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1.
Mol Cell Neurosci ; 125: 103843, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36935047

RESUMO

Proteins must fold into three-dimensional structures to execute their biological functions. Therefore, maintenance of protein homeostasis, proteostasis, including prevention of protein misfolding is essential for cellular activity and health. Molecular chaperones are key actors in proteostasis. BRICHOS domain is an intramolecular chaperone that also interferes with several aggregation-prone proteins including amyloid ß (Aß), involved in Alzheimer's disease (AD). To extend the knowledge about Bri2 BRICHOS interactome we here used recombinant human (rh) Bri2 BRICHOS-mCherry fusion protein to probe for potential binding partners. Firstly, exogenously added Bri2 BRICHOS-mCherry was used to stain brain sections of wildtype and amyloid precursor protein (App) knock-in AD mice exhibiting robust Aß pathology. Unexpectedly, we found that rh Bri2 BRICHOS-mCherry stained the cytoplasm of neurons which are devoid of Aß deposits. To identify these intraneuronal proteins that bind to the rh Bri2 BRICHOS domain, we performed co-immunoprecipitation (co-IP) of mouse brain hippocampi homogenates using the Bri2 BRICHOS-mCherry probe and analyzed co-IP proteins by LC-MS/MS. This identified several cytoskeletal proteins including spectrin alpha and beta chain, drebrin, tubulin ß3, and ß-actin as binding partners. The interactions were confirmed by a second round of pulldown experiments using rh Bri2 BRICHOS linked to magnetic beads. The interaction of rh Bri2 BRICHOS and tubulin ß3 was further investigated by staining both mouse brain sections and SH-SY5Y neuroblastoma cells with rh Bri2 BRICHOS-mCherry and tubulin ß3 immunostaining, which revealed partial co-localization. These data suggest a possible interplay of extracellular chaperone Bri2 BRICHOS domain in the intracellular space including the cytoskeleton.


Assuntos
Doença de Alzheimer , Neuroblastoma , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cromatografia Líquida , Proteínas do Citoesqueleto , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Espectrometria de Massas em Tandem , Tubulina (Proteína)
2.
Acta Neuropathol Commun ; 10(1): 96, 2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35787306

RESUMO

Cerebrospinal fluid (CSF) biomarkers play an important role in diagnosing Alzheimer's disease (AD) which is characterized by amyloid-ß (Aß) amyloidosis. Here, we used two App knock-in mouse models, AppNL-F/NL-F and AppNL-G-F/NL-G-F, exhibiting AD-like Aß pathology to analyze how the brain pathologies translate to CSF proteomes by label-free mass spectrometry (MS). This identified several extracellular matrix (ECM) proteins as significantly altered in App knock-in mice. Next, we compared mouse CSF proteomes with previously reported human CSF MS results acquired from patients across the AD spectrum. Intriguingly, the ECM protein decorin was similarly and significantly increased in both AppNL-F/NL-F and AppNL-G-F/NL-G-F mice, strikingly already at three months of age in the AppNL-F/NL-F mice and preclinical AD subjects having abnormal CSF-Aß42 but normal cognition. Notably, in this group of subjects, CSF-decorin levels positively correlated with CSF-Aß42 levels indicating that the change in CSF-decorin is associated with early Aß amyloidosis. Importantly, receiver operating characteristic analysis revealed that CSF-decorin can predict a specific AD subtype having innate immune activation and potential choroid plexus dysfunction in the brain. Consistently, in AppNL-F/NL-F mice, increased CSF-decorin correlated with both Aß plaque load and with decorin levels in choroid plexus. In addition, a low concentration of human Aß42 induces decorin secretion from mouse primary neurons. Interestingly, we finally identify decorin to activate neuronal autophagy through enhancing lysosomal function. Altogether, the increased CSF-decorin levels occurring at an early stage of Aß amyloidosis in the brain may reflect pathological changes in choroid plexus, present in a subtype of AD subjects.


