Mar Pérez

Polymerization of τ into Filaments in the Presence of Heparin: The Minimal Sequence Required for τ ‐ τ Interaction

Abstract: Paired helical filaments isolated from the brains of patients with Alzheimers disease are composed of a major protein component, the microtubule‐associated protein termed τ, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of τ into filamentous structures, we have analyzed the ability of the whole τ protein or some of its fragments to self‐assemble in the presence of heparin. Different τ fragments, all of them containing some sequences of the tubulin‐binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18‐residue‐long peptide corresponding to the third tubulin‐binding motif of τ. This suggests that the ability of τ for self‐assembly could be localized in a short sequence of amino acids present in the tubulin‐binding repeats of the τ molecule.

A novel GSK-3β inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo

Amyloid deposits, neurofibrillary tangles, and neuronal cell death in selectively vulnerable brain regions are the chief hallmarks in Alzheimers (AD) brains. Glycogen synthase kinase-3 (GSK-3) is one of the key kinases required for AD-type abnormal hyperphosphorylation of tau, which is believed to be a critical event in neurofibrillary tangle formation. GSK-3 has also been recently implicated in amyloid precursor protein (APP) processing/Abeta production, apoptotic cell death, and learning and memory. Thus, GSK-3 inhibition represents a very attractive drug target in AD and other neurodegenerative disorders. To investigate whether GSK-3 inhibition can reduce amyloid and tau pathologies, neuronal cell death and memory deficits in vivo, double transgenic mice coexpressing human mutant APP and tau were treated with a novel non-ATP competitive GSK-3beta inhibitor, NP12. Treatment with this thiadiazolidinone compound resulted in lower levels of tau phosphorylation, decreased amyloid deposition and plaque-associated astrocytic proliferation, protection of neurons in the entorhinal cortex and CA1 hippocampal subfield against cell death, and prevention of memory deficits in this transgenic mouse model. These results show that this novel GSK-3 inhibitor has a dual impact on amyloid and tau alterations and, perhaps even more important, on neuronal survival in vivo further suggesting that GSK-3 is a relevant therapeutic target in AD.

Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3, and beta-catenin in the hippocampus

Estrogens regulate a wide set of neuronal functions such as gene expression, survival and differentiation in a manner not very different from that exerted by neurotrophins or by growth factors. The best-studied hormonal action is the transcriptional activation mediated by estrogen receptors. However, the direct effects of estrogen on growth factor signaling have not been well clarified. The present data show that estradiol, in vivo, induces a transient activation of GSK3 in the adult female rat hippocampus, followed by a more sustained inhibition, as inferred from phosphorylation levels of Tau. Similar data was obtained from cultured hippocampal neurons when treated with the hormone. The transient activation was confirmed by direct measure of GSK3 kinase activity. In addition, our results show a novel complex of estrogen receptor alpha, GSK3, and beta-catenin. The presence of the hormone removes beta-catenin from this complex. There is a second complex, also affected by estradiol, in which Tau is associated with GSK3, beta-catenin, and elements of the PI3 kinase complex. Considering the role of GSK3 in neurodegeneration, our data suggest that part of the neuroprotective effects of estrogen may be due to the control of GSK3.

Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice

Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimers disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein processing and tau alterations in this type of dementia remains controversial. We further investigated the role of beta-amyloid production/deposition in tau pathology and neuronal cell death in the mouse brain by crossing Tg2576 and VLW lines expressing human mutant amyloid precursor protein and human mutant tau, respectively. The resulting double transgenic mice showed enhanced amyloid deposition accompanied by neurofibrillary degeneration and overt neuronal loss in selectively vulnerable brain limbic areas. These findings challenge the idea that tau pathology in Alzheimers disease is merely a downstream effect of amyloid production/deposition and suggest that reciprocal interactions between beta-amyloid and tau alterations may take place in vivo.

Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3.

Prion diseases are characterized by neuronal cell death, glial proliferation and deposition of prion peptide aggregates. An abnormal misfolded isoform of the prion protein (PrP) is considered to be responsible for this neurodegeneration. The PrP 106-126, a synthetic peptide obtained from the amyloidogenic region of the PrP, constitutes a model system to study prion-induced neurodegeneration as it retains the ability to trigger cell death in neuronal cultures. In the present study, we show that the addition of this prion peptide to cultured neurons increases the activity of glycogen synthase kinase 3 (GSK-3), which is accompanied by the enhanced phosphorylation of some microtubule-associated proteins including tau and microtubule-associated protein 2. Prion peptide-treated neurons become progressively atrophic, and die ultimately. Both lithium and insulin, which inhibit GSK-3 activity, significantly decrease prion peptide-induced cell death both in primary neuronal cultures and in neuroblastoma cells. Finally, the overexpression of a dominant-negative mutant of GSK-3 in transfected neuroblastoma cells efficiently prevents prion peptide-induced cell death. These results are consistent with the view that the activation of GSK-3 is a crucial mediator of prion peptide-induced neurodegeneration.

