Roger Nuyens

Okadaic acid-induced apoptosis in neuronal cells : Evidence for an abortive mitotic attempt

Abstract: There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimers research to increase the degree of phosphorylation of various proteins, such as the microtubule‐associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2‐N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B1 and cyclin D1 markers of the cell cycle, and triggers a time‐dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2‐N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up‐regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.

Sodium butyrate induces aberrant tau phosphorylation and programmed cell death in human neuroblastoma cells

Paired helical filaments, one of the major hallmarks of Alzheimers disease brains at autopsy, consist mainly of aberrantly phosphorylated tau. This aberrant tau phosphorylation can be induced in the human neuroblastoma cell line TR14 by a hyperstimulating mixture, consisting of nerve growth factor (NGF), db-cAMP, gangliosides and sodium butyrate (NaBut) [20,23]. Evidence is presented that exposing these cells to increasing concentrations of NaBut alone in the 0.5-2 mM dose-range is sufficient to induce aberrant tau phosphorylation within 24 h, measured by AT-8 immunocytochemistry and Western blotting. This process is associated with increased morphological differentiation. Furthermore, the aberrant tau phosphorylation is followed by neurotoxicity. This neurotoxicity has features of programmed cell death, such as fragmentation on a DNA agarose gel, fragmented nuclei and chromatin condensation and inhibition by the protein synthesis inhibitor cycloheximide. The mechanism by which NaBut induces these modified tau proteins and neurotoxicity are largely unknown but the data suggest an involvement of cytoskeletal proteins.

Sabeluzole stabilizes the neuronal cytoskeleton

There is growing evidence that cytoskeletal instability of neuronal cells is an important step towards tangle formation and subsequent functional disconnection in the AD brain. Sabeluzole, a new drug in clinical trials for Alzheimers disease (AD), has been shown to slow down the clinical progression of the disease. In a search for the mechanism of action of this compound, the effect of sabeluzole on the neuronal cytoskeleton was investigated. Previous studies have shown that in human TR14 neuroblastoma cells and in rat hippocampal neurons a hyperstimulating medium of kinase activators leads to induction of aberrant tau phosphorylation followed by neurotoxicity. This report documents the attenuation of this neurotoxicity by sabeluzole. By selective permeabilization procedures and quantitative immunocytochemistry we show that the compound is found to preferentially increase the fraction of polymerized tubulin. Evidence is presented that the compound differentially modulates a nocodazole-induced depolymerization in contrast to a cold-induced depolymerization. In the mouse, N4 neuroblastoma cells sabeluzole decreases the spontaneous retraction frequency of neurites and lowers the lateral mobility of the cells. We, therefore, propose that sabeluzole exerts its neuroprotective effect by a stabilization of the neuronal cytoskeleton and that this mechanism provides a completely new approach for treatment in Alzheimers disease.

Neuronal kinase stimulation leads to aberrant tau phosphorylation and neurotoxicity

Neurofibrillary tangles in Alzheimers disease brain consist mainly of abnormally phosphorylated tau proteins organised in paired helical filaments. Induction of tau phosphorylation in living neurons by hyperstimulation is monitored by specific monoclonal antibodies, such as AT-8 and PHF-1. By quantitative immunocytochemistry, we show that aberrant phosphorylation at the Ser199/Ser202 epitope (AT-8) and at the Ser 396 epitope (PHF-1) are moderately induced, proportionally to the degree of kinase stimulation. Whereas AT8 expression is prominent after 48 h, cell death becomes significant at 72 h and is related to the degree of stimulation and the expression level of aberrant tau phosphorylation. Time-lapse videomicroscopy of individual neuroblastoma cells suggest that hyperstimulation leads to a form of morphological over-differentiation. Immediately before cell death, some cells tend to display some features of mitosis. The data suggest a strong correlation between the expression of specific PHF-epitopes and subsequent cell death. The extended time scale of toxicity in this model may be appropriate to study in more detail the steps leading to aberrant phosphorylation associated neurotoxicity.

