C. A. F. von Arnim

Neuroprotection and neuronal dysfunction upon repetitive inhibition of oxidative phosphorylation

Repetitive inhibition of oxidative phosphorylation is an established model of neurodegeneration. In contrast, a single mild treatment can be neuroprotective-chemical preconditioning. Repetitive chemical inhibition of oxidative phosphorylation may thus be a tool to study deterioration and improvement of cellular hypoxic tolerance and subsequent differential regulation of cellular responses in the same model. We investigated murine hippocampal function upon repetitive intraperitoneal injections of 3-nitropropionate (3-NP; 20 mg/kg body weight), an inhibitor of mitochondrial complex II. With a 2-day interval of repetitive in vivo treatment with 3-NP, posthypoxic recovery of population spike amplitude was below control. In contrast, even after nine in vivo treatments with 3-NP at 4-day intervals, an almost complete recovery of population spike amplitude was observed. Nerve growth factor (NGF) as assessed by ELISA and expression of beta-amyloid precursor protein (APP) mRNA increased upon nine treatments at 2-day intervals, but remained at control levels with 4-day intervals. In contrast, brain-derived neurotrophic factor (BDNF) as assessed by ELISA increased with the latter treatment. Expression of mRNA for adenosine-A1 and -A3 receptors and endothelial and neuronal nitric oxide synthase remained at control level for both treatment intervals. We conclude that the time interval between mild, subclinical repetitive inhibition of oxidative phosphorylation determines hippocampal neuronal impairment and integrity and modulates NGF and BDNF differently. Decreased hypoxic tolerance and increased APP expression upon repetitive inhibition of oxidative phosphorylation at short time intervals may thus trigger a vicious cycle and be a cofactor for neuronal dysfunction in cerebral hypoxia and neurodegenerative diseases.

Gender-dependent hypoxic tolerance mediated via gender-specific mechanisms

Primary hypoxic tolerance and preconditioning are gender dependent and modulated in females during the estrus cycle. The underlying mechanisms, however, remain to be determined. mRNA of estrogen receptor‐α (EAR), progesterone receptor (PR), and adenosine receptor subtypes A1 and A3 (A1R and A3R) were investigated with reverse transcriptase‐PCR in hippocampi from control male and female mice and animals treated in vivo with a single i.p. injection of 20 mg/kg body weight 3‐nitropropionate (3NP) 1 or 24 hr prior to preparation. Results were analyzed relative to expression in hippocampi from untreated males. mRNA levels of EAR and A1R were alike in males and females and unaltered by preconditioning with 3NP. In contrast, PR mRNA levels were alike in males and females during proestrus but lower during estrus and diestrus (85% ± 15%, P < 0.05; and 80% ± 10%, P < 0.05, respectively). Upon preconditioning, PR mRNA decreased to 67% ± 19% (P < 0.05) and 56% ± 13% (P < 0.05) during proestrus and diestrus, respectively, but was unaltered during estrus and in males. On preconditioning, A3R mRNA decreased from 115% ± 16% to 86% ± 29% (P < 0.05) during diestrus but remained at the control level during proestrus and estrus. With low‐level expression of PRs, as achieved upon preconditioning, hypoxic tolerance is increased. Other than in males, adenosine A3 receptors are not up‐regulated upon preconditioning in females. Thus, not only is net hypoxic tolerance gender dependent but mechanisms conferring hypoxic tolerance are gender specific.

An unbiased, staged, multicentre, validation strategy for Alzheimer's disease CSF tau levels

Newly proposed diagnostic criteria for Alzheimers disease include cerebrospinal fluid (CSF) tau levels as one core supportive criterion. The published high sensitivity and specificity figures for CSF tau levels in Alzheimers disease are offset by the large range of proposed cutoff values (9.6 pg/mL to 1140 pg/mL). This study aimed to provide guidance on how to establish, validate and audit CSF tau cutoff values using an unbiased, two-stage multicentre strategy. Both receiver operator characteristics (ROC) optimised and population-based cutoff values were calculated on a pilot dataset (n=99), validated in a large dataset (n=560) and then compared to the literature. The data suggest using an ROC optimised cutoff level of 323+/-51.7 pg/mL allowing for the published inter-laboratory coefficient of variation of 16%. This cutoff level was confirmed in a prospective audit (n=100). As demand for CSF tau levels will increase globally, the accuracy of local CSF hTau cutoff levels can be compared against this benchmark.

