Javier Díaz-Nido

Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function

Neurons, the basic information processing units of the nervous system, are characterized by a complex polar morphology which is essential for their function. To attain their precise morphology, neurons extend cytoplasmatic processes (axons and dendrites) and establish synaptic connections in a highly regulated way. Additionally, neurons are also subjected to small plastic changes at the adult stage which serve to regulate synaptic transmission. Every step of neuronal development is genetically controlled by endogenous determinants, as well as by environmental signals including intercellular contacts, extracellular matrix and diffusible signals. Cytoskeletal components are among the main protein targets modified in response to most of those extracellular signals which ultimately determine neuronal morphology. One of the major mechanisms controlling the neuronal cytoskeleton is the modification of the phosphorylation state of cytoskeletal proteins via changes in the relative activities of protein kinases and phosphatases within neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins is an abundant group of cytoskeletal components which are predominantly expressed in neurons and serve as substrates for most of protein kinases and phosphatases present in neurons. MAP2 phosphorylation seems to control its association with the cytoskeleton and it is developmentally regulated. Moreover, MAP2 may perform many functions including the nucleation and stabilization of microtubules (and maybe microfilaments), the regulation of organelle transport within axons and dendrites, as well as the anchorage of regulatory proteins such as protein kinases which may be important for signal transduction. These putative functions of MAP2 have also been proposed to play important roles in the outgrowth of neuronal processes, synaptic plasticity and neuronal cell death. Thus, MAP2 constitutes an interesting case to understand the regulation of neuronal function by the alteration of the phosphorylation state of cytoskeletal proteins in response to different extracellular signals. Here we will review the current knowledge about the regulation of MAP2 function through phosphorylation/dephosphorylation and its relevance in the broader context of neuronal functions.

Lithium inhibits Alzheimer's disease-like tau protein phosphorylation in neurons

In Alzheimers disease, tau protein becomes hyperphosporylated, which can contribute to neuronal degeneration. However, the implicated protein kinases are still unknown. Now we report that lithium (an inhibitor of glycogen synthase kinase‐3) causes tau dephosphorylation at the sites recognized by antibodies Tau‐1 and PHF‐1 both in cultured neurons and in vivo in rat brain. This is consistent with a major role for glycogen synthase kinase‐3 in modifying proline‐directed sites on tau protein within living neurons under physiological conditions. Lithium also blocks the Alzheimers disease‐like proline‐directed hyperphosphorylation of tau protein which is observed in neurons treated with a phosphatase inhibitor. These data raise the possibility of using lithium to prevent tau hyperphosphorylation in Alzheimers disease.

Lithium protects cultured neurons against β-amyloid-induced neurodegeneration

The deposition of β‐amyloid peptide (Aβ), the hyperphosphorylation of tau protein and the death of neurons in certain brain regions are characteristic features of Alzheimers disease. It has been proposed that the accumulation of aggregates of Aβ is the trigger of neurodegeneration in this disease. In support of this view, several studies have demonstrated that the treatment of cultured neurons with Aβ leads to the hyperphosphorylation of tau protein and neuronal cell death. Here we report that lithium prevents the enhanced phosphorylation of tau protein at the sites recognized by antibodies Tau‐1 and PHF‐1 which occurs when cultured rat cortical neurons are incubated with Aβ. Interestingly, lithium also significantly protects cultured neurons from Aβ‐induced cell death. These results raise the possibility of using chronic lithium treatment for the therapy of Alzheimers disease.

Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease

Brain tau protein is phosphorylated in vitro by cdc2 and MAP2 kinases, obtained through immunoaffinity purification from rat brain extracts. The phosphorylation sites are located on the tau molecule both upstream and downstream of the tubulin‐binding motifs. A synthetic peptide comprising residues 194–213 of the tau sequence, which contains the epitope recognized by the monoclonal antibody tau‐1, is also efficiently phosphorylated in vitro by cdc2 and MAP2 kinases. Phosphorylation of this peptide markedly reduces its interaction with the antibody tau‐1, as it has been described for tau protein in Alzheimers disease. Both cdc2 and MAP2 kinases are present in brain extracts obtained from Alzheimers disease patients. Interestingly, the level of cdc2 kinase may be increased in patient brains as compared with non‐demented controls. These results suggest a role for cdc2 and MAP2 kinases in phosphorylating tau protein at the tau‐1 epitope in Alzheimers disease.

Chronic lithium treatment decreases mutant tau protein aggregation in a transgenic mouse model.

