Botond Penke

Amyloid β-Induced Neuronal Hyperexcitability Triggers Progressive Epilepsy

Alzheimers disease is associated with an increased risk of unprovoked seizures. However, the underlying mechanisms of seizure induction remain elusive. Here, we performed video-EEG recordings in mice carrying mutant human APPswe and PS1dE9 genes (APdE9 mice) and their wild-type littermates to determine the prevalence of unprovoked seizures. In two recording episodes at the onset of amyloid β (Aβ) pathogenesis (3 and 4.5 months of age), at least one unprovoked seizure was detected in 65% of APdE9 mice, of which 46% had multiple seizures and 38% had a generalized seizure. None of the wild-type mice had seizures. In a subset of APdE9 mice, seizure phenotype was associated with a loss of calbindin-D28k immunoreactivity in dentate granular cells and ectopic expression of neuropeptide Y in mossy fibers. In APdE9 mice, persistently decreased resting membrane potential in neocortical layer 2/3 pyramidal cells and dentate granule cells underpinned increased network excitability as identified by patch-clamp electrophysiology. At stimulus strengths evoking single-component EPSPs in wild-type littermates, APdE9 mice exhibited decreased action potential threshold and burst firing of pyramidal cells. Bath application (1 h) of Aβ1–42 or Aβ25–35 (proto-)fibrils but not oligomers induced significant membrane depolarization of pyramidal cells and increased the activity of excitatory cell populations as measured by extracellular field recordings in the juvenile rodent brain, confirming the pathogenic significance of bath-applied Aβ (proto-)fibrils. Overall, these data identify fibrillar Aβ as a pathogenic entity powerfully altering neuronal membrane properties such that hyperexcitability of pyramidal cells culminates in epileptiform activity.

Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease.

Microglial activation is a key feature in Alzheimer’s disease and is considered to contribute to progressive neuronal injury by release of neurotoxic products. The innate immune receptor Toll-like-receptor 4 (TLR4), localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms. Here, we show that a spontaneous loss-of-function mutation in the Tlr4 gene strongly inhibits microglial and monocytic activation by aggregated Alzheimer amyloid peptide resulting in a significantly lower release of the inflammatory products IL-6, TNFα and nitric oxide. Treatment of primary murine neuronal cells with supernatant of amyloid peptide-stimulated microglia demonstrates that Tlr4 contributes to amyloid peptide-induced microglial neurotoxicity. In addition, stimulation experiments in transfected HEK293 cells allowed to define a tri-molecular receptor complex consisting of TLR4, MD-2 and CD14 necessary for full cellular activation by aggregated amyloid peptide. A clinical relevance of these findings is supported by a marked upregulation of Tlr4 mRNA in APP transgenic mice and by an increased expression of TLR4 in Alzheimer’s disease brain tissue associated with amyloid plaque deposition. Together, these observations provide the first evidence for a role of the key innate immune receptor, TLR4, in neuroinflammation in Alzheimer’s disease.

The LPS receptor (CD14) links innate immunity with Alzheimer's disease

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe‐detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimers disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimers disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimers disease.

Impact of different saturated fatty acid, polyunsaturated fatty acid and cholesterol containing diets on beta-amyloid accumulation in APP/PS1 transgenic mice.

The present study assessed the influence of dietary lipids on accumulation of amyloid beta-peptide (Abeta) in the brain. Seven experimental diets with varying n-6/n-3-ratio, saturated and polyunsaturated fatty acid and cholesterol contents were fed to transgenic APPswe/PS1dE9 mice for 3-4 months beginning at a young adult age (6 months). Hippocampal Abeta levels were determined with ELISA and plaque load by using immunocytochemistry. A typical Western diet with 40% saturated fatty acids and 1% of cholesterol increased, while diets supplemented with docosahexaenoic acid (DHA) decreased Abeta levels compared to regular (soy oil based) diet. DHA diet also decreased the number of activated microglia in hippocampus and increased exploratory activity of transgenic mice, but did not improve their spatial learning in the water maze. The favorable effect of DHA on Abeta production was verified in two different cell lines. Regulation of dietary lipid intake may offer a new tool to reduce the risk of Alzheimers disease at the population level.

