Determination of Cerebrospinal Fluid Proteome Variations by Isobaric Labeling Coupled with Strong Cation-Exchange Chromatography and Tandem Mass Spectrometry.

Cerebrospinal fluid (CSF) is in direct contact with the brain and represents a valuable source of mediators that reflect metabolic processes occurring in the central nervous system (CNS). In this sense, mass spectrometry (MS) methods have proven to be sensitive in quantifying the proteomic profiles of CSF, therefore being able to detect biomarker candidates for neurological disorders. In particular, a key development has been the use of multiplexing technologies to easily identify and quantify complex protein mixtures. This chapter describes a workflow suitable for the analysis of CSF proteome using isobaric labeling coupled to strong cation-exchange chromatography fractionation for its potential use as a biomarker discovery platform. In this case, the isobaric tags for relative and absolute quantitation (iTRAQ) label all proteins in a sample via free amines at the N-terminus and on the side chain of lysine residues. Then, the labeled samples are pooled and chromatographically fractionated. These fractions with the pooled samples are afterward analyzed by tandem mass spectrometry (MS/MS), and proteins are quantified by the relative intensities of the reporter ions in the MS/MS spectra, simultaneously obtaining the amino acid sequence. This method complements the neuroproteomic toolbox to identify new protein biomarkers not only for the early clinical diagnosis and disease staging of CNS-related disorders but also to elucidate the molecular mechanisms related to the pathophysiology of these symptoms.

Monitoring the Cerebrospinal Fluid Cytokine Profile Using Membrane-Based Antibody Arrays.

The brain is the most complex organ of the human body, and the study of the different diseases and injuries that affect it is far behind the ones that affect other organs. Some of these pathologies such as neurodegenerative diseases, physical injuries, and cancer present an important alteration in its inflammatory component, which affects their outcome in a positive or negative way. For this reason, it is important to characterize the joint expression of the cytokines and growth factors (GF) that are part of this inflammatory component. The cerebrospinal fluid (CSF) is in direct contact with the brain and spinal cord, being the best biofluid to study the cytokine and GF secretion patterns of these conditions. Currently, the proteomic workflows based on mass spectrometry (MS) are unable to easily detect these proteins in CSF. In this chapter, we describe a method based on cytokine membrane arrays to characterize, in a straightforward way, the secretion profile of different cytokines and GF at once in CSF.

Deployment of Label-Free Quantitative Olfactory Proteomics to Detect Cerebrospinal Fluid Biomarker Candidates in Synucleinopathies.

Nowadays, diagnosis of neurodegenerative disorders is mainly based on neuroimaging and clinical symptoms, although postmortem neuropathological confirmation remains the gold standard diagnostic technique. Therefore, cerebrospinal fluid (CSF) proteome is considered a valuable molecular repository for diagnosing and targeting the neurodegenerative process. It is well known that olfactory dysfunction is among the earliest features of synucleinopathies such as Parkinson's disease (PD). Consequently, we consider that the application of tissue proteomics in primary olfactory structures is an ideal approach to explore early pathophysiological changes, detecting olfactory proteins that might be tested in CSF as potential biomarkers. Data mining of mass spectrometry-generated datasets has revealed that 30% of the olfactory bulb (OB) proteome is also localized in CSF. In this chapter, we describe a method that utilizes label-free quantitative proteomics and computational analysis to characterize human OB proteomes and potential cerebrospinal fluid (CSF) biomarkers associated with neurodegenerative syndromes. For that, we applied peptide fractionation methods, followed by tandem mass spectrometry (nanoLC-MS/MS), in silico analysis, and semi-quantitative orthogonal techniques in OB derived from PD subjects. After obtaining the differential OB proteome across Lewy-type alpha-synucleinopathy (LTS) stages and further validating the method, this workflow was applied to probe changes in NEGR1 (neuronal growth regulator 1) and GNPDA2 (glucosamine-6-phosphate deaminase 2) protein levels in CSF derived from parkinsonian subjects with respect to controls, observing an inverse correlation between both proteins and α-synuclein, the principal component analysis of Lewy pathology.