Assuntos
Doença de Alzheimer , Amiloidose , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Amiloidose/patologia , Animais , Encéfalo/patologia , Decorina/líquido cefalorraquidiano , Decorina/metabolismo , Humanos , Camundongos , Placa Amiloide/patologia , Proteoma/metabolismo
3.
J Cachexia Sarcopenia Muscle ; 13(4): 2162-2174, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35502572

RESUMO

BACKGROUND: Critical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a by-product of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood; however, loss of mechanical stimuli contributes to critical illness-associated muscle atrophy and weakness. Passive mechanical loading and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care. METHODS: Rats were subjected to 8 days of critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM. RESULTS: Following 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, myosin : actin ratio, and single muscle fibre maximum force normalized to cross-sectional area (CSA; specific force) were reduced by 40-50% (P < 0.0001). ES significantly reduced the loss of soleus muscle fibre CSA and myosin : actin ratio by approximately 30% (P < 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signalling in the soleus muscle following critical care, and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (P < 0.01). ES promoted phosphofructokinase and insulin signalling pathways to control levels (P < 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signalling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (P = 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately two-fold (P = 0.06). Reduction of muscle protein degradation rather than increased synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (P = 0.006) and MuRF1 (P = 0.08) by approximately 50%, downstream of AMPK-FoxO3. CONCLUSIONS: ES maintained GLUT4 translocation through increased AMPK-TBC1D4 signalling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signalling, and reduced muscle protein degradation in CIM.


Assuntos
Estado Terminal , Terapia por Estimulação Elétrica , Transportador de Glucose Tipo 4 , Atrofia Muscular , Doenças Musculares , Proteínas Quinases Ativadas por AMP/metabolismo , Actinas , Animais , Estado Terminal/terapia , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glicogênio/metabolismo , Insulina/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/terapia , Doenças Musculares/etiologia , Doenças Musculares/terapia , Miosinas/metabolismo , Ratos
4.
Clin Appl Thromb Hemost ; 28: 10760296221074347, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35043712

RESUMO

Acute pulmonary embolism (PE) is a common emergency with a high morbidity and mortality. Most clinical presentations are non-specific and there is a lack of suitable biomarkers for PE. For example, the traditional D-dimer tests shows a rather high sensitivity for PE, but yet a rather low positive predictive value due to its lack of specificity. Research on novel biomarkers for PE is thus of interest to improve early diagnostics and reduce the number of unnecessary computed tomography pulmonary angiogram (CTPA) scans performed. In this study we evaluate the feasibility to use label-free quantitative proteomics to discover potential biomarkers for acute PE and to monitor changes in proteins levels in PE patients over time. Blood was collected from 8 patients with CTPA verified PE and from 8 patients presenting with same symptoms but with a negative CTPA. The samples were analyzed by liquid chromatography-mass spectrometry and thirteen protein concentrations were found to be significantly changed in PE patients compared to the CTPA negative controls. This exploratory study shows that proteomic analysis can be used to identify potential biomarkers for PE as well as to monitor changes of protein levels over time.The complement proteins play a part in PE but further studies are needed to clarify their specific role in the pathophysiological process and to look for more specific proteins.


Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Proteômica/métodos , Embolia Pulmonar/sangue , Doença Aguda , Adulto , Idoso , Biomarcadores/sangue , Angiografia por Tomografia Computadorizada/métodos , Estudos de Viabilidade , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/epidemiologia , Fatores de Risco
5.
Curr Res Transl Med ; 70(2): 103323, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34979484

RESUMO

RATIONALE: Extracellular vesicles (EVs) derived exogenously from pluripotent stem cells or endogenously from healthy human serum exert cardioprotective effects after injury. However role of endogenous EVs from myocardial infarction (MI) patients not well understood in this settings. METHODS AND RESULTS: The EVs from plasma of MI patients with preserved or reduced left ventricular ejection fraction (LVEF) and healthy controls (HC) were purified and characterized by flow cytometry, mass spectrometry (MS) and transmission electron microscopy (TEM). HCM and human cardiac microvascular endothelial cells (hCMVECs), under individual culture or co-culture, were used to study functional effects of EVs upon TNFα stimulation. These effects of EVs on HCM and hCMVECs were observed using cell death assays, western blots and confocal microscopy. Higher concentrations of platelet-, leukocyte-, endothelial- and erythrocyte-derived EVs were found in MI patients, both with preserved and reduced LVEF, compared to HC, and MS data on MI EVs proteome displayed alteration in several proteins. MI EVs protected HCM and hCMVECs against staurosporine-induced apoptosis. Furthermore, MI EVs were observed to abrogate TNFα-triggered HCM and hCMVECs death under both individually cultured and co-cultured conditions. MI EVs failed to inhibit TNFα induced hCMVECs and HCM activation when cultured individually, however co-cultured hCMVECs with HCM supported MI EVs capacity to attenuate TNFα induced cells activation. MI CD41+ EVs but not HC EVs were found to be internalized by HCM directly or migrated through hCMVECs to HCM. MI EVs indirectly restores TNFα mediated drop in mitochondrial membrane potential. CONCLUSIONS: Endogenous EVs from MI patients, regardless of severity of the MI exert cardioprotective potential upon TNFα-induced cell death. Patient-derived EVs needs to be further explored to elucidate their potential cardioprotective role during MI.