β-Amyloid neurotoxicity is mediated by a glutamate-triggered excitotoxic cascade in rat nucleus basalis

Whereas a cardinal role for β‐amyloid protein (Aβ) has been postulated as a major trigger of neuronal injury in Alzheimers disease, the pathogenic mechanism by which Aβ deranges nerve cells remains largely elusive. Here we report correlative in vitro and in vivo evidence that an excitotoxic cascade mediates Aβ neurotoxicity in the rat magnocellular nucleus basalis (MBN). In vitro application of Aβ to astrocytes elicits rapid depolarization of astroglial membranes with a concomitant inhibition of glutamate uptake. In vivo Aβ infusion by way of microdialysis in the MBN revealed peak extracellular concentrations of excitatory amino acid neurotransmitters within 20–30 min. Aβ‐triggered extracellular elevation of excitatory amino acids coincided with a significantly enhanced intracellular accumulation of Ca2+ in the Aβ injection area, as was demonstrated by 45Ca2+ autoradiography. In consequence of these acute processes delayed cell death in the MBN and persistent loss of cholinergic fibre projections to the neocortex appear as early as 3 days following the Aβ‐induced toxic insult. Such a sequence of Aβ toxicity was effectively antagonized by the N‐methyl‐d‐aspartate (NMDA) receptor ligand dizocilpine maleate (MK‐801). Moreover, Aβ toxicity in the MBN decreases with advancing age that may be associated with the age‐related loss of NMDA receptor expression in rats. In summary, the present results indicate that Aβ compromises neurons of the rat MBN via an excitotoxic pathway including astroglial depolarization, extracellular glutamate accumulation, NMDA receptor activation and an intracellular Ca2+ overload leading to cell death.

TLR2 Is a Primary Receptor for Alzheimer’s Amyloid β Peptide To Trigger Neuroinflammatory Activation

Microglia activated by extracellularly deposited amyloid β peptide (Aβ) act as a two-edged sword in Alzheimer’s disease pathogenesis: on the one hand, they damage neurons by releasing neurotoxic proinflammatory mediators (M1 activation); on the other hand, they protect neurons by triggering anti-inflammatory/neurotrophic M2 activation and by clearing Aβ via phagocytosis. TLRs are associated with Aβ-induced microglial inflammatory activation and Aβ internalization, but the mechanisms remain unclear. In this study, we used real-time surface plasmon resonance spectroscopy and conventional biochemical pull-down assays to demonstrate a direct interaction between TLR2 and the aggregated 42-aa form of human Aβ (Aβ42). TLR2 deficiency reduced Aβ42-triggered inflammatory activation but enhanced Aβ phagocytosis in cultured microglia and macrophages. By expressing TLR2 in HEK293 cells that do not endogenously express TLR2, we observed that TLR2 expression enabled HEK293 cells to respond to Aβ42. Through site-directed mutagenesis of tlr2 gene, we identified the amino acids EKKA (741–744) as a critical cytoplasmic domain for transduction of inflammatory signals. By coexpressing TLR1 or TLR6 in TLR2-transgenic HEK293 cells or silencing tlrs genes in RAW264.7 macrophages, we observed that TLR2-mediated Aβ42-triggered inflammatory activation was enhanced by TLR1 and suppressed by TLR6. Using bone marrow chimeric Alzheimer’s amyloid precursor transgenic mice, we observed that TLR2 deficiency in microglia shifts M1- to M2-inflammatory activation in vivo, which was associated with improved neuronal function. Our study demonstrated that TLR2 is a primary receptor for Aβ to trigger neuroinflammatory activation and suggested that inhibition of TLR2 in microglia could be beneficial in Alzheimer’s disease pathogenesis.

