Effects of Alzheimer-Like Pathology on Homocysteine and Homocysteic Acid Levels-An Exploratory In Vivo Kinetic Study.

Hyperhomocysteinemia has been suggested potentially to contribute to a variety of pathologies, such as Alzheimer's disease (AD). While the impact of hyperhomocysteinemia on AD has been investigated extensively, there are scarce data on the effect of AD on hyperhomocysteinemia. The aim of this in vivo study was to investigate the kinetics of homocysteine (HCys) and homocysteic acid (HCA) and effects of AD-like pathology on the endogenous levels. The mice received a B-vitamin deficient diet for eight weeks, followed by the return to a balanced control diet for another eight weeks. Serum, urine, and brain tissues of AppNL-G-F knock-in and C57BL/6J wild type mice were analyzed for HCys and HCA using LC-MS/MS methods. Hyperhomocysteinemic levels were found in wild type and knock-in mice due to the consumption of the deficient diet for eight weeks, followed by a rapid normalization of the levels after the return to control chow. Hyperhomocysteinemic AppNL-G-F mice had significantly higher HCys in all matrices, but not HCA, compared to wild type control. Higher serum concentrations were associated with elevated levels in both the brain and in urine. Our findings confirm a significant impact of AD-like pathology on hyperhomocysteinemia in the AppNL-G-F mouse model. The immediate normalization of HCys and HCA after the supply of B-vitamins strengthens the idea of a B-vitamin intervention as a potentially preventive treatment option for HCys-related disorders such as AD.

RELSA-A multidimensional procedure for the comparative assessment of well-being and the quantitative determination of severity in experimental procedures.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1<0.27, L2<0.59, L3<0.79, and L4<3.45. We show further that severity patterns in the contributing variables are time and model-specific and use this information to obtain contextualized between animal-model and subgroup comparisons with the severity of sepsis > surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

The Roles of Long-Term Hyperhomocysteinemia and Micronutrient Supplementation in the AppNL-G-F Model of Alzheimer's Disease.

A causal contribution of hyperhomocysteinemia to cognitive decline and Alzheimer's disease (AD), as well as potential prevention or mitigation of the pathology by dietary intervention, have frequently been subjects of controversy. In the present in vivo study, we attempted to further elucidate the impact of elevated homocysteine (HCys) and homocysteic acid (HCA) levels, induced by dietary B-vitamin deficiency, and micronutrient supplementation on AD-like pathology, which was simulated using the amyloid-based AppNL-G-F knock-in mouse model. For this purpose, cognitive assessment was complemented by analyses of ex vivo parameters in whole blood, serum, CSF, and brain tissues from the mice. Furthermore, neurotoxicity of HCys and HCA was assessed in a separate in vitro assay. In confirmation of our previous study, older AppNL-G-F mice also exhibited subtle phenotypic impairment and extensive cerebral amyloidosis, whereas dietary manipulations did not result in significant effects. As revealed by proximity extension assay-based proteome analysis, the AppNL-G-F genotype led to an upregulation of AD-characteristic neuronal markers. Hyperhomocysteinemia, in contrast, indicated mainly vascular effects. Overall, since there was an absence of a distinct phenotype despite both a significant amyloid-ß burden and serum HCys elevation, the results in this study did not corroborate the pathological role of amyloid-ß according to the "amyloid hypothesis," nor of hyperhomocysteinemia on cognitive performance. Nevertheless, this study aided in further characterizing the AppNL-G-F model and in elucidating the role of HCys in diverse biological processes. The idea of AD prevention with the investigated micronutrients, however, was not supported, at least in this mouse model of the disease.

Improving target assessment in biomedical research: the GOT-IT recommendations.

Academic research plays a key role in identifying new drug targets, including understanding target biology and links between targets and disease states. To lead to new drugs, however, research must progress from purely academic exploration to the initiation of efforts to identify and test a drug candidate in clinical trials, which are typically conducted by the biopharma industry. This transition can be facilitated by a timely focus on target assessment aspects such as target-related safety issues, druggability and assayability, as well as the potential for target modulation to achieve differentiation from established therapies. Here, we present recommendations from the GOT-IT working group, which have been designed to support academic scientists and funders of translational research in identifying and prioritizing target assessment activities and in defining a critical path to reach scientific goals as well as goals related to licensing, partnering with industry or initiating clinical development programmes. Based on sets of guiding questions for different areas of target assessment, the GOT-IT framework is intended to stimulate academic scientists' awareness of factors that make translational research more robust and efficient, and to facilitate academia-industry collaboration.

Impact of Hyperhomocysteinemia and Different Dietary Interventions on Cognitive Performance in a Knock-in Mouse Model for Alzheimer's Disease.

BACKGROUND: Hyperhomocysteinemia is considered a possible contributor to the complex pathology of Alzheimer's disease (AD). For years, researchers in this field have discussed the apparent detrimental effects of the endogenous amino acid homocysteine in the brain. In this study, the roles of hyperhomocysteinemia driven by vitamin B deficiency, as well as potentially beneficial dietary interventions, were investigated in the novel AppNL-G-F knock-in mouse model for AD, simulating an early stage of the disease. METHODS: Urine and serum samples were analyzed using a validated LC-MS/MS method and the impact of different experimental diets on cognitive performance was studied in a comprehensive behavioral test battery. Finally, we analyzed brain samples immunohistochemically in order to assess amyloid-ß (Aß) plaque deposition. RESULTS: Behavioral testing data indicated subtle cognitive deficits in AppNL-G-F compared to C57BL/6J wild type mice. Elevation of homocysteine and homocysteic acid, as well as counteracting dietary interventions, mostly did not result in significant effects on learning and memory performance, nor in a modified Aß plaque deposition in 35-week-old AppNL-G-F mice. CONCLUSION: Despite prominent Aß plaque deposition, the AppNL-G-F model merely displays a very mild AD-like phenotype at the investigated age. Older AppNL-G-F mice should be tested in order to further investigate potential effects of hyperhomocysteinemia and dietary interventions.