Tobias Stahl

Astrocytic expression of the Alzheimer's disease β-secretase (BACE1) is stimulus-dependent

The beta‐site APP‐cleaving enzyme (BACE1) is a prerequisite for the generation of β‐amyloid peptides, which give rise to cerebrovascular and parenchymal β‐amyloid deposits in the brain of Alzheimers disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to β‐amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N‐M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by β‐amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1‐immunoreactive astrocytes in proximity to β‐amyloid plaques in the brains of aged Tg2576 mice and Alzheimers disease patients. GLIA 41:169–179, 2003.

Phylogenetic diversity of the expression of the microtubule-associated protein tau: implications for neurodegenerative disorders.

The microtubule-associated protein tau regulates the dynamic stability of the neuronal cytoskeleton by interacting with microtubules. It is encoded by a single gene, but expressed in a variety of isoforms due to differential RNA splicing. Six isoforms can be found in the human central nervous system. These isoforms differ in their ability to promote the assembly of microtubules as well as in their capacity to stabilize existing microtubule structures. Furthermore, some of the isoforms of tau are specifically involved in the pathogenesis of neurodegenerative disorders. Thus, splicing of tau might critically influence the physiological functions of tau protein as well as the pathogenesis of neurodegenerative diseases with tauopathy. The present study addresses the differential expression of the six isoforms of tau in the central nervous system of 12 mammalian species including Homo sapiens. The occurrence of each of the six tau isoforms was highly variable. However, species that were phylogenetically related expressed a similar pattern of tau isoforms. These results suggest a phylogenetic descent of splicing paradigms, which can be matched with known phylogenetic concepts based on morphological and molecular genetical studies. Especially, the unique expression pattern of tau isoforms in the human central nervous system implicates a possible link to the particular vulnerability of humans to neurodegenerative disorders with tauopathy, namely Alzheimers disease, frontotemporal dementia and Picks disease.

Constitutive overactivation of protein kinase C in guinea pig brain increases α-secretory APP processing without decreasing β-amyloid generation

Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and β‐amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA‐methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester‐stimulated PKC enzyme activity (by 47% and 71%) as compared to age‐matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol‐treated animals, there was increased α‐secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP α‐secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of β‐amyloid peptides 1–40 or 1–42 as measured by ELISA, suggesting that only the α‐secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.

Bepridil and Amiodarone Simultaneously Target the Alzheimer's Disease β- and γ-Secretase via Distinct Mechanisms

The two proteases β-secretase and γ-secretase generate the amyloid β peptide and are drug targets for Alzheimers disease. Here we tested the possibility of targeting the cellular environment of β-secretase cleavage instead of the β-secretase enzyme itself. β-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited β-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit β-secretase. Surprisingly, bepridil and amiodarone also modulated γ-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target β- and γ-secretase through distinct molecular mechanisms. In addition to Alzheimers disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.

Assessment of neuroprotective effects of human umbilical cord blood mononuclear cell subpopulations in vitro and in vivo.

Experimental transplantation of human umbilical cord blood (hUCB) mononuclear cells (MNCs) in rodent stroke models revealed the therapeutic potential of these cells. However, effective cells within the heterogeneous MNC population and their modes of action are still under discussion. MNCs and MNC fractions enriched (CD34+) or depleted (CD34-) for CD34-expressing stem/progenitor cells were isolated from hUCB. Cells were transplanted intravenously following middle cerebral artery occlusion in spontaneously hypertensive rats and directly or indirectly cocultivated with hippocampal slices previously subjected to oxygen and glucose deprivation. Application of saline solution or a human T-cell line served as controls. In vivo, MNCs, CD34+ and CD34- cells reduced neurofunctional deficits and diminished lesion volume as determined by magnetic resonance imaging. MNCs were superior to other fractions. However, human cells could not be identified in brain tissue 29 days after stroke induction. Following direct application on postischemic hippocampal slices, MNCs reduced neural damage throughout a 3-day observation period. CD34+ cells provided transient protection for 2 days. The CD34- fraction, in contrast to in vivo results, failed to reduce neural damage. Direct cocultivation of MNCs was superior to indirect cocultivation of equal cell numbers. Indirect application of up to 10-fold MNC concentrations enhanced neuroprotection to a level comparable to direct cocultivation. After direct application, MNCs migrated into the slices. Flow cytometric analysis of migrated cells revealed that the CD34+ cells within MNCs were preferably attracted by damaged hippocampal tissue. Our study suggests that MNCs provide the most prominent neuroprotective effect, with CD34+ cells seeming to be particularly involved in the protective action of MNCs. CD34+ cells preferentially home to neural tissue in vitro, but are not superior concerning the overall effect, implying that there is another, still undiscovered, protective cell population. Furthermore, MNCs did not survive in the ischemic brain for longer periods without immunosuppression.

