Multiple events case-control study in a prospective cohort to identify systemic, cellular, and molecular biomarkers of obesity-induced accelerated aging in 30-years-olds: the ObAGE study protocol.

BACKGROUND: Aging is characterized by a progressive loss of capacities linked to fundamental alterations/damage in multiple cellular and molecular pathways. It is the most significant risk factor for all non-communicable diseases (NCDs). Another contributing factor to the rise in NCDs is obesity. It has been suggested that obesity not only accelerates the onset of metabolic imbalances but also decreases lifespan and impacts cellular and molecular processes in a manner similar to aging. Obesity might accelerate the pace of aging. Guided by a lifecourse approach, we will explore how exposure to obesity in critical developmental stages disrupt homeostatic resilience mechanisms that preserve physiological integrity, inducing an early expression of aging phenotypes. Also, we will determine whether exposure to early psychosocial adversity influences vulnerability to obesity as a risk factor for accelerated aging. METHODS: Multiple events case-control study embedded in a prospective cohort of Chileans at 30-31y, 50% females, of low- to-middle socioeconomic status, who participated in nutrition research since birth. At 23y, 25% had obesity and cardiometabolic risk was high. We will use a multi-layer approach including: anthropometric assessment; DXA scan for body composition; abdominal ultrasound of the liver; stool samples collection and sequencing of the ribosomal RNA 16S gene to characterize the gut microbiome; determination of age-related pro-inflammatory cytokynes and anti-inflammatory miokynes. For the first time in Chile, we will address age-related epigenetic changes using the Horvath´s epigenetic clock. In a subset we will conduct a controlled physical challenge to characterize physical resilience (autophagy). DISCUSSION: ObAGE is in an excellent position to: approach aging as a process whose expression involves multiple factors from the early stages of a person's life; understand how longitudinal changes in health trajectories impact the biological mechanisms of aging; identify potential resilience mechanisms that help prevent unhealthy aging. Because SLS participants are still young, our research setting combined with advanced scientific techniques may identify individuals or groups at risk of early onset health issues. Results from ObAGE may pave the way to address the contribution of obesity to aging through lifespan from cells to systems and might be instrumental to developing interventions to improve health span in the Chilean population. TRIAL REGISTRATION: The proposed study does not consider any health care intervention on human participants.

A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases.

The universal second messenger cAMP is generated upon stimulation of Gs protein-coupled receptors, such as the ß2 -adreneoceptor, and leads to the activation of PKA, the major cAMP effector protein. PKA oscillates between an on and off state and thereby regulates a plethora of distinct biological responses. The broad activation pattern of PKA and its contribution to several distinct cellular functions lead to the introduction of the concept of compartmentalization of cAMP. A-kinase anchoring proteins (AKAPs) are of central importance due to their unique ability to directly and/or indirectly interact with proteins that either determine the cellular content of cAMP, such as ß2 -adrenoceptors, ACs and PDEs, or are regulated by cAMP such as the exchange protein directly activated by cAMP. We report on lessons learned from neurons indicating that maintenance of cAMP compartmentalization by AKAP5 is linked to neurotransmission, learning and memory. Disturbance of cAMP compartments seem to be linked to neurodegenerative disease including Alzheimer's disease. We translate this knowledge to compartmentalized cAMP signalling in the lung. Next to AKAP5, we focus here on AKAP12 and Ezrin (AKAP78). These topics will be highlighted in the context of the development of novel pharmacological interventions to tackle AKAP-dependent compartmentalization.

Cyclin-dependent kinase 5 activator p35 over-expression and amyloid beta synergism increase apoptosis in cultured neuronal cells.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuronal loss, dementia and pain. Two main protein aggregates, extracellular (senile plaques, SP) and intracellular (neurofibrillary tangles, NFT), are associated with AD. NFT are mainly composed of hyperphosphorylated microtubule-associated protein tau. Nowadays several protein kinases have been implicated in the phosphorylation of tau, including glycogen synthase kinase 3 beta (GSK3beta), MAP kinase, protein kinase A and cyclin-dependent kinase 5 (Cdk5). A deregulation in the activity of Cdk5 has been postulated to participate in the abnormal tau hyperphosphorylation in AD. Activation of Cdk5 occurs after its association with p35, a neuron-specific activator, predominantly in the nervous system. Therefore, in this study we used the tetracycline transactivator system to increase p35/GFP in neuronal cells, treated with amyloid beta 1-42 (Abeta(1-42)) peptide. These cells showed an increase of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3 staining, indicating increased apoptosis of neuronal cells. This effect could be reversed by the addition of tetracycline in the culture medium, suggesting synergistic effects of p35 over-expression and Abeta treatment in the apoptosis of neuronal cells. These results represent a linkage between amyloidogenic and cdk5 pathways leading to apoptosis of neuronal cells.

Role of the JAKs/STATs pathway in the intracellular calcium changes induced by interleukin-6 in hippocampal neurons.

