Adriana Ferreira - US grants

We intend to establish the relationship between senile plaques and neurofibrillary tangles, the two hallmark lesions in Alzheimer's Disease. Recently, we have obtained data suggesting a link between these two lesions in hippocampal neurons. Our work builds on those models in which fibrillar beta-amyloid (Abeta) added to neurons in culture results in neurotoxicity. The addition of Abeta to mature hippocampal cultures resulted in severe alterations of neuronal morphology, including the progressive degeneration of axons and dendrites, and the selective hyperphosphorylation of adult tau isoforms. Therefore, the specific aims of this proposal are directed to obtain further and novel insights into the mechanistic link between Abeta deposition, tau hyperphosphorylation and neurite degeneration. The specific aims will address the following hypotheses: 1) the deposition of Abeta results in the integrin-mediated activation of the mitogen-activated protein kinase (MAPK) signal transduction pathway in mature central neurons; and 2) this activation of MAPK contributes to the enhanced phosphorylation of adult tau isoforms which in term, plays a key role in the mechanism underlying neurite degeneration. We shall determine: 1) whether the activation of MAPK by fibrillar Abeta is mediated by integrins. We will attempt to block the activation of MAPK induced by Abeta by pretreating the cells with antibodies known to inhibit the function of different integrins. We will repeat these experiments using hippocampal neurons depleted of different integrins by means of homologous recombination techniques or antisense oligonucleotides. 2) The participation of MAPK in the selective phosphorylation of adult human tau isoforms induced by Abeta. The experiments will be using mature hippocampal cultures prepared from wild type, tau knockout and human tau transgenic mice treated with soluble or fibrillar Abeta. The activity of MAPK will be suppressed by means specific inhibitors. Tau phosphorylation will be determined using tau antibodies and by phosphorylation assays. The presence of signs of neurite degeneration will be assessed at the light and electron microscopy levels.

DESCRIPTION (provided by applicant) Cyclin-dependent kinase 5 (Cdk5), a small serine-threonine kinase is required for proper development of the mammalian nervous system, and abnormal activation of this kinase is involved in the pathogenesis of cytoskeletal abnormalities and neuronal death in neurodegenerative disorders. To be activated, Cdk5 has to associate with regulatory subunits, such as p35 or p39. The experiments of the present proposal are directed to analyze the participation of these two highly related Cdk5 activators in neuronal polarization and in B-amyloid induced neurodegeneration. The specific hypotheses to be tested are that: 1) p35 and p39 have different expression patterns and subcellular localization, targeting Cdk5 to different substrates and hence having different functional roles during neuronal development. In this regard, we specifically propose that p35 is mainly involved in the regulation of cytoskeletal dynamics during axon formation, while p39 in synaptogenesis; and 2) Activation of the MAPK pathway by extracellular matrix molecules, such as laminin, or by B-amyloid enhances Cdk5 activity by promoting the synthesis of p35 and/or p39. These experiments will be carried out using as a model system cultured hippocampal pyramidal and several state of the art techniques in cellular and molecular biology. The results that we expect to obtain will certainly contribute to a better understanding of the mechanisms underlying neurite polarization and degeneration in central neurons.

DESCRIPTION (provided by applicant): In the past two decades, a great deal of attention has been directed to the study of synaptogenesis in the mammalian central nervous system. Many of these studies are driven by the belief that the diagnosis and the prevention of synapse loss associated with several neurodegenerative diseases can be achieved through a better understanding of the basic mechanisms of synapse formation in central neurons. Although these mechanisms are not completely understood, it is tempting to speculate that proteins, such as agrin, that play a key role in the formation of the neuromuscular junction might also play an important role in the formation of synaptic contacts between neurons. Recently, we have shown that agrin differentially regulates the rates of axonal and dendritic elongation as well as synapse formation in hippocampal neurons. However, the agrin receptor(s) has yet to be identified in these neurons. The experiments proposed here are intended to test the following hypothesis: ror proteins, two tyrosine kinase receptors considered "orphan receptors", could mediate the effects of agrin on early stages of development in central neurons. A combination of techniques including: Western blot analysis, immunocytochemistry, RT-PCR, the generation of null mutations, binding assays, and immunoprecipitation will be used to analyze: 1) the pattern of expression and localization of ror 1 and ror 2 in central neurons; 2) the role of these tyrosine kinase receptors in neurite elongation and synapse formation; and 3) the participation of ror 1 and ror 2 in the agrin-signaling pathway.