Docosahexaenoic Acid Reduces Amyloid β Production via Multiple Pleiotropic Mechanisms

Alzheimer disease is characterized by accumulation of the β-amyloid peptide (Aβ) generated by β- and γ-secretase processing of the amyloid precursor protein (APP). The intake of the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been associated with decreased amyloid deposition and a reduced risk in Alzheimer disease in several epidemiological trials; however, the exact underlying molecular mechanism remains to be elucidated. Here, we systematically investigate the effect of DHA on amyloidogenic and nonamyloidogenic APP processing and the potential cross-links to cholesterol metabolism in vivo and in vitro. DHA reduces amyloidogenic processing by decreasing β- and γ-secretase activity, whereas the expression and protein levels of BACE1 and presenilin1 remain unchanged. In addition, DHA increases protein stability of α-secretase resulting in increased nonamyloidogenic processing. Besides the known effect of DHA to decrease cholesterol de novo synthesis, we found cholesterol distribution in plasma membrane to be altered. In the presence of DHA, cholesterol shifts from raft to non-raft domains, and this is accompanied by a shift in γ-secretase activity and presenilin1 protein levels. Taken together, DHA directs amyloidogenic processing of APP toward nonamyloidogenic processing, effectively reducing Aβ release. DHA has a typical pleiotropic effect; DHA-mediated Aβ reduction is not the consequence of a single major mechanism but is the result of combined multiple effects.

Immunohistochemical visualization of a metabotropic glutamate receptor.

The immunocytochemical localization of the recently cloned metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was demonstrated with a C-terminus specific antibody in rat cerebellar cortex. This antibody detects a 138-140 kDa major, and a 46 kDa minor band in membrane preparations of rat cortex and cerebellum. mGluR1 alpha immunoreactivity (mGRi) was present in Purkinje and basket cells. Purkinje cell dendritic spines and their postsynaptic membranes showed selective labelling. Presynaptic membranes, parallel fibres and glial processes were devoid of mGRi. It is suggested that the selective postsynaptic localization of this receptor at the dendritic spines of Purkinje cells serves as the morphological basis for long term depression processes in the molecular layer of the cerebellar cortex.

The cocaine-induced elevation of plasma corticosterone is mediated by endogenous corticotropin-releasing factor (CRF) in rats

The role of endogenous corticotropin-releasing factor (CRF) in the cocaine-induced corticosterone response was investigated by using the immunoneutralization and receptor blockade of endogenous CRF. Pretreatment with different dilutions (1:5, 1:10 and 1:20, i.c.v.) of CRF antibody and different doses of an antagonist for CRF receptors, alpha-helical CRF9-41 (alpha h-CRF, 0.001-1.0 micrograms, i.c.v.), dose-dependently prevented the cocaine-induced increase in corticosterone level. These results support the hypothesis that the activation of the hypothalamo-pituitary-adrenal (HPA) axis by cocaine is mediated through the release of endogenous CRF.

Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells

Reliable in vitro assays are essential for study of the effects of neurotoxic compounds such as beta-amyloid peptides (Abeta). The MTT assay has been used in cultures of different cells, e.g. SH-SY5Y neuroblastoma cells, for the quantitative measurement of Abeta toxicity. In our laboratory differentiated SH-SY5Y cells were used in the MTT assay. Cell differentiation with 10 microM all-trans-retinoic acid resulted in a constant cell number. The cells possess highly developed neurites and exhibit high sensitivity against Abeta. Owing to the constant cell number in differentiated SH-SY5Y cultures the decrease of the redox activity is directly proportional to the neurotoxicity of the substances, no correction is needed. The results of the MTT assay of Abeta peptides on differentiated SH-SY5Y cells displayed a good correlation also with the in vivo results. The present experiments reveal an effective assay for the study of potentially neurotoxic compounds.