Biomedical subjects

R Nuydens

Publications and source records attributed to R Nuydens.

At least 19 recordsLinked to original sources

PubMed2005

A multiparametric assay for quantitative nerve regeneration evaluation.

We introduce an assay for the semi-automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high-resolution mosaic images representing the entire nerve. These are analysed by a dedicated image-processing package that computes nerve-specific features (e.g. nerve area, fibre count, myelinated area) and fibre-specific features (area, perimeter, myelin sheet thickness). The assay's performance and correlation of the automatically computed data with visually obtained data are determined on a set of 140 semithin sections from the distal part of a rat tibial nerve from four different experimental treatment groups (control, sham, sutured, cut) taken at seven different time points after surgery. Results show a high correlation between the manually and automatically derived data, and a high discriminative power towards treatment. Extra value is added by the large feature set. In conclusion, the assay is fast and offers data that currently can be obtained only by a combination of laborious and time-consuming tests.

Animals↗

PubMed2002

Coexpression of GSK-3beta corrects phenotypic aberrations of dorsal root ganglion cells, cultured from adult transgenic mice overexpressing human protein tau.

Coexpression of constitutively active GSK-3beta[S9A] rescued the axonal pathology induced by overexpression of human tau in transgenic mice (Spittaels et al., (2000) J. Biol. Chem. 275, 41340-41349). We isolated dorsal root ganglion (DRG) neuronal cultures from adult tau4R- and tau4R x GSK-3beta-transgenic mice to define the mechanisms at the cellular and subcellular level. DRG from tau4R-transgenics showed a reduced sprouting capacity while density and stability of microtubules in the axonal processes were significantly increased. Video-enhanced contrast microscopy demonstrated a dramatic inhibition of fast axonal transport. Coexpression of GSK-3beta increased tau phosphorylation and reversed the effects on microtubule stability and saltatory motion. In DRG from GSK-3beta single transgenics, increased tau phosphorylation was evident without any major effects on microtubule stability or axonal transport. These observations support the hypothesis that excess tau competed with motor-proteins for binding to microtubules and/or that a rigid microtubular system inhibits axonal transport.

Acetylation↗

PubMed2000

Bcl-2 protects neuronal cells against taxol-induced apoptosis by inducing multi-nucleation.

Taxol-induced peripheral neuropathy is a commonly-occurring side-effect in the treatment of cancer patients with taxoteres or taxanes. Taxol is known to induce apoptosis in a number of tumor cells. This report documents that, similar to proliferating cells, taxol induces apoptosis in NGF-differentiated PC12 cells, as assessed by exogenous FITC-annexin-V binding and nuclear fragmentation. It is shown that PC12 cells that stably overexpress Bcl-2 are protected against the toxic effect of taxol, as evidenced by the XTT assay and by a decreased fraction of propididum iodide positive cells in a dye exclusion test. Also the number of annexin-V-positive cells and the number of fragmented nuclei are lower in the Bcl-2 transfected cells. The effect is similar to the protective effect of Bcl-2 against NGF deprivation in differentiated PC12 cells. Although taxol forced both wild-type and Bcl-2-overexpressing cells into a mitotic state, only in Bcl-2-overexpressing cells did this lead to the appearance of metabolically active, multi-nucleated cells. This suggests that Bcl-2 is able to induce an alternative escape pathway, downstream of the G2/M block, in taxol-treated differentiated PC12 cells.

Animals↗

PubMed2000

Bcl-2 protects against apoptosis-related microtubule alterations in neuronal cells.

Bcl-2 is a gene with clear anti-apoptotic properties in neurodegenerative conditions. One of the earliest hallmarks of degeneration in neuronal cell cultures is the loss of neurite morphology. Therefore the effect of Bcl-2 on neuronal morphology and microtubule stability was studied in nerve growth factor differentiated PC12 cells. Microtubule dynamics were modulated using the microtubule stabilizer taxol and the microtubule destabilizer, okadaic acid, a protein phosphatase inhibitor. It was shown that Bcl-2 protects against both taxol- and okadaic acid induced neurite retraction. Bcl-2 overexpression also significantly reduced the increased ratio of acetylated tubulin over total tubulin induced by taxol treatment. Interestingly, Bcl-2 attenuates the decrease of the same ratio after exposure to okadaic acid, suggesting that Bcl-2 is able to normalize the level of acetylated tubulin. In addition, cell death and nuclear fragmentation, induced by okadaic acid, were reduced in Bcl-2 overexpressing cells. This protection is either downstream or independent of tau phosphorylation as quantitative immunocytochemistry with AT8 showed that Bcl-2 did not modify the level of tau phosphorylation. The data suggest that the protective effect of Bcl-2 on the neuronal cytoskeleton is probably linked to changes in the post-translational modification of tubulin.