Assuntos
Vesículas Extracelulares , Infarto do Miocárdio , Animais , Apoptose , Modelos Animais de Doenças , Células Endoteliais , Humanos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Volume Sistólico , Fator de Necrose Tumoral alfa/metabolismo , Função Ventricular Esquerda
6.
Cancers (Basel) ; 13(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070587

RESUMO

Molecular screening programs for cervical cancer detect the presence of human papilloma virus (HPV) in cell material or vaginal fluids. Persistent infection with high-risk HPV is a necessary pre-requisite, but the majority of infections do not lead to pathological states. Additional biomarkers are needed to increase the specificity of the molecular tests. Here, we have investigated the possibility of detecting protein biomarkers using mass spectrometry from dried self-sampled cervico-vaginal fluid deposited on FTA cards. We found significant intra-individual correlations (p < 2.2 × 10-16), although heterogenous protein profiles were obtained between individuals. Out of 3699 proteins found in total, 169 were detected in at least 95% of the samples. Using a discovery/replication design, 18 proteins were found to be significant in the discovery cohort, with higher values in those cases compared to controls. All of these were found to also have higher levels among the cases in the replication cohort, with one protein (DEAD-Box Helicase) remaining statistically significant. Finally, a predictive 7-protein multivariate model was developed with a sensitivity and specificity of 0.90 and 0.55, respectively. Our results demonstrate that robust measurements of protein biomarkers can be obtained from self-sampled dried CVF and that these could be used to predict cervical cancer pre-stages.

7.
Sci Rep ; 11(1): 7595, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828141

RESUMO

Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer (NMIBC), and T2/T3-muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.


Assuntos
Inclusão em Parafina/métodos , Manejo de Espécimes/métodos , Fixação de Tecidos/métodos , Biomarcadores Tumorais/genética , Cromatografia Líquida/métodos , Fixadores/química , Formaldeído/química , Humanos , Proteínas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Preservação de Tecido/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/genética
8.
Vaccines (Basel) ; 9(3)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803302

RESUMO

The induction of long-lasting clinical and virological protection is needed for a successful vaccination program against the bovine respiratory syncytial virus (BRSV). In this study, calves with BRSV-specific maternally derived antibodies were vaccinated once, either with (i) a BRSV pre-fusion protein (PreF) and MontanideTM ISA61 VG (ISA61, n = 6), (ii) BRSV lacking the SH gene (ΔSHrBRSV, n = 6), (iii) a commercial vaccine (CV, n = 6), or were injected with ISA61 alone (n = 6). All calves were challenged with BRSV 92 days later and were euthanized 13 days post-infection. Based on clinical, pathological, and proteomic data, all vaccines appeared safe. Compared to the controls, PreF induced the most significant clinical and virological protection post-challenge, followed by ΔSHrBRSV and CV, whereas the protection of PreF-vaccinated calves was correlated with BRSV-specific serum immunoglobulin (Ig)G antibody responses 84 days post-vaccination, and the IgG antibody titers of ΔSHrBRSV- and CV-vaccinated calves did not differ from the controls on this day. Nevertheless, strong anamnestic BRSV- and PreF-specific IgG responses occurred in calves vaccinated with either of the vaccines, following a BRSV challenge. In conclusion, PreF and ΔSHrBRSV are two efficient one-shot candidate vaccines. By inducing a protection for at least three months, they could potentially improve the control of BRSV in calves.