Mechanisms of hypoxic tolerance in presymptomatic APP23 transgenic mice

In the B6-Tg (ThylAPP)23Sdz (APP23tg) transgenic mouse model of Alzheimers disease hypoxic tolerance is impaired prior to amyloid deposition. We therefore investigated mechanisms known to mediate resistance to hypoxic episodes in presymptomatic APP23tg and appropriate control strains. The mRNA expression levels in the hippocampus of adenosine receptor subtypes A1 and A3, estrogen receptors alpha and beta, progesterone receptor, and neuronal and endothelial nitric oxide synthase were investigated with semi-quantitative RT-PCR. Mice were pretreated in vivo with a low dose of 3-nitropropionate, an inhibitor of succinic dehydrogenase, known to mediate hypoxic tolerance within 1h. We found increased expression levels in presymptomatic, untreated APP23tg animals of adenosine A3 receptor mRNA and estrogen receptor alpha mRNA. In addition, we observed an increase in nNOS expression levels upon mild cellular hypoxia induced by 3-NP in transgenic but not in wild-type animals. We conclude that overexpression of human APP results in differential expression of receptors conferring hypoxic tolerance prior to amyloid deposition. Up-regulation of nNOS expression levels upon hypoxic challenge in APP23tg transgenic animals may therefore reflect a selective vulnerability in these animals even before amyloid deposition.

Regional selectivity of amyloid mRNA expression and neurotrophins on repetitive inhibition of oxidative phosphorylation

Summary.Previously we showed that repetitive inhibition of oxidative phosphorylation impairs synaptic transmission and induces overexpression of amyloid precursor protein mRNA (APP-mRNA) in the hippocampus (Hellweg et al., 2003). Here we show that APP-mRNA remains alike in murine frontal cortex and cerebellum on repetitive treatment with 3-nitropropionate. However, nerve growth factor and brain-derived neurotrophic factor decreased by 28 to 38% in frontal cortex. Taken together, the pattern of change resembles genetic models of Alzheimer’s disease with less susceptibility for overexpression of amyloid mRNA in frontal cortex than in hippocampus and reduced neurotrophin levels in frontal cortex. Given the similarity of this pattern to the one observed in human Alzheimer’s disease the present model in future may give further insight into the pathophysiology of sporadic Alzheimer’s disease.

Strain dependence of receptor regulation on chemical preconditioning in mice hippocampus

While one current focus for studying mechanisms of disease is investigation of transgenic mice confounding effects of the background strain often are neglected. We investigated mRNA expression of known markers of hypoxic tolerance by a semiquantitative RT-PCR (adenosine receptors (A1 and A3), nitric oxide synthases (eNOS and nNOS), APP production, progesterone receptor, and estrogen receptors alpha and beta) in CD-1, C3H, and B6 mice. We found differences in the baseline mRNA expression of adenosine A3 receptors in C3H mice and neuronal NOS in B6 mice as well as a distinct regulation of adenosine A3 receptors and estrogen receptor beta (no changes in C3H and B6 compared to upregulation in CD-1) on treatment of animals with a low dosage of 3-nitropropionate (20mg/kg body weight, i.p.). We conclude that the choice of background strain may confound interpretation of the effects of specific transgens in the study of the mechanisms of primary and induced hypoxic tolerance.

