Mike Francke

Glutaminyl cyclase inhibition attenuates pyroglutamate Aβ and Alzheimer's disease–like pathology

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Aβ peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimers disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimers disease and correlated with the appearance of pE-modified Aβ. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Aβ3(pE)–42 burden in two different transgenic mouse models of Alzheimers disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Aβx–40/42, diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Aβ3(pE)–42 acts as a seed for Aβ aggregation by self-aggregation and co-aggregation with Aβ1–40/42. Therefore, Aβ3(pE)–40/42 peptides seem to represent Aβ forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Aβ by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimers disease and provides implications for other amyloidoses, such as familial Danish dementia.

Role of retinal glial cells in neurotransmitter uptake and metabolism

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, gamma-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.

Role of glial K(+) channels in ontogeny and gliosis: a hypothesis based upon studies on Müller cells.

The electrophysiological properties of Müller cells, the principal glial cells of the retina, are determined by several types of K+ conductances. Both the absolute and the relative activities of the individual types of K+ channels undergo important changes in the course of ontogenetic development and during gliosis. Although immature Müller cells express inwardly rectifying K+ (KIR) currents at a very low density, the membrane of normal mature Müller cells is predominated by the KIR conductance. The KIR channels mediate spatial buffering K+ currents and maintain a stable hyperpolarized membrane potential necessary for various glial‐neuronal interactions. During “conservative” (i.e., non‐proliferative) reactive gliosis, the KIR conductance of Müller cells is moderately reduced and the cell membrane is slightly depolarized; however, when gliotic Müller cells become proliferative, their KIR conductances are dramatically down‐regulated; this is accompanied by an increased activity of Ca2+‐activated K+ channels and by a conspicuous unstability of their membrane potential. The resultant variations of the membrane potential may increase the activity of depolarization‐activated K+, Na+ and Ca2+ channels. It is concluded that in respect to their K+ current pattern, mature Müller cells pass through a process of dedifferentiation before proliferative activity is initiated. GLIA 29:35–44, 2000.

Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

Loss of inwardly rectifying potassium currents by human retinal glial cells in diseases of the eye

We compared the inward K+ currents of Müller glial cells from healthy and pathologically changed human retinas. To this purpose, the whole‐cell voltage‐clamp technique was performed on noncultured Müller cells acutely isolated from human retinas. Cells originated from retinas of four healthy organ donors and of 24 patients suffering from different vitreoretinal and chorioretinal diseases. Müller cells from organ donors displayed inward K+ currents in the whole‐cell mode similar to those found in other species. In contrast, this pattern was clearly changed in the Müller cells from patient retinas. In whole‐cell recordings many Müller cells had strongly decreased inward K+ current amplitudes or lost these currents completely. Thus, the mean input resistance of Müller cells from patients was significantly increased to 1,129 ± 812 MΩ, compared to 279 ± 174 MΩ in Müller cells from healthy organ donor retinas. Accordingly, since the membrane potential is mainly determined by the K+ inward conductance in healthy Müller cells, a large amount of Müller cells from patient retinas had a membrane potential which was significantly lower than that of Müller cells from control eyes. The mean membrane potentials were −37 ± 24 mV and −63 ± 25 mV for patient and donor Müller cells, respectively. The newly described membrane characteristic changes of Müller cells from patient eyes are assumed to interfere severely with normal retinal function: (1) the retinal K+ homeostasis, which is partly regulated by the Müller cell‐mediated spatial buffering, should be disturbed, and (2) the diminished membrane potential should influence voltage‐dependent transporter systems of the Müller cells, e.g., the Na+‐dependent glutamate uptake. GLIA 20:210–218, 1997.

Selective Staining by Vital Dyes of Muller Glial Cells in Retinal Wholemounts

Müller glial cells within the retina may respond to different signaling molecules with an elevation of their intracellular free calcium. To prove the localization of the recorded calcium responses in Müller cells within acutely isolated retinal wholemounts, retinal pieces from adult animals and humans were exposed to different vital dyes just after the calcium imaging records were finished. The dyes, Mitotracker Orange, Mitotracker Green, Celltracker Orange, Celltracker Green, and monochlorobimane, are all selectively taken up by Müller glial cells, while neuronal cells remain largely devoid of the dyes. By using this method, it can be demonstrated that the free calcium alterations within the wholemounts indeed occur within Müller cells. Moreover, the cross‐sectional areas of (dye‐filled) Müller glial cell bodies, as well as of (dye‐free) neuronal cell bodies, can be measured in retinal wholemounts, and the spatial densities of both types of cells can be determined. The vital dye loading of Müller cells may facilitate investigations of stimulus‐induced alterations of retinal glial cell physiology and morphology.