Early-Onset Molecular Derangements in the Olfactory Bulb of Tg2576 Mice: Novel Insights Into the Stress-Responsive Olfactory Kinase Dynamics in Alzheimer's Disease.

The olfactory bulb (OB) is the first processing station in the olfactory pathway. Despite smell impairment, which is considered an early event in Alzheimer's disease (AD), little is known about the initial molecular disturbances that accompany the AD development at olfactory level. We have interrogated the time-dependent OB molecular landscape in Tg2576 AD mice prior to the appearance of neuropathological amyloid plaques (2-, and 6-month-old), using combinatorial omics analysis. The metabolic modulation induced by overproduction of human mutated amyloid precursor protein (APP) clearly differs between both time points. Besides the progressive perturbation of the APP interactome, functional network analysis unveiled an inverse regulation of downstream extracellular signal-regulated kinase (ERK1/2), and p38 mitogen-activated protein kinase (MAPK) routes in 2-month-old Tg2576 mice with respect to wild-type (WT) mice. In contrast, Akt and MAPK kinase 4 (SEK1)/ stress-activated protein kinase (SAPK) axis were parallel activated in the OB of 6-months-old-Tg2576 mice. Furthermore, a survival kinome profiling performed during the aging process (2-, 6-, and 18-month-old) revealed that olfactory APP overexpression leads to changes in the activation dynamics of protein kinase A (PKA), and SEK1/MKK4-SAPK/JNK between 6 and 18 months of age, when memory deficits appear and AD pathology is well established in transgenic mice. Interestingly, both olfactory pathways were differentially activated in a stage-dependent manner in human sporadic AD subjects with different neuropathological grading. Taken together, our data reflect the early impact of mutated APP on the OB molecular homeostasis, highlighting the progressive modulation of specific signaling pathways during the olfactory amyloidogenic pathology.

The olfactory bulb proteotype differs across frontotemporal dementia spectrum.

Mild olfactory dysfunction has been observed in frontotemporal dementias (FTD). However, the underlying molecular mechanisms associated to this deficit are poorly understood. We applied quantitative proteomics to analyze pathological effects on the olfactory bulb (OB) from progressive supranuclear palsy (PSP) and frontotemporal lobar degeneration (FTLD-TDP43) subjects respect to elderly non-FTD group. Our data revealed: i) a mitochondrial and calcium homeostasis impairment in PSP and ii) a disruption of protein synthesis and vesicle trafficking in FTLD-TDP43. Although differential OB proteomes clearly differ between both FTD phenotypes, functional analyses pointed out an imbalance in survival signaling in both pathologies. A common alteration of olfactory mitogen-activated protein kinases (MAPKs), calcium/calmodulin dependent protein kinase II (CAMKII), and protein kinase C (PKC) signaling pathways was observed in PSP and FTLD subjects. In contrast, a specific shut off in mitogen-activated protein kinase kinase 4 (SEK1/MKK4)/stress-activated protein kinase (SAPK) axis was exclusively observed in PSP, whereas a specific phosphoinositide-dependent protein kinase 1 (PDK1) inactivation was observed in FTLD-TDP43. In summary, our data contribute to a better understanding of the molecular mechanisms that are modulated in PSP and FTLD-TDP43 at olfactory level, highlighting cross-disease similarities and differences in the regulation of survival pathways across FTD spectrum. SIGNIFICANCE: This work reflects differential olfactory molecular disarrangements in PSP and FTLD-TDP43, two clinically similar FTD disorders, but with different neuropathological signature. Besides FTDs present mild olfactory dysfunction, our data provide basic information for understanding the implication of the OB in the pathophysiology of FTDs.

Unveiling the olfactory proteostatic disarrangement in Parkinson's disease by proteome-wide profiling.

