Biology subjects

Freichel, M.

Publications and source records attributed to Freichel, M..

6 recordsLinked to original sources

bioRxiv2021

Deep metabolic profiling assessment of tissue extraction protocols for three model organisms

Metabolic profiling harbors the potential to better understand various disease entities such as cancer, diabetes, Alzheimers, Parkinsons disease or COVID-19. Deciphering these intricate pathways in human studies requires large sample sizes as a means of reducing variability. While such broad human studies have discovered new associations between a given disease and certain affected metabolites, i.e. biomarkers, they often provide limited functional insights. To design more standardized experiments, reduce variability in the measurements and better resolve the functional component of such dynamic metabolic profiles, model organisms are frequently used. Standardized rearing conditions and uniform sampling strategies are prerequisites towards a successful metabolomic study. However, further aspects such as the choice of extraction protocol and analytical technique can influence the outcome drastically. Here, we employed a highly standardized metabolic profiling assay analyzing 630 metabolites across three commonly used model organisms (Drosophila, mouse and Zebrafish) to find the optimal extraction protocols for various matrices. Focusing on parameters such as metabolite coverage, metabolite yield and variance between replicates we compared seven extraction protocols. We found that the application of a combination of 75% ethanol and methyl tertiary-butyl ether (MTBE), while not producing the broadest coverage and highest yields, was the most reproducible extraction protocol. We were able to determine up to 530 metabolites in mouse kidney samples, 509 in mouse liver, 422 in Zebrafish and 388 in Drosophila and discovered a core overlap of 261 metabolites in these four matrices. To enable other scientists to search for the most suitable extraction protocol in their experimental context and interact with this comprehensive data, we have integrated our data set in the open-source shiny app "MetaboExtract". This will enable scientists to search for their metabolite or metabolite class of interest, compare it across the different tested extraction protocols and sample types as well as find reference concentrations.

biochemistry↗

bioRxiv2021

Induction of mitochondrial heat shock proteins and mitochondrial biogenesis in endothelial cells upon acute methylglyoxal stress: Evidence for hormetic autofeedback

Increased metabolic flux produces potentially harmful side-products, such as reactive dicarbonyl and oxygen species. The reactive dicarbonly methylglyoxal (MG) can impair oxidative capacity, which is downregulated in type 2 diabetes. Heat shock proteins (HSPs) of subfamily A (Hsp70s) promote ATP-dependent processing of damaged proteins during MG exposure which also involve mitochondrial proteins. Since the protection of mitochondrial proteins could promote higher production of reactive metabolites due to increased substrate flux, tight regulation of HspA-mediated protein handling is important. We hypothesized that stress-inducible HspAs (HspA1A/HspA1B) are pivotal for maintaining mitochondrial biogenesis during acute MG-stress. To analyze the role of stress-inducible HspA1A/HspA1B for maintenance of mitochondrial homeostasis during acute MG exposure, we knocked out HSPA1A/HSPA1B in mouse endothelial cells. HSPA1A/HSPA1B KO cells showed upregulation of the mitochondrial chaperones HspA9 (mitochondrial Hsp70/mortalin) and HspD1 (Hsp60) as well as induction of mitochondrial biogenesis upon MG exposure. Increased mitochondrial biogenesis was reflected by elevated mitochondrial branching, total count and area as well as by upregulation of mitochondrial proteins and corresponding transcription factors. Our findings suggest that mitochondrial HspA9 and HspD1 promote mitochondrial biogenesis during acute MG stress, which is counterregulated by HspA1A/HspA1B to prevent mitochondrial overstimulation and to maintain balanced oxidative capacity under metabolic stress conditions. These data support an important role of HSPs in MG-induced hormesis.

cell biology↗

bioRxiv2021

A novel non-genetic murine model of hyperglycemia and hyperlipidemia-associated accelerated atherosclerosis

ObjectiveAtherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s). Methods and ResultsWild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less -SMA-positive area) and had more inflammation (increased plasma IL-1{beta} level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks. ConclusionWe established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

pharmacology and toxicology↗

bioRxiv2021

Efficient single copy integration via homology-directed repair (scHDR) by 5'modification of large DNA donor fragments in mice

CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5biotinylation. Thus, we suggest that a combination of a 5biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.

molecular biology↗

bioRxiv2021

hei-tag: a highly efficient tag to boost targeted genome editing

Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems1. While active in all species tested so far, editing efficiencies still leave room for improvement. To reach its target, the bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion of nuclear localization signals (NLSs) to the Cas9 protein2. Additional domains such as FLAG- or myc-tags are added for immediate detection or straight-forward purification3. To avoid steric hinderance impacting on activity, amino acid linkers are employed connecting Cas9 and additional domains. We present the hei-tag (high efficiency-tag), boosting the activity of the wide variety of CRISPR/Cas genome editing tools. The addition of the hei-tag to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency in model systems ranging from fish to mammals, including tissue culture applications. This allows to instantly upgrade existing and potentially highly adapted systems as well as establish novel highly efficient tools.

genetics↗

bioRxiv2020

Enhancing mitochondrial activity in neurons protects against neurodegeneration in CNS inflammation

Central nervous system (CNS) inflammation in multiple sclerosis (MS) drives neuro-axonal loss resulting in irreversible disability. While transcripts of mitochondrial genes are strongly suppressed in neurons during CNS inflammation, it is unknown whether this results in mitochondrial dysfunction and whether interventions that increase mitochondrial function can rescue neurodegeneration. Here we show that suppression of mitochondrial gene transcripts in inflamed neurons was predominantly affecting genes of the electron transport chain resulting in impaired mitochondrial complex IV activity. This was associated with posttranslational inactivation of the transcriptional co-regulator peroxisome proliferator-activated receptor gamma co-activator 1- (PGC-1). Neuronal overexpression of Pgc-1 led to increased numbers of mitochondria, complex IV activity and elevated maximum respiratory capacity. Moreover, Pgc-1- overexpressing neurons showed a higher mitochondrial membrane potential that related to an improved calcium buffering capacity. Accordingly, neuronal deletion of Pgc-1 aggravated neurodegeneration during experimental autoimmune encephalomyelitis (EAE), while neuronal overexpression of Pgc-1 ameliorated EAE disease course and preserved neurons. Our study provides systemic insights into mitochondrial dysfunction in neurons during inflammation and commends elevation of mitochondrial activity as a promising neuroprotective strategy.

neuroscience↗