Ricardo Maccioni

... Cristóbal Maccioni, María E. Arzola, Lorena Mujica, Ricardo Maccioni. ... Los ONFs comprenden una red filamentosa compacta formada por los filamentos pareados helicoidales (PHFs), los cuales constituyen agregados de la proteína tau hiperfosforilada (7, 8). Estas estructuras ...

In this study we have used the transgenic mouse model Tg2576 to analyze the involvement of anomalous loss of regulation of cdk5 and the stress kinases JNK and p38 in brain neuronal death as related to neurodegenerative disorders such as Alzheimer's disease. Previous studies on hippocampal cells led us to the discovery that the cdk5/p35 complex is activated in neurodegeneration, a finding that was confirmed later in the transgenic mouse model. Here we show a link between the cdk5 system and JNK and p38 phosphoproteins, as an alternative pathway to neuronal death. Brains of the Tg2576 transgenic mice overexpressing amyloid precursor protein exhibited immunoreactivity with the phosphoproteins p-JNK, p-p38 and the GTPase protein Rac1 surrounding neuritic plaques. A significant increase in the immunodetection of JNK and p38 phosphoproteins in the Tg2576 mouse compared with wild type controls confirmed these findings. The significant increase in co-immunoprecipitation of p-JNK, p-p38 and Rac1 proteins with cdk5 in the transgenic mouse provided evidence for these interactions. At the cellular level, p-JNK and cdk5 colocalized in the cytoplasm of the cell bodies and neurites of brain cortical areas of the transgenic mouse. The present evidence suggests a cellular link between the cdk5 system and the stress kinase JNK and p38 pathways in an in vivo model. This study sheds new light on the pathogenesis of neuronal degeneration processes such as those occurring in Alzheimer's disease.

The treatment of tubulin with subtilisin resulted in a significant decrease in the ability of tubulin to assemble. The addition of taxol reduced the effect of subtilisin on the assembly of digested protein. Limited proteolysis of tubulin by subtilisin affected simultaneously both alpha- and beta-subunits, and it resulted in the appearance of two major cleavage fragments (32 and 20 kilodaltons) or an alternative pattern yielding two fragments (48 and 4 kilodaltons). The smallest peptide (4 kilodaltons) and also the 20-kilodalton fragment are localized in the C-terminal region of the tubulin alpha-subunit. Digested tubulin can assemble into sheet-shaped polymers, which cannot incorporate MAP2. On the other hand, the isolated C-terminal fragments can bind to MAP2. These results suggest that the carboxyl-terminal domain of the tubulin molecule is the site for the MAP2 interaction.

Limited proteolysis of phosphocellulose-pu-rifled tubulin with subtilisin resulted in cleavage of both a and ,B tubulin subunits, with the formation of two major fragments (S., and Sp,48 kDa) and a small peptide (4 kDa) containing the carboxyl-terminal region of tubulin. ...

José Tomás Egaña1,a, Cristian Zambrano1,a, Marco Tulio Nuñez2, Christian Gonzalez-Billault1 & Ricardo B. Maccioni1,∗ 1Laboratory of Cellular and Molecular Biology and 2Laboratory of Membrane Physiology, Millennium Institute for Advanced Studies in Cell Biology and ...

Alzheimer's disease, the cause of one of the most common types of dementia, is a brain disorder affecting the elderly and is characterized by the formation of two main protein aggregates: senile plaques and neurofibrillary tangles, which are involved in the process leading to progressive neuronal degeneration and death. Neurodegeneration in Alzheimer's disease is a pathologic condition of cells rather than an accelerated way of aging. The senile plaques are generated by a deposition in the human brain of fibrils of the beta-amyloid peptide (Abeta), a fragment derived from the proteolytic processing of the amyloid precursor protein (APP). Tau protein is the major component of paired helical filaments (PHFs), which form a compact filamentous network described as neurofibrillary tangles (NFTs). Experiments with hippocampal cells in culture have indicated a relationship between fibrillary amyloid and the cascade of molecular signals that trigger tau hyperphosphorylations. Two main protein kinases have been shown to be involved in anomalous tau phosphorylations: the cyclin-dependent kinase Cdk5 and glycogen synthase kinase GSK3beta. Cdk5 plays a critical role in brain development and is associated with neurogenesis as revealed by studies in brain cells in culture and neuroblastoma cells. Deregulation of this protein kinase as induced by extracellular amyloid loading results in tau hyperphosphorylations, thus triggering a sequence of molecular events that lead to neuronal degeneration. Inhibitors of Cdk5 and GSK3beta and antisense oligonucleotides exert protection against neuronal death. On the other hand, there is cumulative evidence from studies in cultured brain cells and on brains that oxidative stress constitutes a main factor in the modification of normal signaling pathways in neuronal cells, leading to biochemical and structural abnormalities and neurodegeneration as related to the pathogenesis of Alzheimer's disease. This review is focused on the main protein aggregates responsible for neuronal death in both sporadic and familial forms of Alzheimer's disease, as well as on the alterations in the normal signaling pathways of functional neurons directly involved in neurodegeneration. The analysis is extended to the action of neuroprotective factors including selective inhibitors of tau phosphorylating protein kinases, estrogens, and antioxidants among other molecules that apparently prevent neuronal degeneration.

The diagnosis of Alzheimer's disease (AD) is mainly performed by excluding other disorders with similar clinical features. In addition, an analysis of symptoms and signs, blood studies and brain imaging are major ingredients of the clinical diagnostic work-up. However, the diagnosis based on these instruments is unsatisfactory, indicating the need of a highly sensitive and reliable approaches, selective for AD and based on biological markers. Ideally, such markers should reflect the pathophysiological mechanisms of AD, which according to the current hypotheses, derive from the actions of two major protein aggregates, the extracellular beta-amyloid (Abeta) plaques and the neurofibrillary tangles. Since AD is a multifactorial disease, other factors that cause neuronal insult and that contribute to neuronal degeneration in AD include free radical and oxidative stress promoting molecules, proinflammatory cytokines and neurotoxic agents. In this context, the search for anomalous levels or changes in the molecular patterns of Abeta(1-42) or Abeta(1-40), hyperphosphorylated tau isoforms, oxidation products in the cell or cytokines such as interleukin-1 or 6 facilitates the selection of biomarkers in AD. There is clear evidence that the cerebrospinal fluid (CSF) levels of beta(1-42) are significantly reduced in AD patients as compared with senile controls, while increased levels of tau have been revealed. The CSF levels of these proteins reflect their metabolism in the central nervous system. Approaches using ELISA and immunochemical methods for the quantification of these markers in CSF have been preferentially used. Diagnosis criteria and number of patients exhibits variations in the different reports, while clinico-pathological studies are scarce. An increasing number of studies suggest that supplementary use of these CSF markers preferably in combination, adds to the accuracy of an AD diagnosis.

The cdk5/p35 complex has been implicated in a variety of functions related to brain development, including axonal outgrown and neuronal migration. In this study, by co-immunoprecipitation and pull-down experiments, we have shown that the cdk5/p35 complex associates with ...