Acetylation↗

PubMed1999

Nimodipine and flunarizine have different effects on survival and morphology of PC12 cells during nerve growth factor deprivation.

The purpose of this study was to examine the effect of antagonists of different subtypes of Ca(2+) channels (nimodipine and flunarizine) and two types of Ca(2+) chelating agents (the cell permeant Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethylester (BAPTA-AM) and the cell non-permeant Ca(2+) chelator EGTA) on neurite retraction and cell death of nerve growth factor (NGF)-differentiated PC12 cells after NGF deprivation. We demonstrated that flunarizine and nimodipine, but not BAPTA-AM and EGTA, provided protection against cell death due to NGF deprivation. Using time-lapse videomicroscopy and quantitative image analysis, we found that retraction of neurites was an early and fast phenomenon after removal of NGF. None of the compounds tested (flunarizine, nimodipine, BAPTA-AM, EGTA) could prevent the retraction of neurites.

Animals↗

PubMed1999

A rapid method for the evaluation of compounds with mitochondria-protective properties.

Mitochondrial dysfunction has been implicated in a number of neurodegenerative diseases, such as ischemia and Parkinson's disease. We present here a method that allows the rapid quantification of interventions, aimed at inhibiting the effect of mitochondrial membrane potential uncouplers, based on the ratioing properties of the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), by using currently available 96-well fluorescent plate readers. A method is presented for evaluation of cross-talk between the two excitation/emission channels. Further characterization of the probe shows that the effect of plasma membrane potential changes on JC-1 fluorescence ratio are negligible, but that the signal is very sensitive to pH. One of the most exciting applications is the possibility to perform end-point measurements, thanks to the ratioing properties of the probe. The system is tested in different culture types with different mitochondrial uncouplers. As an example of a quantitative evaluation, we show that flunarizine is able to inhibit, dose-dependently, FCCP mediated JC-1 signal increase. The procedure is simple and allows for the fast screening of mitochondria-protective compounds.

Animals↗

PubMed1999

Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).

Animals↗

PubMed1998

Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells.

We describe a rapid and reliable method to quantitate the extent of apoptosis in neuronal cell cultures. Based on their annexin V-affinity, resulting from phosphatidylserine (PS) exposure at the outer leaflet of the plasma membrane, apoptotic cells can be distinguished from annexin V-negative living cells, by using microscopic and flow cytometric procedures. When combined with propidium iodide (PI) the double labeling procedure allows a further distinction of necrotic (annexin V+/PI+), apoptotic (annexin V+/PI-) cells. Furthermore, when the cells are incubated with annexin V prior to harvesting, the former cell populations can be separated from cells damaged during isolation (annexin V-/PI+). In the present paper, we show that the annexin V-binding assay is also applicable to differentiated neuronal cells with fragile neurite outgrowths.

Annexin A5↗

PubMed1998

Altered [Ca2+] homeostasis in PC12 cells after nerve growth factor deprivation.

[Ca2+]i homeostasis in individual PC12 cells after elevated [K+]o was studied by ratiometric microscopy, during nerve growth factor (NGF) deprivation. A significantly lower number of cells responded with an increased [Ca2+]i in the NGF deprived condition. Moreover, the responding cells were more deficient in regulating their [Ca2+]i back to control levels, after the transient peak. This suggests that differentiated neurons do not traverse the apoptotic program homogeneously with regard to their [Ca2+]i regulation and that NGF deprived PC12 cells have more difficulties to reduce their [Ca2+]i after influx of [Ca2+]o.

Animals↗

PubMed1998

Neuronal differentiation is accompanied by NSP-C expression.