Polymerization of tau peptides into fibrillar structures. The effect of FTDP-17 mutations

The peptides corresponding to the four repeats found in the microtubule binding region of tau protein were synthesized and their ability for self‐aggregation in presence of heparin or chondroitin sulfate was measured. Mainly, only the peptide containing the third tau repeat is able to form polymers in a high proportion. Additionally, the peptide containing the second repeat aggregates with a very low efficiency. However, when this peptide contains the mutation (P301L), described in a fronto temporal dementia, it is able to form polymers at a higher extent. Finally, it is suggested to have a role for the first and fourth tau repeats. It could be to decrease the ability of the third tau repeat for self‐aggregation in the presence of heparin.

Phosphorylated, but not native, tau protein assembles following reaction with the lipid peroxidation product, 4-hydroxy-2-nonenal

A correlation between hyperphosphorylation of tau protein and its aberrant assembly into paired helical filaments has lead to suggestions that phosphorylation controls assembly, but lacked a mechanistic basic. In this work, we have found that phosphorylated, but not native, tau protein is able to form polymers after the reaction with 4‐hydroxy‐2‐nonenal, a highly toxic product of lipid peroxidation. Phosphorylation of tau by both proline or non‐proline directed kinases, was able to assemble it into polymers.

Tau Dephosphorylation at Tau-1 Site Correlates with its Association to Cell Membrane*

It has been considered that tau protein is mainly a cytoplasmic protein since it is a microtubule associated protein. However, it has also been suggested that tau could be located in the cell nucleus and membrane. In our work, the cellular distribution of tau has been studied by immunofluorescence and western blot analysis, after subcellular fractionation in neuroblastoma cells and in tau-transfected non neural cells using, mainly, two types of tau antibodies; antibody 7.51 (that recognizes tau independent of its phosphorylation level); and antibody Tau-1 (that recognizes tau only in its dephosphorylated form). Also, tau was expressed in COS-1 cells to test for the features involved in the sorting of tau to different cell localizations. Our results show that tau associated to cell membrane has a lower phosphorylation level in its proline-rich region. Additionally, in differentiated neuroblastoma cells, tau phosphorylation, at that region, decreases and the amount of tau associated to cell membrane increases.

GSK3β-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling

A major determinant of neuronal morphology is the cytoskeleton. And one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of protein kinases and phosphatases in neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins are abundant cytoskeletal components predominantly expressed in neurons and have been found to be substrates for most of protein kinases and phosphatases present in neurons, including glycogen-synthase kinase 3 (GSK3). It has been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could control neuronal development. We have previously shown that MAP2 is phosphorylated in vitro and in situ by GSK3 at Thr1620 and Thr1623, located in the proline-rich region of MAP2 and recognized by antibody 305. However, the function of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-1 cells have been co-transfected with cDNAs encoding MAP2C and either wild type or mutated GSK3beta to analyze possible effects on microtubule stability and on the association of MAP2 with microtubules. We have found that GSK3beta phosphorylates MAP2C in co-transfected cells. Moreover, this phosphorylation is inhibited by the specific GSK3 inhibitor lithium chloride. Additionally, the formation of microtubule bundles, which is observed after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3beta wild type. Microtubule bundles were not observed in cells expressing MAP2C phosphorylated at the site recognized by antibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3beta wild type transfected cells with lithium chloride. Highly phosphorylated MAP2C species, which were phosphorylated at the site recognized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3beta wild type. Interestingly, these MAP2C species were enriched in cytoskeleton-unbound protein preparations. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulate microtubule stability.

Characterization of a double (amyloid precursor protein-tau) transgenic: tau phosphorylation and aggregation.

A double transgenic mouse expressing the amyloid precursor protein, bearing the Swedish mutations, and expressing tau protein containing three of the mutations present in frontotemporal dementia linked to chromosome 17 (FTDP-17), has been characterized. In the double transgenic mouse an increase in tau phosphorylation at serine S262 and S422 was observed compared with that found in simple transgenic mice. The phosphorylation at S262 was also found, in a much lower level, in the single transgenic mouse expressing amyloid precursor protein (APP), and it was absent in that overexpressing tau variant. Additionally, in the double transgenic mouse a slight increase in the amount of sarkosyl insoluble tau polymers was observed in comparison with that found in single transgenic tau mouse. Also, wider tau filaments were found in the double transgenic mouse compared with those found in the single transgenic mouse. Our results suggest that beta-amyloid peptide could facilitate the phosphorylation of tau at a site not directed by proline, such as serine 262, and that modification could facilitate tau aberrant aggregation. Also, they suggest that different types of tau filamentous polymers can occur in different mouse models for tauopathies, like those used for Alzheimers disease or FTDP-17.