Multiwavelength FLIM: new applications and algorithms

The combination of time-resolved and spectral resolved techniques as achieved by SLIM (spectrally resolved fluorescence lifetime imaging) improves the analysis of complex situations, when different fluorophores have to be distinguished. This could be the case when endogenous fluorophores of living cells and tissues are observed to identify the redox state and oxidative metabolic changes of the mitochondria. Other examples are FRET (resonant energy transfer) measurements, when different donor/acceptor pairs are observed simultaneously. SLIM is working in the time domain employing excitation with short light pulses and detection of the fluorescence intensity decay in many cases with time-correlated single photon counting (TCSPC). Spectral resolved detection is achieved by a polychromator in the detection path and a 16-channel multianode photomultiplier tube with the appropriate routing electronics. Within this paper special attention will be focused on FRET measurements with respect to protein interactions in Alzheimers disease. Using global analysis as the phasor plot approach or integration of the kinetic equations taking into account the multidimensional datasets in every spectral channel we could demonstrate considerable improvement of our calculations.

SLIM: multispectral FLIM with wide applications in cell biology

The fluorescence decay of a fluorophore in many cases does not show a simple monoexponential profile. A very complex situation arises, when more than one compound must be analyzed. A considerable improvement of the measurement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated. SLIM (spectral fluorescence lifetime imaging) is a new technique, which combines both. Time-correlated single photon counting (TCSPC) enables high counting efficiency for biomedical applications. For spectral resolved detection a polychromator in the detection path together with a 16-channel multianode photomultiplier tube and appropriate TCSPC routing electronics are used as a highly sophisticated system. The various possibilities which SLIM offers to improve molecular imaging in living cells will be discussed as well as successfully realized applications. These include FRET (resonant energy transfer) measurements for protein interactions, related to Alzheimers disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase (BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.

Correlated oxygen-sensing PLIM, cell metabolism FLIM and applications

Correlated imaging of phosphorescence and fluorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis, a process known as “Warburg” effect. Also during stem cell differentiation a switch in cell metabolism is observed. Mitochondrial dysfunction associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimer´s disease, cardiac ischemia/reperfusion injury, tissue inflammation and cancer. Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect oxygen concentration and distribution are therefore of prominent interest. Moreover, they offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways, whereas oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NAD(P)H and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.

Interaction of Engulfment Adaptor Protein (GULP) and Amyloid Precursor Protein (APP) decreases Aβ40/42 secretion

Background: The frequent co-occurrence of Alzheimer’s disease (AD) pathology in patients with clinically diagnosed normal pressure hydrocephalus suggests a possible link between ventricular dilation and AD. If enlarging ventricles serve as a marker of potentially faulty cerebrospinal fluid (CSF) clearance mechanisms, then a relationship may be demonstrable between increasing ventricular volume and decreasing levels of A-beta in CSF in preclinical and early AD. Methods: CSF biomarker data (A-beta, tau, and phosphorylated tau) as well as direct measurements of whole brain and ventricular volume data were obtained from the Alzheimer’s Disease Neuroimaging Initiative dataset. The ratio of ventricular volume to whole brain volume was derived as a secondary independent measure. Baseline data were used for the group analyses of 819 subjects classified as being either normal (n1⁄4229), having the syndrome of mild cognitive impairment (n1⁄4397), or mild AD (n1⁄4193). Linear regression models were derived for each biomarker as the dependent variable, using the MRI volume measures and age as independent variables. Subjects in each diagnostic group were further stratified by ApoE genotype. Results: For controls, ventricular volume was negatively associated with A-beta in E4 positive subjects, independent of age. Ventricular volume as well as age was negatively associated with tau in E4 negative control subjects. A significant reversed pattern was seen in AD subjects, in whom ventricular volume was negatively associated with tau, but not A-beta, in E4 positive subjects, independent of age. Abeta and tau were not significantly related to whole brain volume in either controls or AD; however, tau was negatively related to whole brain volume in MCI subjects. Conclusions: Increased ventricular volume may be associated with decreased levels of CSF A-beta in preclinical AD. The basis for the apparent effect of ApoE4 genotype on the relationship of ventricular volume to A-beta and tau levels is unknown, but could involve altered CSF-bloodbrain barrier function early in the course of the disease. Further experimental evidence with animal models of hydrocephalus and ApoE may shed light on the exact nature of these relationships.