Olfactory dysfunction is one of the earliest features in Lewy-type alpha-synucleinopathies (LTSs) such as Parkinson's disease (PD). However, the underlying molecular mechanisms associated to smell impairment are poorly understood. Applying mass spectrometry-based quantitative proteomics in postmortem olfactory bulbs across limbic, early-neocortical, and neocortical LTS stages of parkinsonian patients, a proteostasis impairment, was observed, identifying 268 differentially expressed proteins between controls and PD phenotypes. In addition, network-driven proteomics revealed a modulation in ERK1/2, MKK3/6, and PDK1/PKC signaling axes. Moreover, a cross-disease study of selected olfactory molecules in sporadic Alzheimer's disease (AD) cases revealed different protein derangements in the modulation of secretagogin (SCGN), calcyclin-binding protein (CACYBP), and glucosamine 6 phosphate isomerase 2 (GNPDA2) between PD and AD. An inverse correlation between GNPDA2 and α-synuclein protein levels was also reflected in PD cerebrospinal fluid. Interestingly, PD patients exhibited significantly lower serum GNPDA2 levels than controls (n = 82/group). Our study provides important avenues for understanding the olfactory bulb proteostasis imbalance in PD, deciphering mechanistic clues to the equivalent smell deficits observed in AD and PD pathologies.

Oncolytic adenovirus Delta-24-RGD induces a widespread glioma proteotype remodeling during autophagy.

Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. SIGNIFICANCE: The current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma.

Spatial and temporal proteome dynamics of glioma cells during oncolytic adenovirus Delta-24-RGD infection.

Glioblastoma multiforme (GBM) is the most common and aggressive type of malignant glioma. Oncolytic adenoviruses are being modified to exploit the aberrant expression of proteins in tumor cells to increase the antiglioma efficacy. E1A mutant adenovirus Delta-24-RGD (DNX-2401) has shown a favorable toxicity profile and remarkable efficacy in a first-in-human phase I clinical trial. However, the comprehensive modulation of glioma metabolism in response to Delta-24-RGD infection is poorly understood. Integrating mass spectrometry based-quantitative proteomics, physical and functional interaction data, and biochemical approaches, we conducted a cell-wide study of cytosolic, nuclear, and secreted glioma proteomes throughout the early time course of Delta-24-RGD infection. In addition to the severe proteostasis impairment detected during the first hours post-infection (hpi), Delta-24-RGD induces a transient inhibition of signal transducer and activator of transcription 3 (STAT3), and transcription factor AP-1 (c-JUN) between 3 and 10hpi, increasing the nuclear factor kappa B (NF-κB) activity at 6hpi. Furthermore, Delta-24-RGD specifically modulates the activation dynamics of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) pathways early in infection. At extracellular level, Delta-24-RGD triggers a time -dependent dynamic production of multitasking cytokines, and chemotactic factors, suggesting potential pleiotropic effects on the immune system reactivation. Taken together, these data help us to understand the mechanisms used by Delta-24-RGD to exploit glioma proteome organization. Further mining of this proteomic resource may enable design and engineering complementary adenoviral based-vectors to increase the specificity and potency against glioma.

Network-Driven Proteogenomics Unveils an Aging-Related Imbalance in the Olfactory IκBα-NFκB p65 Complex Functionality in Tg2576 Alzheimer's Disease Mouse Model.

Olfaction is often deregulated in Alzheimer's disease (AD) patients, and is also impaired in transgenic Tg2576 AD mice, which overexpress the Swedish mutated form of human amyloid precursor protein (APP). However, little is known about the molecular mechanisms that accompany the neurodegeneration of olfactory structures in aged Tg2576 mice. For that, we have applied proteome- and transcriptome-wide approaches to probe molecular disturbances in the olfactory bulb (OB) dissected from aged Tg2576 mice (18 months of age) as compared to those of age matched wild-type (WT) littermates. Some over-represented biological functions were directly relevant to neuronal homeostasis and processes of learning, cognition, and behavior. In addition to the modulation of CAMP responsive element binding protein 1 (CREB1) and APP interactomes, an imbalance in the functionality of the IκBα-NFκB p65 complex was observed during the aging process in the OB of Tg2576 mice. At two months of age, the phosphorylated isoforms of olfactory IκBα and NFκB p65 were inversely regulated in transgenic mice. However, both phosphorylated proteins were increased at 6 months of age, while a specific drop in IκBα levels was detected in 18-month-old Tg2576 mice, suggesting a transient activation of NFκB in the OB of Tg2576 mice. Taken together, our data provide a metabolic map of olfactory alterations in aged Tg2576 mice, reflecting the progressive effect of APP overproduction and ß-amyloid (Aß) accumulation on the OB homeostasis in aged stages.