Comparative studies on mammalian Muller (retinal glial) cells

Muller cells from 22 mammalian species were subjected to morphological and electrophysiological studies. In the ‘mid-periphery’ of retinae immunocytochemically labeled for vimentin, estimates of Muller cell densities per unit retinal surface area, and of neuron-to-(Muller) glia indices were performed. Muller cell densities were strikingly similar among the species studied (around 8000–11000 mm−2) with the extremes of the horse (≤5000 mm−2) and the tree shrew (≥20000 mm−2). By contrast, the number of neurons per Muller cell varied widely, being clustered at 6–8 (in retinae with many cones), at about 16, and at up to more than 30 (in strongly rod-dominated retinae). Isolated Muller cell volumes were estimated morphometrically, and cell surface areas were calculated from membrane capacities. Muller cells isolated from thick vascularized retinae (carnivores,rats, mice, ungulates) were longer and thinner, and had smaller volumes but higher surface-to-volume ratios than cells from thin paurangiotic (i.e. with blood vessels only near the optic disc) or avascular retinae (rabbits, guinea pigs, horses, zebras). In whole-cell voltage-clamp studies, Muller cells from all mammals studied displayed two dominant K+ conductances, inwardly rectifying currents and delayed rectifier currents. TTX-sensitive Na+ currents were recorded only in some species. Based on these data, the following hypotheses are presented, (a) neuron-to-(Muller) glia indices are determined by precursor cell proliferation rather than by metabolic demands; (b) Muller cell volumes depend on available space rather than on the number of supported neurons; and (c) it follows that, the specific metabolic activities of Muller cells must differ greatly between species, a difference that may contribute to distinct patterns of retinal vascularization.

Early Activation of Inflammation-and Immune Response-Related Genes after Experimental Detachment of the Porcine Retina

PURPOSE To determine early alterations in retinal gene expression in a porcine model of rhegmatogenous retinal detachment. METHODS Local detachment was created in eyes of adult pigs by subretinal application of sodium hyaluronate. The gene expression in control tissues and retinas detached for 24 hours was analyzed with a pig genome microarray. Genes with at least three-fold expression changes were detected in the detached retina and in the attached retinal tissue surrounding the local detachment in situ. Structural alterations of the retina were examined by light and electron microscopy. RESULTS Identified were 85 genes that were upregulated and 7 that were downregulated in the detached retina. Twenty-eight genes were identified as upregulated in the nondetached retina of the surgical eyes. The genes upregulated in detached retinas were related to inflammation and immune responses (n = 52), antioxidants and metal homeostasis (n = 7), intracellular proteolysis (n = 6), and blood coagulation/fibrinolysis (n = 4). The upregulation of at least 15 interferon-stimulated genes indicates elevated interferon levels after detachment. Gene expression of blue-sensitive opsin was not detectable in the detached retinal tissue, suggesting an early reduction in phototransduction, especially in blue cones. Electron microscopy revealed an accumulation of microglial cells in the inner retinal tissue and of polymorphonuclear leukocytes in the vessels of detached and peridetached retinas. CONCLUSIONS Differentially expressed genes in the retina early after experimental detachment are mainly related to inflammation and immune responses, intracellular proteolysis, and protection against oxidative stress. A local immune and inflammatory response may represent a major causative factor for reactive changes in the retina after detachment. The inflammatory response is not restricted to the detached retina but is also observed in the nondetached retina; this response may underlie functional changes in these regions described in human subjects.

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.

Spatial mapping of the mechanical properties of the living retina using scanning force microscopy

The retina is an active soft material, in which mechanosensitive cells are thought to respond to the local mechanical heterogeneity they encounter during development and adult physiological functioning. The retina is also constantly exposed to mechanical stress with shear and traction forces acting at its inner surface. Consequences of these forces depend on the tissues resistance to deformation, which is characterized by its stiffness. However, currently there is a lack of high-resolution data on retinal mechanical properties. Here, we used scanning force microscopy to determine the apparent elastic modulus K of the retinal inner surface along the length of the eye with sub-millimetre resolution, and compared characteristic K values of the retinal quadrants. We found that the inner retina is a mechanically inhomogeneous tissue. Most elastic moduli were in the range of 940 to 1800 Pa; significant differences were found between areas less than 50 µm apart. To identify the origin of this mechanical inhomogeneity, we investigated the size and distribution of structures comprising the retinal surface: large cell bodies in the ganglion cell layer, nerve fibers, inner limiting membrane, and Muller cell endfeet. Our data suggest that the distribution of compliant nerve fiber bundles and stiff neuronal cell bodies contributes most to the mechanical properties of the inner retina. These data offer a basis for understanding cellular mechanoresistivity and -sensitivity in the retina as a mechanically active tissue, and they may help to understand mechanisms and consequences of a variety of retino-pathological processes and their surgical treatment.