Recent studies show that inflammation has an active role in the onset of neurodegenerative diseases. It is known that in response to extracellular insults microglia and/or astrocytes produce inflammatory agents. These contribute to the neuropathological events in the aging process and neuronal degeneration. Interleukin-6 (IL-6) has been involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Here, we show that IL-6 treatment of rat hippocampal neurons increases the calcium influx via NMDA-receptor, an effect that is prevented by the specific NMDA receptor antagonist MK-801 (dizocilpine). We also show that this calcium influx is mediated by the JAKs/STATs pathway, since the inhibitor of JAKs/STATs pathway, JAK 3 inhibitor, blocks calcium influx even in the presence of IL-6. This increase in calcium signal was dependent on external sources, since this signal was not observed in the presence of EGTA. Additional studies indicate that the increase in cytosolic calcium induces tau protein hyperphosphorylation, as revealed by using specific antibodies against Alzheimer phosphoepitopes. This anomalous tau hyperphosphorylation was dependent on both the JAKs/STATs pathway and NMDA receptor. These results suggest that IL-6 would induce a cascade of molecular events that produce a calcium influx through NMDA receptors, mediated by the JAKs/STATs pathway, which subsequently modifies the tau hyperphosphorylation patterns.

Participation of structural microtubule-associated proteins (MAPs) in the development of neuronal polarity.

Several lines of evidence have indicated that changes in the structure of neuronal cytoskeleton provide the support for the dramatic morphological changes that occur during neuronal differentiation. It has been proposed that microtubule-associated proteins can contribute to the development of this phenomenon by controlling the dynamic properties of microtubules. In this report we have characterized the effect of the combined suppression of MAP1B and tau, and MAP1B and MAP2 on neuronal polarization in cultured hippocampal cells grown on a laminin-containing substrate. We have taken advantage of the use of a mouse line deficient in MAP1B expression obtained by the gene trapping approach. In addition to this engineered mice line we used the antisense oligonucleotide approach to induce the suppression of tau or MAP2, in wild type and MAP1B-deficient neurons. Together these results show a synergistic role for MAP1B/MAP2 and MAP1B/TAU.

Evidence for the role of MAP1B in axon formation.

Cultured neurons obtained from a hypomorphous MAP1B mutant mouse line display a selective and significant inhibition of axon formation that reflects a delay in axon outgrowth and a reduced rate of elongation. This phenomenon is paralleled by decreased microtubule formation and dynamics, which is dramatic at the distal axonal segment, as well as in growth cones, where the more recently assembled microtubule polymer normally predominates. These neurons also have aberrant growth cone formation and increased actin-based protrusive activity. Taken together, this study provides direct evidence showing that by promoting microtubule dynamics and regulating cytoskeletal organization MAP1B has a crucial role in axon formation.

Perinatal lethality of microtubule-associated protein 1B-deficient mice expressing alternative isoforms of the protein at low levels.

Microtubule-associated protein 1B (MAP1B) has been implicated in axogenesis in cultured cells. To gain insight into the functions that MAP1B plays in vivo, we analyzed a strain of Map1B mutant mice generated by a gene trapping approach. Homozygous mice die on the first day after birth, probably due to a severe abnormal development of the nervous system. They present alterations in the structure of several brain regions. The normal Map1B gene yields different protein isoforms from alternatively spliced transcripts. The smaller isoforms were present in wild type, hetero-, and homozygous mice, but their expression was higher in the mutants than in the wild-type. Moreover, trace amounts of MAP1B protein were also observed in Map1B homozygous mutants, indicating an alternative splicing around the gene trap insertion. Thus, the Map1B gene trapped mutation reported in this work did not generated a null mutant, but a mouse with a drastic deficiency in MAP1B expression. Analyses of these mice indicate the presence of several neural defects and suggest the participation of MAP1B in neuronal migration.

Molecular genetic approaches to microtubule-associated protein function.

Protein function in vivo can be studied by deleting (knock-out) the gene that encodes it, and search for the consequences. This procedure involves different technologies, including recombinant DNA procedures, cell biology methods and histological and immunocytochemical analysis. In this work we have reviewed these procedures when they have been applied to ascertain the function of several microtubule-associated proteins. These proteins have been previously involved, through in vitro experiments, in having a role in the microtubule stabilization. Here, we will summarize the generation and characterization of different microtubule-associated protein knock-out mice. Special attention will be paid to MAP1B knock-out mice. Amongst the different MAPs knock-out mice these show the strongest phenotype, the most likely for being MAP1B, the MAP that is expressed earliest in neurogenesis. Molecular genetics could be considered as a valid and useful procedure to truly establish the in vivo functions of a protein, although it is necessary to be aware of possible artifacts such as the generation of some kinds of RNA alternative splicing. To avoid this the best strategy to be used must consider the deletion of the exon that contains the functional domains of the protein.

Immunological characterization of epitopes on tau of Alzheimer's type and chemically modified tau.

The microtubule-associated protein tau is the main structural component of paired helical filaments (PHFs), which in turn are one of the major aberrant polymers found in Alzheimer's disease. Immunological studies were carried out using site-directed monoclonal and polyclonal antibodies that recognize tubulin binding epitopes on tau, to further understand the mechanisms of tau self-association into PHFs. Tau protein was subjected to either carbamoylation with potassium cyanate (KCNO) or glycation with glucose, and the immunoreactivity of the chemically-modified protein with these antibodies was compared with tau derived from paired helical filaments and with normal brain tau. The data on the immunoblot patterns of tau isoforms and the ELISA titration curves revealed significant differences between the modified tau and normal controls. However, the Western blot patterns of immunoreactive tau from the chemically-modified protein and from Alzheimer brains were similar. The data on the differences in the electrophoretic profiles and Western blots of normal brain tau as compared with solubilized paired helical filaments, insoluble tangles and tau proteins of the Alzheimer's type, provide new clues to understand the anomalous interactions of tau in Alzheimer's disease.