Olfactory bulb neuroproteomics reveals a chronological perturbation of survival routes and a disruption of prohibitin complex during Alzheimer's disease progression.

Olfactory dysfunction is among the earliest features of Alzheimer's disease (AD). Although neuropathological abnormalities have been detected in the olfactory bulb (OB), little is known about its dynamic biology. Here, OB- proteome analysis showed a stage-dependent synaptic proteostasis impairment during AD evolution. In addition to progressive modulation of tau and amyloid precursor protein (APP) interactomes, network-driven proteomics revealed an early disruption of upstream and downstream p38 MAPK pathway and a subsequent impairment of Phosphoinositide-dependent protein kinase 1 (PDK1)/Protein kinase C (PKC) signaling axis in the OB from AD subjects. Moreover, a mitochondrial imbalance was evidenced by a depletion of Prohibitin-2 (Phb2) levels and a specific decrease in the phosphorylated isoforms of Phb1 in intermediate and advanced AD stages. Interestingly, olfactory Phb subunits were also deregulated across different types of dementia. Phb2 showed a specific up-regulation in mixed dementia, while Phb1 isoforms were down-regulated in frontotemporal lobar degeneration (FTLD). However, no differences were observed in the olfactory expression of Phb subunits in progressive supranuclear palsy (PSP). To sum up, our data reflect, in part, the missing links in the biochemical understanding of olfactory dysfunction in AD, unveiling Phb complex as a differential driver of neurodegeneration at olfactory level.

An early dysregulation of FAK and MEK/ERK signaling pathways precedes the ß-amyloid deposition in the olfactory bulb of APP/PS1 mouse model of Alzheimer's disease.

UNLABELLED: Olfactory dysfunction is an early event of Alzheimer's disease (AD). However, the mechanisms associated to AD neurodegeneration in olfactory areas are unknown. Here we used double-transgenic amyloid precursor protein/presenilin 1 (APPswe/PS1dE9) mice and label-free quantitative proteomics to analyze early pathological effects on the olfactory bulb (OB) during AD progression. Prior to ß-amyloid plaque formation, 9 modulated proteins were detected on 3-month-old APP/PS1 mice while 16 differential expressed proteins were detected at 6months, when ß-amyloid plaques appear, indicating a moderate imbalance in cytoskeletal rearrangement, and synaptic plasticity in APP/PS1 OBs. Moreover, ß-amyloid induced an inactivation of focal adhesion kinase (FAK) together with a transient activation of MEK1/2, leading to inactivation of ERK1/2 in 6-months APP/PS1 OBs. In contrast, the analysis of human OBs revealed a late activation of FAK in advanced AD stages, whereas ERK1/2 activation was enhanced across AD staging respect to controls. This survival potential was accompanied by the inhibition of the proapototic factor BAD in the OB across AD phenotypes. Our data contribute to a better understanding of the early molecular mechanisms that are modulated in AD neurodegeneration, highlighting significant differences in the regulation of survival pathways between APP/PS1 mice and sporadic human AD. SIGNIFICANCE: Loss of smell is involved in early stages of Alzheimer's disease (AD), usually preceding classic disease symptoms. However, the mechanisms governing this dysfunction are still poorly understood, losing its potential as a useful tool for clinical diagnosis. Our study characterizes potential AD-associated molecular changes in APP/PS1 mice olfactory bulb (OB) using MS-quantitative proteomics, revealing early cytoskeletal disruption and synaptic plasticity impairment. Moreover, an opposite pattern was found when comparing the activation status of specific survival pathways between APP/PS1 OBs and OBs derived from sAD subjects with different neuropathological grading. Our data reflect, in part, the progressive effect of APP overproduction and Aß accumulation on the OB proteome during AD progression.