9.
Mov Disord ; 36(2): 481-491, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33247616

RESUMO

OBJECTIVE: Identifying molecular changes that contribute to the onset and progression of Huntington's disease (HD) is of importance for the development and evaluation of potential therapies. METHODS: We conducted an unbiased mass-spectrometry proteomic analysis on the cerebrospinal fluid of 12 manifest HD patients (ManHD), 13 pre-manifest (preHD), and 38 controls. A biologically plausible and significant possible biomarker was validated in samples from a separate cohort of patients and controls consisting of 23 ManHD patients and 23 controls. RESULTS: In ManHD compared to preHD, 10 proteins were downregulated and 43 upregulated. Decreased levels of proenkephalin (PENK) and transthyretin were closely linked to HD symptom severity, whereas levels of 15 upregulated proteins were associated with symptom severity. The decreased PENK levels were replicated in the separate cohort where absolute quantitation was performed. CONCLUSIONS: We hypothesize that declining PENK levels reflect the degeneration of medium spiny neurons (MSNs) that produce PENK and that assays for PENK may serve as a surrogate marker for the state of MSNs in HD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Assuntos
Doença de Huntington , Biomarcadores , Progressão da Doença , Encefalinas , Humanos , Neurônios , Precursores de Proteínas , Proteômica
10.
Arterioscler Thromb Vasc Biol ; 40(9): 2293-2309, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32757648

RESUMO

OBJECTIVE: Extracellular vesicles (EVs) have the potential to act as intercellular communicators. The aims were to characterize circulating EVs in patients with pulmonary arterial hypertension (PAH) and to explore whether these EVs contribute to endothelial activation and angiogenesis. Approach and Results: Patients with PAH (n=70) and healthy controls (HC; n=20) were included in this cross-sectional study. EVs were characterized and human pulmonary endothelial cells (hPAECs) were incubated with purified EVs. Endothelial cell activity and proangiogenic markers were analyzed. Tube formation analysis was performed for hPAECs, and the involvement of PSGL-1 (P-selectin glycoprotein ligand 1) was evaluated. The numbers of CD62P+, CD144+, and CD235a EVs were higher in blood from PAH compared with HC. Thirteen proteins were differently expressed in PAH and HC EVs, where complement fragment C1q was the most significantly elevated protein (P=0.0009) in PAH EVs. Upon EVs-internalization in hPAECs, more PAH compared with HC EVs evaded lysosomes (P<0.01). As oppose to HC, PAH EVs stimulated hPAEC activation and induced transcription and translation of VEGF-A (vascular endothelial growth factor A; P<0.05) and FGF (fibroblast growth factor; P<0.005) which were released in the cell supernatant. These proangiogenic proteins were higher in patient with PAH plasma compered with HC. PAH EVs induced a complex network of angiotubes in vitro, which was abolished by inhibitory PSGL-1antibody. Anti-PSGL-1 also inhibited EV-induced endothelial cell activation and PAH EV dependent increase of VEGF-A. CONCLUSIONS: Patients with PAH have higher levels of EVs harboring increased amounts of angiogenic proteins, which induce activation of hPAECs and in vitro angiogenesis. These effects were partly because of platelet-derived EVs evasion of lysosomes upon internalization within hPAEC and through possible involvement of P-selectin-PSGL-1 pathway.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Neovascularização Fisiológica , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Idoso , Estudos de Casos e Controles , Células Cultivadas , Estudos Transversais , Células Endoteliais/ultraestrutura , Endotélio Vascular/fisiopatologia , Endotélio Vascular/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Selectina-P/metabolismo , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/ultraestrutura , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
11.
J Pain ; 21(9-10): 1075-1084, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32553624

RESUMO

The main cause of trigeminal neuralgia (TN) is compression of a blood vessel at the root entry zone of the trigeminal nerve. However, a neurovascular conflict does not seem to be the only etiology and other mechanisms are implicated in the development of the disease. We hypothesized that TN patients may have distinct protein expression in the CSF. In this study, lumbar CSF from TN patients (n = 17), scheduled to undergo microvascular decompression, and from controls (n = 20) was analyzed and compared with in depth mass spectrometry TMTbased quantitative proteomics. We identified 2552 unique proteins, of which 46 were significantly altered (26 increased, and 20 decreased, q-value < .05) in TN patients compared with controls. An over-representation analysis showed proteins involved in high-density lipoprotein, such as Apolipoprotein A4, Apolipoprotein M, and Apolipoprotein A1, and the extracellular region, including proteins involved in the complement cascade to be over-represented. We conclude that TN patients have distinct protein expression in the CSF compared to controls. The pathophysiological background of the protein alterations found in this study warrants further investigation in future studies. PERSPECTIVE: In this article, cerebrospinal fluid from patients with trigeminal neuralgia was analyzed using in depth shotgun proteomics, revealing 46 differentially expressed proteins compared to controls. Among these, apolipoproteins and proteins involved in the complement system were elevated and significantly over-represented, implying an inflammatory component in the pathophysiology of the disease.