Electrophysiological alterations and upregulation of ATP receptors in retinal glial Müller cells from rats infected with the Borna disease virus.

Infection with the neurotropic Borna disease virus (BDV) causes an immune‐mediated neurological disease in a broad range of species. In addition to encephalitis, BDV‐infected Lewis rats develop a retinitis histologically characterized by the loss of most retinal neurons. By contrast, the dominating retinal macroglia, the Müller cells, do not degenerate. It is known from several models of neurodegeneration that glial cells may survive but undergo significant alterations of their physiological parameters. This prompted us to study the electrophysiology and ATP‐induced changes of intracellular Ca2+‐concentration ([Ca2+]i) in Müller cells from BDV‐infected rat retinae. Freshly isolated cells were used for whole‐cell patch‐clamp recordings. Whereas neither zero current potentials nor membrane resistances showed significant alterations, the membrane capacitance increased in cells from BDV‐infected rats during survival times of up to 8 months. This process was accompanied by a decrease in K+ current densities. Müller cells from BDV‐infected rats were characterized by expression of a prominent fast‐inactivating A‐type K+ current which was rarely found in control cells. Moreover, the number of cells displaying Na+ currents was slightly increased after BDV‐infection. ATP evoked increases in [Ca2+]i in Müller cells within retinal wholemounts of both control and BDV‐infected animals. However, the number of ATP‐responding isolated cells increased from 24% (age‐matched controls) to 78% (cells from animals ≥18 weeks after infection). We conclude that in BDV‐induced retinopathy, reactive rat Müller cells change their physiological parameters but these changes are different from those in Müller cells during proliferative vitreoretinopathy in man and rabbit. GLIA 35:213–223, 2001.

Neuronal hypoxia in vitro: investigation of therapeutic principles of HUCB-MNC and CD133+ stem cells.

BackgroundThe therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood.MethodsA human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC), a stem cell enriched fraction (CD133+, 97.38% CD133-positive cells) and a stem cell depleted fraction (CD133-, 0.06% CD133-positive cells) of HUCB-MNC by either direct or indirect co-cultivation with post-hypoxic neuronal cells (differentiated SH-SY5Y). Over three days, development of apoptosis and necrosis of neuronal cells, chemotaxis of MNC and production of chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9) and growth factors (G-CSF, GM-CSF, VEGF, bFGF) were analyzed using fluorescence microscopy, FACS and cytometric bead array.ResultstMNC, CD133+ and surprisingly CD133- reduced neuronal apoptosis in direct co-cultivations significantly to levels in the range of normoxic controls (7% ± 3%). Untreated post-hypoxic control cultures showed apoptosis rates of 85% ± 11%. tMNC actively migrated towards injured neuronal cells. Both co-cultivation types using tMNC or CD133- reduced apoptosis comparably. CD133- produced high concentrations of CCL3 and neuroprotective G-CSF within indirect co-cultures. Soluble factors produced by CD133+ cells were not detectable in direct co-cultures.ConclusionOur data show that heterogeneous tMNC and even CD133-depleted fractions have the capability not only to reduce apoptosis in neuronal cells but also to trigger the retaining of neuronal phenotypes.

Neuron-glia interactions in the rat retina infected by Borna disease virus.