Factores asociados a letalidad de intentos de suicidio en sujetos con trastorno depresivo mayor / Factors associated with suicide attempts lethality in subjects with mayor depressive disorder

Objetivo: Identificar en una muestra de población antioqueña con trastorno depresivo mayor y antecedente de conducta suicida las características que se asocian a intentos de suicidio de alta letalidad. Método: Se evaluaron 140 sujetos con diagnóstico de trastorno depresivo mayor y antecedente de, al menos, un intento de suicidio, por medio de una entrevista diagnóstica semiestructurada. De acuerdo con las características del intento de suicidio más serio, los sujetos se dividieron en grupos de menor letalidad (letalidad leve y moderada) y mayor letalidad (letalidad alta y extrema), los cuales se compararon en variables sociodemográficas y clínicas. Resultados: Los sujetos con intentos de suicidio de letalidad alta y extrema con mayor frecuencia vivían solos, tenían antecedente de más de un intento de suicidio y presentaban dependencia a nicotina y a sustancias psicoactivas diferentes al alcohol. En el análisis multivariado se estableció que vivir solo y la dependencia a sustancias psicoactivas se asociaron de manera independiente con los intentos de suicidios de letalidad alta y extrema. Conclusiones: Los hallazgos del presente estudio se corresponden con las características clínicas descritas en estudios previos en asociación con intentos de suicidio de alta letalidad. Los presentes resultados se proponen como factores para tener en cuenta en la valoración del riesgo suicida en sujetos con trastorno depresivo mayor...

Objective: To identify the characteristics associated with high-lethality suicide attempts in a population sample IN Antioquia with major depressive disorder and suicidal behavior history. Method: A semi-structured diagnostic interview was used to assess 140 subjects diagnosed with major depressive disorder and who had made at least one suicide attempt. According to the characteristics of the most serious suicide attempt, the subjects were classified into two groups—low-lethality suicide attempters (low and moderate lethality) and high-lethality suicide attempters (high and very high lethality)—which were compared for clinical and demographic variables. Results: It was found that high- and very high-lethality suicide attempters lived alone more often, had made more than one suicide attempt, and exhibited greater dependence on nicotine and psychoactive substances, other than alcohol, than low-lethality attempters. The multivariate analysis established that living alone and being dependent on psychoactive substances were independently associated with very high- and high-lethality attempts. Conclusion: These findings confirm the clinical characteristics associated with high-lethality suicide attempts described in previous studies. The present results are proposed as factors to be taken into account for the assessment of suicidal risk in patients with major depressive disorder...

Evaluación de los programas de control prenatal para adolescentes en el valle de Aburrá 2003 / Evaluation of Prenatal Control Programs offered to teenagers in Valle de Aburrá, Colombia, 2003

Objetivo: La investigación que se presenta a continuación tuvo como objetivo identificar y evaluar los programas de control prenatal para adolescentes ofrecidos en el valle de Aburrá, Antioquia, Colombia, durante el año 2003.Materiales y métodos: Se revisaron varias fuentes primarias y se elaboró un listado de todas las instituciones de salud del valle de Aburrá. Se hicieron entrevistas telefónicas con los respectivos directores para establecer cuáles instituciones tenían un programa de control prenatal exclusivo para adolescentes y a estas instituciones se les aplicó un formato de evaluación para determinar si cumplían con los estándares internacionales descritos en la literatura como esenciales en un programa de control prenatal para adolescentes.Resultados: Sólo una de las 101 instituciones de salud que ofrecían control prenatal tenía un programa exclusivo para adolescentes. Sin embargo, a la luz de la evaluación, dicho programa no cumplía con los estándares de un programa de control prenatal para adolescentes.Conclusión: A pesar de que en Colombia el embarazo en adolescentes es un problema de salud pública, los programas de control prenatal para adolescentes no se han implementado en el valle de Aburrá. Establecerlos constituye una de las estrategias para resolver esta problemática.