Sabeluzole, a memory-enhancing molecule, increases fast axonal transport in neuronal cell cultures

Morphological rearrangements, such as synapse number changes, have been observed in the adult mammalian brain after various experimental paradigms of learning and behavioral experience. The role of axonal transport in the physical translocation of material during this form of brain plasticity has not been fully appreciated. We show here by quantitative video microscopy that sabeluzole (R58735), a new memory-enhancing drug in humans, effectively increases fast axonal transport in rat neuronal cell cultures. Long-term incubation (24 hr) with sabeluzole in the concentration range between 0.1 and 1 microM increases both velocity and jump length of saltatory movements maximally by 20-30% in embryonic hippocampal neurons. Acute treatment only increases the velocity by 15-20%. Furthermore, the inhibition of axonal transport by 0.1 mM vanadate in N4 neuroblastoma cells is reversed by 1 microM sabeluzole. Observations on the kinesin-induced microtubule mobility in a reconstituted system show a 10% enhancement by sabeluzole at an optimal concentration of 2 microM, but no increase in kinesin ATPase activity. To our knowledge, this is the first pharmacological compound shown to increase fast axonal transport. The mechanism of fast axonal transport enhancement is discussed as a rationale for new therapeutic treatment in neuropathology.

The fast axonal transport in hippocampal neurones is acutely enhanced by db-cAMP

It has been observed that neurones have a certain capacity for the upregulation of fast axonal transport, for instance during nerve regeneration or reactive sprouting. However, the molecular regulation of this transport system is largely unknown. We show here by quantitative video-microscopy of endogenous organelles that application of 1 mM db-cAMP increases the velocity of fast axonal transport maximally by 32% within 60 minutes in neonatal hippocampal cells. At the same time, the jump length of the saltatory motions remains largely unchanged. The data suggest that activation of protein kinase A plays a role in the immediate upregulation of axonal transport.

Sabeluzole accelerates neurite outgrowth in different neuronal cell lines

The outgrowth of neurites in neuronal cell cultures reflects the intrinsic capacity for neurite regeneration and morphological rearrangements after axotomy and in plasticity. The role of fast axonal transport in these neurite outgrowth responses has not been investigated. We have recently shown that sabeluzole (R58735), a new neuro-active compound increases fast axonal transport in cultures of hippocampal neurons. In rat hippocampal neurons, N4 neuroblastoma cells and adult rat dorsal root ganglion cultures, incubation with sabeluzole at an optimal concentration of between 0.1 μM and 0.5 μM enhances neurite outgrowth between 10 and 30%. The relative number of cells with neurite length greater than twice the cell body, is also increased dose-dependently. Time-dependent studies further indicate that the rate of neurite elongation is markedly enhanced during the first 24-48 h. This neurite enhancing effect of sabeluzole is discussed in relation to the enhancement of fast axonal transport.

Automatic quantification of fast axonal transport in neuronal cell cultures

A method is presented which allows the automatic quantification of the fast axonal transport of endogenous organelles in neurites of cultured neuronal cells. Stretches of videotape recordings from Allen video enhanced contrast (AVEC) microscopy are digitized by currently available image processor hardware and analysed off-line on a MicroVAX II. Movements along the axon are calculated in great detail, allowing statistically significant changes to be detected. Interaction from the operator is minimised, thereby bypassing tedious manual analysis. This paper further reports the application of this system to the effect of vanadate treatment on axonal transport in cultures of rat embryonic hippocampal neurons.

Quantification of Fast Axonal Transport by Video Microscopy

Publisher Summary Recent advances in neuronal cell culture together with technological achievements in high-resolution light microscopy and image processing make it possible to obtain a detailed quantification of fast axonal transport. This chapter describes two complementary techniques based on high-resolution video microscopy. The first technique uses Allen video-enhanced Nomarski interference contrast to visualize small vesicles (∼100 nm) in neurites of living cultured neurons. An advanced image processing system detects individual moving vesicles and calculates parameters related to axonal transport. A major drawback, however, is the unknown content of these vesicles. The chapter presents an analysis of this technique involving evaluation of the effect of vanadate on axonal transport in embryonic hippocampal neurons. The second technique, called nanovid microscopy, partially overcomes the problem of selectivity. By coupling small colloidal gold probes (30 nm) to antibodies against specific epitopes and detecting these probes individually, the motion of specific and discrete proteins can be followed.

The Dynamic Study of Cell Surface Organization by Nanoparticle Video Microscopy

To follow individual, well-defined molecular structures in living cells has always been a dream of cell biologists. This is an important step in the evolution towards a biochemistry in situ. Indeed, quantifying the localization and dynamics of molecular biological activity should illuminate details of the biochemical reaction.