The experiments performed during the first funded period of this project (2000-2004) led to the first evidence of a direct link between senile plaques and tau pathology in an Alzheimer's disease (AD) cell model system (Rapoport et al., 2002). This study showed that neurons expressing human tau degenerated in the presence of preaggregated beta amyloid (A[unreadable]), while no signs of degeneration were detected in A[unreadable]-treated tau-depleted neurons (Rapoport et al., 2002). Data obtained during the second funded period (2005-2009) have identified calpain-mediated tau cleavage leading to the generation of a 17 kDa fragment as an important step in neuronal degeneration (Park and Ferreira 2005, Park et al., 2007). More recently, we have obtained preliminary evidence suggesting that the levels of this tau neurotoxic fragment might be elevated not only in AD but also in other tauopathies. However, the mechanisms by which this tau fragment could induce neurotoxicity remain poorly understood. We speculate that the generation of a 17 kDa tau fragment is a conserved mechanism leading to neuronal degeneration in different tauopathies. To test this hypothesis, we will determine: 1) the presence of the 17 kDa tau fragment in brain samples obtained from AD and other tauopathies patients;and 2) we will generate a transgenic animal model in which this toxic fragment is over expressed. This animal model could provide an essential tool for future studies on the neurotoxic effects of this fragment in central neurons that develop in situ. These experiments will be performed by means of a combination of techniques including: Western blot analysis, immunocytochemistry, calpain activity assays, real-time RT-PCR, and homologous recombination techniques. The data obtained could provide useful for the diagnosis, prevention, and eventually the treatment of AD and other tauopathies.

? DESCRIPTION (provided by applicant): The mechanisms underlying tau pathology in neurodegenerative diseases have not been completely elucidated. However, a growing body of evidence suggests that tau posttranslational modifications, other than phosphorylation, might play an important role in those mechanisms. Recently, we have shown that one of such modifications is calpain-mediated tau cleavage. This cleavage leads to the generation of the 17 kDa tau45-230 fragment in the context of Alzheimer's disease (AD) and other tauopathies. In addition, our data indicated that this fragment induced progressive degeneration in cultured hippocampal neurons. Conversely, conditions that prevented the generation of this fragment were associated with enhanced neuronal survival in central neurons. Furthermore, increased neuronal death, synapse loss, and behavioral abnormalities have been detected in transgenic mice expressing tau45-230. Together, these data provide evidence indicating that tau cleavage into this neurotoxic fragment is a conserved molecular pathogenic pathway of neurodegeneration. On the other hand, the mechanisms by which tau45-230 induces degeneration and cell death remain unknown. Based on our preliminary results, we hypothesize that tau45-230 could exert its neurotoxic effects through a dual mechanism: 1) tau45-230 could modulate the aggregation of full-length tau inducing the formation of smaller and more toxic aggregates; and 2) tau45-230 could interfere, either as a monomer or as small aggregates, with full-length tau's biological functions. To test these hypotheses, we propose to: 1) assess the presence and toxicity of tau45-230 aggregates in brain samples obtained from AD and other tauopathy subjects; 2) determine to what extent tau45-230 modulates full-length tau aggregation; and 3) identify the mechanisms by which tau45-230 alters tau function in central neurons. These experiments will be performed by means of a combination of techniques including: Western blot analysis, immunocytochemistry, in vitro polymerization assays, aggregate purification, homologous recombination techniques, microtubule-binding assays, live cell microscopy, and cell viability assays. These studies could lead to the identification of a novel molecular pathway of degeneration. In addition, they could provide useful for the diagnosis, prevention, and eventually the treatment of AD and other tauopathies.