Rony Nuydens

High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method.

Abstract A new, simple procedure is described for the production of 5 nm colloidal gold/secondary antibody reagents. Utilizing them with antitubulin shows 1) that they can be used for high resolution ultrastructural localization studies and 2) that this can be done with retention of satisfactory preservation of cell structure. The same, simple procedure can be used to prepare 20 nm colloidal gold/antibody reagents. These can be used for the high resolution light microscopic visualization of microtubules in interphase and mitotic cells. Colloidal gold labelled serum or monoclonal antibodies can be used in a new, general purpose immunocytochemical technique: the IGS (immuno gold staining) method.

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.

Systemic Immune Activation Leads to Neuroinflammation and Sickness Behavior in Mice

Substantial evidence indicates an association between clinical depression and altered immune function. Systemic administration of bacterial lipopolysaccharide (LPS) is commonly used to study inflammation-associated behavioral changes in rodents. In these experiments, we tested the hypothesis that peripheral immune activation leads to neuroinflammation and depressive-like behavior in mice. We report that systemic administration of LPS induced astrocyte activation in transgenic GFAP-luc mice and increased immunoreactivity against the microglial marker ionized calcium-binding adapter molecule 1 in the dentate gyrus of wild-type mice. Furthermore, LPS treatment caused a strong but transient increase in cytokine levels in the serum and brain. In addition to studying LPS-induced neuroinflammation, we tested whether sickness could be separated from depressive-like behavior by evaluating LPS-treated mice in a panel of behavioral paradigms. Our behavioral data indicate that systemic LPS administration caused sickness and mild depressive-like behavior. However, due to the overlapping time course and mild effects on depression-related behavior per se, it was not possible to separate sickness from depressive-like behavior in the present rodent model.

Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy

We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection.

Novel Role for Vascular Endothelial Growth Factor (VEGF) Receptor-1 and Its Ligand VEGF-B in Motor Neuron Degeneration

Although vascular endothelial growth factor-B (VEGF-B) is a homolog of the angiogenic factor VEGF, it has only minimal angiogenic activity, raising the question of whether this factor has other (more relevant) biological properties. Intrigued by the possibility that VEGF family members affect neuronal cells, we explored whether VEGF-B might have a role in the nervous system. Here, we document that the 60 kDa VEGF-B isoform, VEGF-B186, is a neuroprotective factor. VEGF-B186 protected cultured primary motor neurons against degeneration. Mice lacking VEGF-B also developed a more severe form of motor neuron degeneration when intercrossed with mutant SOD1 mice. The in vitro and in vivo effects of VEGF-B186 were dependent on the tyrosine kinase activities of its receptor, Flt1, in motor neurons. When delivered intracerebroventricularly, VEGF-B186 prolonged the survival of mutant SOD1 rats. Compared with a similar dose of VEGF, VEGF-B186 was safer and did not cause vessel growth or blood–brain barrier leakiness. The neuroprotective activity of VEGF-B, in combination with its negligible angiogenic/permeability activity, offers attractive opportunities for the treatment of neurodegenerative diseases.

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).

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.

Mitochondrial abnormality in sensory, but not motor, axons in paclitaxel-evoked painful peripheral neuropathy in the rat

The dose-limiting side effect of the anti-neoplastic agent, paclitaxel, is a chronic distal symmetrical peripheral neuropathy that produces sensory dysfunction (hypoesthesia and neuropathic pain) but little or no distal motor dysfunction. Similar peripheral neuropathies are seen with chemotherapeutics in the vinca alkaloid, platinum-complex, and proteasome inhibitor classes. Studies in rats suggest that the cause is a mitotoxic effect on axonal mitochondria. If so, then the absence of motor dysfunction may be due to mitotoxicity that affects sensory axons but spares motor axons. To investigate this, paclitaxel exposure levels in the dorsal root, ventral root, dorsal root ganglion, peripheral nerve, and spinal cord were measured, and the ultrastructure and the respiratory function of mitochondria in dorsal roots and ventral roots were compared. Sensory and motor axons in the roots and nerve had comparably low exposure to paclitaxel and exposure in the spinal cord was negligible. However, sensory neurons in the dorsal root ganglion had a very high and remarkably persistent (up to 10 days or more after the last injection) exposure to paclitaxel. Paclitaxel evoked a significant increase in the incidence of swollen and vacuolated mitochondria in the myelinated and unmyelinated sensory axons of the dorsal root (as seen previously in the peripheral nerve) but not in the motor axons of the ventral root. Stimulated mitochondrial respiration in the dorsal root was significantly depressed in paclitaxel-treated animals examined 2-4 weeks after the last injection, whereas respiration in the ventral root was normal. We conclude that the absence of motor dysfunction in paclitaxel-evoked peripheral neuropathy may be due to the absence of a mitotoxic effect in motor neuron axons, whereas the sensory dysfunction may be due to a mitotoxic effect resulting from the primary afferent neurons cell body being exposed to high and persistent levels of paclitaxel.

The interaction between microtubules and intermediate filaments in cultured cells treated with taxol and nocodazole.

Using double-label immunofluorescence and electron microscopy we studied the interaction between microtubules (MT) and intermediate filaments (IF) in MO cells treated with various combinations of taxol and nocodazole. With taxol, the organized MT of cultured cells are replaced by free MT and MT bundles. This rearrangement of MT is followed by a rearrangement of the IF. As in untreated cells a close association between these two filamentous systems is observed. In cells pretreated with nocodazole followed by addition of taxol, to induce the bundles of free MT, the preexisting IF coils disappear and IF associate with the MT. From these experiments we conclude that an interaction between MT and IF exists independent of the normal organisation of the MT system. The redistribution of IF always follows the redistribution of MT. The data show that MT determine the spatial distribution of IF which most probably involves some kind of physicochemical link.

Bone cancer pain model in mice: evaluation of pain behavior, bone destruction and morphine sensitivity

The primary aim of the study was to correlate pain development during bone cancer growth with objectively obtained tumor-induced changes in bone morphology. Additionally morphine sensitivity of this bone pain was evaluated. Mice were injected into the femur with osteolytic NCTC2472 cells, and behaviorally followed during a 3-week period. During the observation period increasing pain behavior was observed in tumor-bearing animals. Tumor mice exhibited spontaneous and movement-evoked lifting, the latter evoked through non-noxious palpation of the tumor. Limb use during forced ambulation on a rotarod decreased to substantial non-use of the affected limb by day 23. On day 23, micro-computer tomography scans of the tumor-bearing bones were evaluated for bone destruction. Different bone parameters indicative of osteolysis or fragmentation were significantly correlated with pain behavior. In a separate group of mice the effects of different morphine doses on pain behavior were evaluated on days 17 and 21 of tumor growth. Spontaneous lifting and movement-evoked lifting were sensitive to morphine treatment, although stress-induced analgesia due to repeated restraint might minimize movement-evoked lifting in mice. Limb use during forced ambulation was only slightly ameliorated by high morphine doses.