Assuntos
Apolipoproteínas/líquido cefalorraquidiano , Proteínas do Sistema Complemento/líquido cefalorraquidiano , Proteômica/métodos , Neuralgia do Trigêmeo/líquido cefalorraquidiano , Idoso , Apolipoproteínas/genética , Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida/métodos , Estudos de Coortes , Proteínas do Sistema Complemento/genética , Feminino , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Mapas de Interação de Proteínas/genética , Neuralgia do Trigêmeo/diagnóstico , Neuralgia do Trigêmeo/genética
12.
Vaccines (Basel) ; 8(2)2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32443437

RESUMO

Achieving safe and protective vaccination against respiratory syncytial virus (RSV) in infants and in calves has proven a challenging task. The design of recombinant antigens with a conformation close to their native form in virus particles is a major breakthrough. We compared two subunit vaccines, the bovine RSV (BRSV) pre-fusion F (preF) alone or with nanorings formed by the RSV nucleoprotein (preF+N). PreF and N proteins are potent antigenic targets for neutralizing antibodies and T cell responses, respectively. To tackle the challenges of neonatal immunization, three groups of six one-month-old calves with maternally derived serum antibodies (MDA) to BRSV received a single intramuscular injection of PreF, preF+N with MontanideTM ISA61 VG (ISA61) as adjuvant or only ISA61 (control). One month later, all calves were challenged with BRSV and monitored for virus replication in the upper respiratory tract and for clinical signs of disease over one week, and then post-mortem examinations of their lungs were performed. Both preF and preF+N vaccines afforded safe, clinical, and virological protection against BRSV, with little difference between the two subunit vaccines. Analysis of immune parameters pointed to neutralizing antibodies and antibodies to preF as being significant correlates of protection. Thus, a single shot vaccination with preF appears sufficient to reduce the burden of BRSV disease in calves with MDA.

13.
PLoS Genet ; 15(12): e1008455, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31800589

RESUMO

SLC18B1 is a sister gene to the vesicular monoamine and acetylcholine transporters, and the only known polyamine transporter, with unknown physiological role. We reveal that Slc18b1 knock out mice has significantly reduced polyamine content in the brain providing the first evidence that Slc18b1 is functionally required for regulating polyamine levels. We found that this mouse has impaired short and long term memory in novel object recognition, radial arm maze and self-administration paradigms. We also show that Slc18b1 KO mice have altered expression of genes involved in Long Term Potentiation, plasticity, calcium signalling and synaptic functions and that expression of components of GABA and glutamate signalling are changed. We further observe a partial resistance to diazepam, manifested as significantly lowered reduction in locomotion after diazepam treatment. We suggest that removal of Slc18b1 leads to reduction of polyamine contents in neurons, resulting in reduced GABA signalling due to long-term reduction in glutamatergic signalling.


Assuntos
Encéfalo/metabolismo , Proteínas de Transporte de Cátions/genética , Memória de Longo Prazo , Memória de Curto Prazo , Poliaminas/metabolismo , Animais , Sinalização do Cálcio , Técnicas de Inativação de Genes , Ácido Glutâmico/metabolismo , Aprendizagem em Labirinto , Camundongos , Plasticidade Neuronal , Ácido gama-Aminobutírico/metabolismo
14.
J Alzheimers Dis ; 67(2): 639-651, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30614806

