Hugo Geerts

Color invariance

This paper presents the measurement of colored object reflectance, under different, general assumptions regarding the imaging conditions. We exploit the Gaussian scale-space paradigm for color images to define a framework for the robust measurement of object reflectance from color images. Object reflectance is derived from a physical reflectance model based on the Kubelka-Munk theory for colorant layers. Illumination and geometrical invariant properties are derived from the reflectance model. Invariance and discriminative power of the color invariants is experimentally investigated, showing the invariants to be successful in discounting shadow, illumination, highlights, and noise. Extensive experiments show the different invariants to be highly discriminative, while maintaining invariance properties. The presented framework for color measurement is well-founded in the physics of color as well as in measurement science. Hence, the proposed invariants are considered more adequate for the measurement of invariant color features than existing methods.

Prominent Axonopathy in the Brain and Spinal Cord of Transgenic Mice Overexpressing Four-Repeat Human tau Protein

Mutations in the human tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some mutations, including mutations in intron 10, induce increased levels of the functionally normal four-repeat tau protein isoform, leading to neurodegeneration. We generated transgenic mice that overexpress the four-repeat human tau protein isoform specifically in neurons. The transgenic mice developed axonal degeneration in brain and spinal cord. In the model, axonal dilations with accumulation of neurofilaments, mitochondria, and vesicles were documented. The axonopathy and the accompanying dysfunctional sensorimotor capacities were transgene-dosage related. These findings proved that merely increasing the concentration of the four-repeat tau protein isoform is sufficient to injure neurons in the central nervous system, without formation of intraneuronal neurofibrillary tangles. Evidence for astrogliosis and ubiquitination of accumulated proteins in the dilated part of the axon supported this conclusion. This transgenic model, overexpressing the longest isoform of human tau protein, recapitulates features of known neurodegenerative diseases, including Alzheimers disease and other tauopathies. The model makes it possible to study the interaction with additional factors, to be incorporated genetically, or with other biological triggers that are implicated in neurodegeneration.

Glycogen Synthase Kinase-3β Phosphorylates Protein Tau and Rescues the Axonopathy in the Central Nervous System of Human Four-repeat Tau Transgenic Mice

Protein tau filaments in brain of patients suffering from Alzheimers disease, frontotemporal dementia, and other tauopathies consist of protein tau that is hyperphosphorylated. The responsible kinases operating in vivo in neurons still need to be identified. Here we demonstrate that glycogen synthase kinase-3β (GSK-3β) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3β and GSK-3β × human tau40 transgenic mice. Phosphorylated protein tau migrates slower during electrophoretic separation and is revealed by phosphorylation-dependent anti-tau antibodies in Western blot analysis. In addition, its capacity to bind to re-assembled paclitaxel (Taxol®)-stabilized microtubules is reduced, compared with protein tau isolated from mice not overexpressing GSK-3β. Co-expression of GSK-3β reduces the number of axonal dilations and alleviates the motoric impairment that was typical for single htau40 transgenic animals (Spittaels, K., Van den Haute, C., Van Dorpe, J., Bruynseels, K., Vandezande, K., Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A., Loos, R., and Van Leuven, F. (1999) Am. J. Pathol. 155, 2153–2165). Although more hyperphosphorylated protein tau is available, neither an increase in insoluble protein tau aggregates nor the presence of paired helical filaments or tangles was observed. These findings could have therapeutic implications in the field of neurodegeneration, as discussed.

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.

Robust autofocusing in microscopy

BACKGROUND A critical step in automatic microscopy is focusing. This report describes a robust and fast autofocus approach useful for a wide range of microscopic modalities and preparations. METHODS The focus curve is measured over the complete focal range, reducing the chance that the best focus position is determined by dust or optical artifacts. Convolution with the derivative of a Gaussian smoothing function reduces the effect of noise on the focus curve. The influence of mechanical tolerance is accounted for. RESULTS The method is shown to be robust in fluorescence, bright-field and phase contrast microscopy, in fixed and living cells, as well as in fixed tissue. The algorithm was able to focus accurately within 2 or 3 s, even under extremely noisy and low contrast imaging conditions. CONCLUSIONS The proposed method is generally applicable in light microscopy, whenever the image information content is sufficient. The reliability of the autofocus method allows for unattended operation on a large scale.

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.

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.

Neonatal neuronal overexpression of glycogen synthase kinase-3β reduces brain size in transgenic mice

Glycogen synthase kinase-3beta (GSK-3beta) is important in neurogenesis. Here we demonstrate that the kinase influenced post-natal maturation and differentiation of neurons in vivo in transgenic mice that overexpress a constitutively active GSK-3beta[S9A]. Magnetic resonance imaging revealed a reduced volume of the entire brain, concordant with a nearly 20% reduction in wet brain weight. The reduced volume was most prominent for the cerebral cortex, without however, disturbing the normal cortical layering. The resulting compacted architecture was further demonstrated by an increased neuronal density, by reduced size of neuronal cell bodies and of the somatodendritic compartment of pyramidal neurons in the cortex. No evidence for apoptosis was obtained. The marked overall reduction in the level of the microtubule-associated protein 2 in brain and in spinal cord, did not affect the ultrastructure of the microtubular cytoskeleton in the proximal apical dendrites. The overall reduction in size of the entire CNS induced by constitutive active GSK-3beta caused only very subtle changes in the psychomotoric ability of adult and ageing GSK-3beta transgenic mice.

Coexpression of human cdk5 and its activator p35 with human protein tau in neurons in brain of triple transgenic mice.

The potential contribution of cyclin-dependent protein kinase 5 (cdk5) to hyperphosphorylate protein tau, as claimed in Alzheimers disease, was investigated in vivo. We generated single, double, and triple transgenic mice that coexpress human cdk5 and its activator p35 as well as human protein tau in cerebral neurons. Whereas expression and increased cdk5-kinase activity was obtained, as measured in vitro and demonstrated in vivo, neither murine nor human protein tau was appreciably phosphorylated in the brain of double and triple transgenic mice. These mice behaved and reproduced normally. Silver impregnation and immunohistochemistry of brain sections demonstrated that neurofilament proteins became redistributed in apical dendrites of cortical neurons. This suggested a cytoskeletal effect, but no other relevant brain pathology became apparent. These observations indicate that cdk5/p35 is not a major protein tau kinase and that cdk5/p35 did not cause neurodegeneration in mouse brain, as opposed to cdk5/p25.