Jesús Avila

Glycogen synthase kinase 3: a drug target for CNS therapies

Glycogen synthase kinase3 (GSK3) is emerging as a prominent drug target in the CNS. The most exciting of the possibilities of GSK3 lies within the treatment of Alzheimers disease (AD) where abnormal increases in GSK3 levels and activity have been associated with neuronal death, paired helical filament tau formation and neurite retraction as well as a decline in cognitive performance. Abnormal activity of GSK3 is also implicated in stroke. Lithium, a widely used drug for affective disorders, inhibits GSK3 at therapeutically relevant concentrations. Thus while the rationale remains testable, pharmaceutical companies are investing in finding a selective inhibitor of GSK3. In the present review, we summarize the properties of GSK3, and discuss the potential for such a therapy in AD, and other CNS disorders.

Microtubule Reduction in Alzheimer's Disease and Aging Is Independent of τ Filament Formation

Biochemical studies show that phosphorylated tau, like that found in paired helical filaments (PHFs), does not promote microtubule assembly leading to the view that PHF formation leads to microtubule deficiency in Alzheimers disease (AD). However, although this issue is one of the most important aspects to further understanding the cell biology of AD, no quantitative examination of microtubule diminution in AD and its relationship with PHFs has been performed. To examine this issue directly, we undertook a morphometric study of brain biopsy specimens from AD and control cases. Ultrastructural analysis of neurons was performed to compare the microtubule assembly state in neurons of diseased and control cases and to examine the effect of PHF accumulation. We found that both number and total length of microtubules were significantly and selectively reduced in pyramidal neurons from AD in comparison to control cases (P = 0.000004) but that this decrement in microtubule density was surprisingly unrelated to PHFs (P = 0.8). Further, we found a significant age-dependent decrease in microtubule density with aging in the control cases (P = 0.016). These findings suggest that reduction in microtubule assembly is not dependent on tau abnormalities of AD and aging.

High molecular weight neurofilament proteins are physiological substrates of adduction by the lipid peroxidation product hydroxynonenal

Protein adducts of the lipid peroxidation producttrans-4-hydroxy-2-nonenal (HNE) are features of oxidative damage in neuronal cell bodies in Alzheimers disease but are also seen in axons of normal as well as diseased individuals. In this study, focusing on the axons of the mouse sciatic nerve, we found that HNE adducts characterize axons of mice from birth to senility. Immunoblots of axonal proteins showed that HNE adducts are only detected in neurofilament heavy subunit (NFH) and, to a lesser extent, neurofilament medium subunit (NFM), both lysine-rich proteins, consistent with the adducts being limited to lysine residues. In vitro, HNE treatment of permeabilized sciatic nerve showed the same specificity, i.e. NFH and NFM are the only proteins that reacted with HNE, providing they are phosphorylated. Quantitative immunoblot analysis of two strains of mice ages 1–33 months showed that the levels of HNE adducts on NFH are consistent throughout life. Additionally, mice transgenic for human superoxide dismutase-1 with G85R mutation show no difference in HNE adduction to NFH compared with controls. Taken together, these studies indicate that HNE adduction to NFH is physiological, and its constancy from birth to senility as well as its dependence on phosphorylation argues that NFH and NFM modification may play a role in protecting the membrane-rich axon from toxic aldehydes resulting from oxidative damage.

Oxidative imbalance in Alzheimer's disease.

Oxidative stress is a striking feature of susceptible neurons in the Alzheimer’s disease brain. Importantly, because oxidative stress is an early event in Alzheimer’s disease, proximal to the development of hallmark pathologies, it likely plays an important role in the pathogenesis of the disease. Investigations into the cause of such oxidative stress show that interactions between abnormal mitochondria and disturbed metal metabolism are, at least in part, responsible for cytoplasmic oxidative damage observed in these susceptible neurons, which could ultimately lead to their demise. Oxidative stress not only temporally precedes the pathological lesions of the disease but could also contribute to their formation, which, in turn, could provide some protective mechanism to reduce oxidative stress and ensure that neurons do not rapidly succumb to oxidative insults. In this review, we present the evidence for oxidative stress in Alzheimer’s disease and its likely sources and consequence in relation to other pathological changes.

MAP1B Is Required for Netrin 1 Signaling in Neuronal Migration and Axonal Guidance

BACKGROUND The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. RESULTS Here we show that Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. We also show that map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. CONCLUSIONS These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway.

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.

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.

Regulation of phosphorylation of neuronal microtubule-associated proteins MAP1b and MAP2 by protein phosphatase-2A and -2B in rat brain

The function of the neuronal high molecular weight microtubule-associated proteins (MAPs) MAP1b and MAP2 is regulated by the degree of their phosphorylation, which in turn is controlled by the activities of protein kinases and protein phosphatases (PP). To investigate the role of PP in the regulation of the phosphorylation of MAP1b and MAP2, we used okadaic acid and cyclosporin A to selectively inhibit PP2A and PP2B activities, respectively, in metabolically competent rat brain slices. The alteration of the phosphorylation levels of MAP1b and MAP2 was examined by Western blots using several phosphorylation-dependent antibodies to these proteins. The inhibition of PP2A, and to a lesser extent of PP2B, was found to induce an increased phosphorylation of MAP1b and inhibit its microtubule binding activity. Immunocytochemically, a marked increase in neuronal staining in inhibitor-treated tissue was observed with antibodies to the phosphorylated MAP1b. The inhibition of PP2A but not of PP2B also induced phosphorylation of MAP2 at multiple sites and impaired its microtubule binding activity. These results suggest that PP2A might be the major PP that participates in regulation of the phosphorylation of MAP1b and MAP2 and their biological activities.

The role of GSK3 in Alzheimer disease.

Mutations in app, ps-1 and ps-2 genes result in the appearance of Familial Alzheimer disease (FAD). Although, in many cases, those mutations result in an increase of the amount of beta amyloid peptide, there is not a clear correlation between that amount and the time of the onset of the disease. Thus, other factors may explain how mutations in those genes result in the appearance of neurodegeneration. In this minireview we propose that GSK3 could be one of those factors.

Tramiprosate, a drug of potential interest for the treatment of Alzheimer's disease, promotes an abnormal aggregation of tau

Alzheimers disease (AD) is characterized by the presence of two histopathological hallmarks; the senile plaques, or extracellular deposits mainly composed of amyloid-β peptide (Aβ), and the neurofibrillary tangles, or intraneuronal inclusions composed of hyperphosphorylated tau protein.Since Aβ aggregates are found in the pathological cases, several strategies are under way to develop drugs that interact with Aβ to reduce its assembly. One of them is 3-amino-1-propane sulfonic acid (Tramiprosate, 3-APS, Alzhemed™), that was developed as a sulfated glycosaminoglycan mimetic, that could interact with Aβ peptide, preventing its aggregation.However, little is known about the action of 3-APS on tau protein aggregation. In this work, we have tested the action of 3-APS on cell viability, microtubule network, actin organization and tau aggregation. Our results indicate that 3-APS favours tau aggregation, in tau transfected non-neuronal cells, and in neuronal cells. We also found that 3-APS does not affect the binding of tau to microtubules but may prevent the formation of tau-actin aggregates. We like to emphasize the importance of testing on both types of pathology (amyloid and tau) the potential drugs to be used for AD treatment.