RESUMO

BACKGROUND: Alzheimer's disease (AD) is diagnosed based on a clinical evaluation as well as analyses of classical biomarkers: Aß42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF). Although the sensitivities and specificities of the classical biomarkers are fairly good for detection of AD, there is still a need to develop novel biochemical markers for early detection of AD. OBJECTIVE: We explored if integration of novel proteins with classical biomarkers in CSF can better discriminate AD from non-AD subjects. METHODS: We applied ELISA, mass spectrometry, and multivariate modeling to investigate classical biomarkers and the CSF proteome in subjects (n = 206) with 76 AD patients, 74 mild cognitive impairment (MCI) patients, 11 frontotemporal dementia (FTD) patients, and 45 non-dementia controls. The MCI patients were followed for 4-9 years and 21 of these converted to AD, whereas 53 remained stable. RESULTS: By combining classical CSF biomarkers with twelve novel markers, the area of the ROC curves (AUROCS) of distinguishing AD and MCI/AD converters from non-AD were 93% and 96%, respectively. The FTDs and non-dementia controls were identified versus all other groups with AUROCS of 96% and 87%, respectively. CONCLUSIONS: Integration of new and classical CSF biomarkers in a model-based approach can improve the identification of AD, FTD, and non-dementia control subjects.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/diagnóstico , Diagnóstico Diferencial , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Demência Frontotemporal/líquido cefalorraquidiano , Demência Frontotemporal/diagnóstico , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteoma , Sensibilidade e Especificidade , Proteínas tau/líquido cefalorraquidiano
15.
J Mol Cell Biol ; 11(5): 356-370, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29868881

RESUMO

The muscle RING-finger protein-1 (MuRF1) is an E3 ubiquitin ligase expressed in skeletal and cardiac muscle tissues and it plays important roles in muscle remodeling. Upregulation of MuRF1 gene transcription participates in skeletal muscle atrophy, on contrary downregulation of protein expression leads to cardiac hypertrophy. MuRF1 gene point mutations have been found to generate protein aggregate myopathies defined as muscle disorder characterized by protein accumulation in muscle fibers. We have discovered that MuRF1 turned out to be also a target for a new post-translational modification arbitrated by conjugation of SUMO1 and it is mediated by the SUMO ligases E2 UBC9 and the E3 PIASγ/4. SUMOylation takes place at lysine 238 localized at the second coiled-coil protein domain that is required for efficient substrate interaction for polyubiquitination. We provided evidence that SUMOylation is essential for MuRF1 nuclear translocation and its mitochondria accumulation is enhanced in hyperglycemic conditions delivering a stabilization of the overall SUMOylated proteins in cultured myocytes. Thus, our findings add this SUMO1 post-translational modification as a new concept to understand muscle disorders related to the defect in MuRF1 activity.


Assuntos
Proteínas Musculares/metabolismo , Proteína SUMO-1/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Glucose/farmacologia , Humanos , Lisina/química , Lisina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células Musculares/citologia , Células Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/química , Proteína SUMO-1/genética , Especificidade por Substrato , Sumoilação/efeitos dos fármacos , Proteínas com Motivo Tripartido/química , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
16.
Sci Rep ; 8(1): 6807, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29717219

RESUMO

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.


Assuntos
Lesões Encefálicas Traumáticas/genética , Encéfalo/metabolismo , Hidrocefalia/genética , Proteoma/genética , Adulto , Idoso , Biópsia , Encéfalo/patologia , Lesões Encefálicas Traumáticas/diagnóstico , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Estudos de Coortes , Feminino , Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/metabolismo , Humanos , Hidrocefalia/diagnóstico , Hidrocefalia/metabolismo , Hidrocefalia/patologia , Pressão Intracraniana , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Neurogranina/genética , Neurogranina/isolamento & purificação , Neurogranina/metabolismo , Peroxirredoxina III/genética , Peroxirredoxina III/isolamento & purificação , Peroxirredoxina III/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/isolamento & purificação , Peroxirredoxinas/metabolismo , Proteoma/classificação , Proteoma/isolamento & purificação , Proteoma/metabolismo , Proteômica/métodos , Índices de Gravidade do Trauma , Proteínas tau/genética , Proteínas tau/isolamento & purificação , Proteínas tau/metabolismo
17.
J Alzheimers Dis ; 60(1): 305-321, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28826183