Tau protein hyperphosphorylation and aggregation into neurofibrillary tangles are characteristic features of several neurodegenerative disorders referred to as tauopathies. Among them, frontotemporal dementia and Parkinsonism linked to chromosome 17 may be caused by dominant missense mutations in the tau gene. Transgenic mice expressing mutant tau serve as valid model systems to study the ethiopathogenesis of these diseases and assay possible therapeutic interventions. Here we report that chronic lithium treatment of a transgenic mouse strain expressing human tau with three missense mutations results in decreased glycogen synthase kinase-3-dependent-tau phosphorylation and a reduction of filamentous aggregates. These data indicate that lithium, presumably acting through the inhibition of glycogen synthase kinase 3, may be useful to curb neurodegeneration in tauopathies.

Glycosaminoglycans and β-amyloid, prion and tau peptides in neurodegenerative diseases

Protein aggregation into dense filamentous inclusions is a characteristic feature of many etiologically diverse neurodegenerative disorders including Alzheimers disease (AD), spongiform encephalopathies, and tauopathies. Thus, beta-amyloid peptide (Abeta) accumulates within senile amyloid plaques in AD, protease-resistant prion protein constitutes the amyloid deposits in spongiform encephalopathies and tau protein gives rise to neurofibrillary tangles (NFT) both in AD and in tauopathies. Curiously, these abnormal protein inclusions contain, in addition to their major peptide components, some associated sulfated glycosaminoglycans (sGAG). Here we discuss the proposal that the binding of sGAG to aggregate-forming peptides may modify the pathogenic process depending on their subcellular localization.

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.

Effect of the lipid peroxidation product acrolein on tau phosphorylation in neural cells.

A hallmark of several neurodegenerative disorders, including Alzheimers disease and tauopathies, is the hyperphosphorylation of the microtubule‐associated protein tau. Tau phosphorylation by proline‐directed and non‐proline‐directed protein kinases has been tested using antibodies PHF1 and 12E8, respectively. The effect of the lipid peroxidation product acrolein on these modes of phosphorylation has been assayed. We have found that acrolein, a peroxidation product from arachidonic acid, increases the phosphorylation of tau at the site recognized by PHF‐1 both in human neuroblastoma cells and in primary cultures of mouse embryo cortical neurons. Whereas the basal phosphorylation of tau protein at the PHF1 site seems to be largely mediated by glycogen synthase kinase‐3 (which is also activated in response to Aβ peptide), the acrolein‐induced tau hyperphosphorylation at the same site is also due to p38 stress‐activated kinase. These results support the view that oxidative stress and subsequent formation of lipid peroxidation products may contribute to tau protein phosphorylation in Alzheimers disease and tauopathies.

Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2.

The molecular mechanisms used by olfactory ensheathing cells (OECs) to promote repair in the damaged adult mammalian CNS remain unknown. Thus, we used microarrays to analyze three OEC populations with different capacities to promote axonal regeneration in cultured rat retinal neurons. Gene expression in “long-term cultured OECs” that do not stimulate adult axonal outgrowth was compared with that of “primary olfactory ensheathing cells” and the immortalized OEC cell line TEG3. In this way, we identified a number of candidate genes that might play a role in promoting adult axonal regeneration. Among these genes, it was striking that both the matrix metalloproteinase 2 (MMP2) and an inhibitor of this protease were represented. The disruption of MMP2 activity in TEG3 cells impaired their capacity to trigger axon regeneration in cultured adult retinal neurons. Furthermore, the MMP2 protein was detected in grafts of OECs that elicited robust axonal regeneration in the injured spinal cord of adult rats in vivo. These data suggest that MMP2 does indeed participate in adult axonal regeneration induced by OECs.

The inhibition of phosphatidylinositol-3-kinase induces neurite retraction and activates GSK3

It has been extensively described that neuronal differentiation involves the signalling through neurotrophin receptors to a Ras‐dependent mitogen‐activated protein kinase (MAPK) cascade. However, signalling pathways from other neuritogenic factors have not been well established. It has been reported that cAMP may activate protein kinase (PKA), and it has been shown that PKA‐mediated stimulation of MAPK pathway regulates not only neuritogenesis but also survival. However, extracellular regulated kinases (ERKs) mediated pathways are not sufficient to explain all the processes which occur in neuronal differentiation. Our present data show that: in cAMP‐mediated neuritogenesis, using the SH‐SY5Y human neuroblastoma cell line, there exists a link between the activation of PKA and stimulation of phosphatidylinositol 3‐kinase (PI3K). Both kinase activities are essential to the initial elongation steps. Surprisingly, this neuritogenic process appears to be independent of ERKs. While the activity of PI3K is essential for elongation and maintenance of neurites, its inhibition causes retraction. In this neurite retraction process, GSK3 is activated. Using both a pharmacological approach and gene transfer of a dominant negative form of GSK3, we conclude that this induced retraction is a GSK3‐dependent process which in turn appears to be a common target for transduction pathways involved in lysophosphatidic acid‐mediated and PI3K‐mediated neurite retraction.