Summary. Neuron-glia interactions in the Borna disease virus (BDV)-infected rat retina were investigated with emphasis on the ultrastructural characterization of degenerative alterations in the ganglion cell and photoreceptor layer. Immuno- and cytochemical techniques were applied to label microglia, macrophages and Müller (macroglial) cells. Four weeks after intracerebral infection of adult rats, the total thickness of the retina was considerably diminished, primarily due to the loss of photoreceptor segments and ganglion cells. A gradual reduction of both plexiform layers was also observed. There was a remarkable increase in the number of microglial cells, predominantly in the ganglion cell and the inner plexiform layers. Ultrastructural analysis confirmed that microglia, but also macrophages, were involved in phagocytosis accompanying severe neuronal degeneration in the ganglion cell and the photoreceptor layer. In contrast, Müller cells showed moderate morphological and cytochemical alterations, indicating that Müller cells play only a minor role in early stages of BDV-induced retinitis. Monitoring neuron-glia interactions in BDV-induced retinopathy, combined with the application of different protocols of immunosuppression effecting the BDV virus and/or the microglia, might help to establish specific strategies to suppress BDV-induced neuronal degeneration.

Viral‐induced inflammation is accompanied by β‐amyloid plaque reduction in brains of amyloid precursor protein transgenic Tg2576 mice

Amyloid plaques, one of the neuropathological hallmarks of Alzheimers disease, and their main constituent, the amyloid β‐peptide (Aβ), are triggers of the activation of innate inflammatory mechanisms involving the activation of microglia. To dissect the effects of a non‐Aβ‐specific microglial activation on the Aβ metabolism, we employed a viral infection‐based model. Transgenic mice expressing a mutated form of the human amyloid precursor protein (Tg2576) were used. In preceding experiments, 2‐week‐old transgenic mice and non‐transgenic littermates were infected intracerebrally with the neurotropic Borna disease virus and investigated at 2, 4 and 14 weeks post‐infection. The Borna disease virus‐inoculated mice showed a persisting, subclinical infection of cortical and limbic brain areas characterized by slight T‐cell infiltrates, expression of cytokines and a massive microglial activation in the hippocampus and neocortex. Viral‐induced effects reached their peak at 4 weeks post‐infection. In 14‐month‐old Tg2576 mice, characterized by the deposition of diffuse and dense‐core amyloid plaques in cortical brain regions, Borna disease virus‐induced microglial activation in the vicinity of Aβ deposits was used to investigate the influence of a local inflammatory response on these deposits. At 4 weeks post‐infection, histometric analyses employing Aβ immunohistochemistry revealed a decrease of the cortical and hippocampal Aβ‐immunopositive area. This overall decrease was accompanied by a decrease of parenchymal thioflavin‐S‐positive amyloid deposits and an increase of such deposits in the walls of cerebral vessels, which indicates that the elicitation of a non‐Aβ‐specific microglial activation may contribute to a reduction of Aβ in the brain parenchyma.

Sterile Inflammation after Permanent Distal MCA Occlusion in Hypertensive Rats

The pathophysiology of stroke is governed by immune reactions within and remote from the injured brain. Hypertension, a major cause and comorbidity of stroke, entails systemic vascular inflammation and may influence poststroke immune responses. This aspect is, however, underestimated in previous studies. Here we aimed to delineate the sequence of cellular inflammation after stroke in spontaneously hypertensive (SH) rats. Spontaneously hypertensive rats were subjected to permanent middle cerebral artery occlusion and killed after 1 or 4 days. Immune cells of the peripheral blood and those which have infiltrated the injured brain were identified and quantified by flow cytometry. The spatial distribution of myeloid cells and T lymphocytes, and the infarct volume were assessed by histology. We observed a concerted infiltration of immune cells into the ischemic brain of SH rats. At day 1, primarily neutrophils, monocytes, macrophages, and myeloid dendritic cells entered the brain, whereas the situation at day 4 was dominated by microglia, macrophages, lymphatic dendritic cells, and T cells. Postischemic inflammation did not cause secondary tissue damage during the subacute stage of experimental stroke in SH rats. Considering the intrinsic vascular pathology of SH rats, our study validates this strain for further translational research in poststroke inflammation.