RESUMO

Extracellular vesicles (EVs), including exosomes and larger microvesicles, have been implicated to play a role in several conditions, including Alzheimer's disease (AD). Since the EV content mirrors the intracellular environment, it could contribute with important information about ongoing pathological processes and may be a useful source for biomarkers, reflecting the disease progression. The aim of the present study was to analyze the protein content of EVs specifically released from a mixed co-culture of primary astrocytes, neurons, and oligodendrocytes treated with synthetic amyloid-ß (Aß42) protofibrils. The EV isolation was performed by ultracentrifugation and validated by transmission electron microscopy. Mass spectrometry analysis of the EV content revealed a total of 807 unique proteins, of which five displayed altered levels in Aß42 protofibril exposed cultures. The most prominent protein was apolipoprotein E (apoE), and by western blot analysis we could confirm a threefold increase of apoE in EVs from Aß42 protofibril exposed cells, compared to unexposed cells. Moreover, immunoprecipitation studies demonstrated that apoE was primarily situated inside the EVs, whereas immunocytochemistry indicated that the EVs most likely derived from the astrocytes and the neurons in the culture. The identified Aß-induced sorting of apoE into EVs from cultured neuroglial cells suggests a possible role for intercellular transfer of apoE in AD pathology and encourage future studies to fully elucidate the clinical relevance of this event.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Apolipoproteínas E/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Neuroglia/citologia , Neurônios/citologia , Fragmentos de Peptídeos/farmacologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Contagem de Células , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Vesículas Extracelulares/ultraestrutura , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Proteínas/metabolismo , Fatores de Tempo , Tubulina (Proteína)/metabolismo
18.
J Alzheimers Dis ; 54(4): 1671-1686, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27636840

RESUMO

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disorder accounting for more than 50% of all dementia cases. AD neuropathology is characterized by the formation of extracellular plaques and intracellular neurofibrillary tangles consisting of aggregated amyloid-ß and tau, respectively. The disease mechanism has only been partially elucidated and is believed to also involve many other proteins. OBJECTIVE: This study intended to perform a proteomic profiling of post mortem AD brains and compare it with control brains as well as brains from other neurological diseases to gain insight into the disease pathology. METHODS: Here we used label-free shotgun mass spectrometry to analyze temporal neocortex samples from AD, other neurological disorders, and non-demented controls, in order to identify additional proteins that are altered in AD. The mass spectrometry results were verified by antibody suspension bead arrays. RESULTS: We found 50 proteins with altered levels between AD and control brains. The majority of these proteins were found at lower levels in AD. Pathway analyses revealed that several of the decreased proteins play a role in exocytic and endocytic pathways, whereas several of the increased proteins are related to extracellular vesicles. Using antibody-based analysis, we verified the mass spectrometry results for five representative proteins from this group of proteins (CD9, HSP72, PI42A, TALDO, and VAMP2) and GFAP, a marker for neuroinflammation. CONCLUSIONS: Several proteins involved in exo-endocytic pathways and extracellular vesicle functions display altered levels in the AD brain. We hypothesize that such changes may result in disturbed cellular clearance and a perturbed cell-to-cell communication that may contribute to neuronal dysfunction and cell death in AD.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Micropartículas Derivadas de Células/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Líquido Extracelular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , Micropartículas Derivadas de Células/genética , Micropartículas Derivadas de Células/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas/fisiologia , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismo
19.
PLoS One ; 11(3): e0150672, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26950848

RESUMO

Alzheimer's disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer's disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer's disease patients and non-demented controls to identify potential biomarkers for Alzheimer's disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer's disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p<0.05). Consequently, global normalization was found to be less accurate compared to using spiked-in chicken ovalbumin for normalization. In addition, we determined that Sieve and OpenMS resulted in the highest reproducibility and PEAKS was the programs with the highest identification performance. Finally, we successfully verified significantly lower levels (p<0.05) of eight proteins (A2GL, APOM, C1QB, C1QC, C1S, FBLN3, PTPRZ, and SEZ6) in Alzheimer's disease compared to controls using an antibody-based detection method. These proteins are involved in different biological roles spanning from cell adhesion and migration, to regulation of the synapse and the immune system.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Proteômica , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Estudos de Casos e Controles , Humanos , Masculino , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes , Software
20.
Biochim Biophys Acta ; 1854(7): 705-17, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25680928

RESUMO

Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.


Assuntos
Espectrometria de Massas/métodos , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteômica/métodos , Animais , Biomarcadores/metabolismo , Humanos , Doenças Neurodegenerativas/diagnóstico
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