison to static conditions, finding that SH-SY5Y cells cultured in 3D scaffold are susceptible to the oxidative damage triggered by a mitochondrial-targeting toxin (6-OHDA) and that the same cells cultured in dynamic conditions kept their basic capacity to secrete PRGN in exosomes once recovered from the bioreactor and plated in standard 2D conditions. We think that a further improvement of our microfluidic system may help in providing a full device where assessing basic FTLD-related features (including PRGN dynamic secretion) that may be useful for monitoring disease progression over time or evaluating therapeutic interventions.

20.
Diabetologia ; 59(7): 1542-1548, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27121168

RESUMO

AIMS/HYPOTHESIS: We investigated the significance of microangiopathy in the development of foot ulcer, which is still disputed. METHODS: We assessed microangiopathy by histological analysis of the capillary ultrastructure using transmission electron microscopy and capillary density and arteriolar morphology in paraffin-embedded sections from the skin of type 2 diabetic patients: 30 neuroischaemic patients (Isc) revascularised with peripheral angioplasty and 30 neuropathic patients (Neu) with foot ulcer, compared with ten non-diabetic volunteers. RESULTS: In the diabetic patients, capillaries in the dermal papillary layer were fewer (-22.2%, 159 ± 43 vs 205 ± 52 mm(2) in non-diabetic volunteers, p < 0.01). They also showed detrimental remodelling, with a 2.2-fold increase in capillary basement membrane thickness (3.44 ± 1.19 vs 1.53 ± 0.34 µm in non-diabetic volunteers, p < 0.001) and a 57.7% decrease in lumen area (14.6 ± 11.1 vs 34.7 ± 27.5 µm(2), p < 0.001). No differences were observed between the diabetic Isc or Neu patients. Isc were more prone to develop arteriolar occlusion than Neu (16.8 ± 6.9% vs 6.7 ± 3.7%, respectively, p < 0.001). No patient had been amputated at 30 days and healing time was significantly longer in Isc (180 ± 120 vs 64 ± 50 days in Neu, p < 0.001). CONCLUSIONS/INTERPRETATION: Capillary microangiopathy is present in equal measure in neuroischaemic and neuropathic diabetic foot skin. The predominance of arteriolar occlusions with neuroischaemia indicated the existence of an additional 'small vessel disease' that did not affect an effective revascularisation and did not worsen the prognosis of major amputations but slowed the healing process of the neuroischaemic foot ulcer. TRIAL REGISTRATION: ClinicalTrials.gov NCT02610036.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Angiopatias Diabéticas/patologia , Úlcera do Pé/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Estudos Prospectivos
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