Neuroendocrine-specific protein (NSP) reticulons are expressed in neural and neuroendocrine tissues and cell cultures derived therefrom, while most other cell types lack NSP-reticulons. Three major subtypes have been identified so far, designated NSP-A, NSP-B, and NSP-C. We have investigated the correlation between the degree of neuronal differentiation, determined by morphological and biochemical criteria, and NSP-reticulon subtype expression. For this purpose, several human neuroblastoma cell lines, exhibiting different degrees of neuronal differentiation, were examined immuno(cyto)chemically. It became obvious that the expression of NSP-C, as detected by immunofluorescence microscopy and Western blotting, is most prominent in cell lines with a high degree of neuronal differentiation, such as LA-N-5. Such highly differentiated cells also express other neural and neuroendocrine markers, such as neural cell adhesion molecule (NCAM), neurofilament proteins, synaptophysin, and chromogranin. NSP-A was observed in all cell lines to a different extent. However, no clear correlation was observed with the degree of neuronal differentiation as defined by other neuronal and neuroendocrine markers or morphology. NSP-B could not be detected. The induction of neuronal differentiation with nerve growth factor, dbcAMP, and retinoic acid in the rat pheochromocytoma cell line PC12 and the human teratocarcinoma cell line hNT2, respectively, induced the expression of NSP-A and NSP-C in these cell lines parallel to the induction of neurofilament protein expression. It is concluded that NSP-C expression, in particular, is strongly correlated with neuronal differentiation.

Animals↗

PubMed1998

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

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 Alzheimer's 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.

Anaphase↗

PubMed1997

Aberrant tau phosphorylation and neurite retraction during NGF deprivation in PC12 cells.

Recently apoptotic markers have been found in Alzheimer's Disease (AD) brain. To investigate the relation between tau phosphorylation and apoptosis, immunocytochemistry of AT8 (indicating the degree of phosphorylation at the tau Ser202/Thr205 site) was quantitatively determined the degree of tau phosphorylation at the Ser202 site was monitored during neuronal apoptosis in differentiated PC12 cells after nerve growth factor (NGF) deprivation. During this programmed cell death a prominent retraction of neurites took place that was associated with a clear increase in the level of AT8 signalaberrant phosphorylated tau at the Ser202 site. The broad spectrum kinase inhibitor staurosporine attenuated both this increase in tau phosphorylation, neurite retraction, and apoptosis. We suggest that at some point during programmed cell death, kinases with tau as substrate become activated and that the resulting loss of cytoskeletal integrity leads to neurite instability.

Animals↗

PubMed1995

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

Paired helical filaments, one of the major hallmarks of Alzheimer's 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.

Antibodies, Monoclonal↗

PubMed1993

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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1991

Nanovid microscopy.

By combining small colloidal gold probes with video-enhanced quantitative microscopy, the intracellular dynamics of specific proteins in living cells can now be studied.

Axonal Transport↗

PubMed1991

Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy.

A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.

Adenosine Triphosphate↗

PubMed1991

Laminin binding and internalization by human and murine mammary gland cell lines in vitro.

We have studied the binding and internalization of Engelbreth-Holm-Swarm mouse sarcoma laminin labeled with colloidal gold (LN-G40) by human and murine mammary gland cell lines. Interactions between the LN-G40 probe and the cells spread on a glass coverslip were monitored with video-enhanced contrast microscopy (Nanovid). Transmission electron microscopy allowed the quantitation of the LN-G40 probe at various cellular locations. During the first 15 min, a homogeneous binding of LN-G40 probe to the cell surface was observed with all cell lines. This binding did not occur with gold particles that were not conjugated to laminin. Then, the LN-G40 probe began to cluster on the cell surface and was, during the following 20 h, internalized by pits that were not coated. In the cells, the LN-G40 probe sometimes showed saltatory movements along linear tracks. The LN-G40 probe was intracellularly found in vesicles, multivesicular bodies, cisternal structures, and lysosomes, suggesting the degradation of the internalized laminin. However, not all cell surface-bound LN-G40 probe was internalized after 20 h. Differences between the cell lines were quantitative, but no clear correlation could be made between migration of cells on laminin and internalization of laminin.

Animals↗