A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock-in via CRISPR/Cas9 Technology.

Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery.

Comprehensive posttranslational modifications in the testis-specific histone variant H3t protein validated in tagged knock-in mice.

During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo.

Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice.

Parkinson's disease (PD) is typically a sporadic late-onset disorder, which has made it difficult to model in mice. Several transgenic mouse models bearing mutations in SNCA, which encodes alpha-Synuclein (α-Syn), have been made, but these lines do not express SNCA in a physiologically accurate spatiotemporal pattern, which limits the ability of the mice to recapitulate the features of human PD. Here, we generated knock-in mice bearing the G51D SNCA mutation. After establishing that their motor symptoms begin at 9 mo of age, we then sought earlier pathologies. We assessed the phosphorylation at Serine 129 of α-Syn in different tissues and detected phospho-α-Syn in the olfactory bulb and enteric nervous system at 3 mo of age. Olfactory deficit and impaired gut transit followed at 6 mo, preceding motor symptoms. The SncaG51D mice thus parallel the progression of human PD and will enable us to study PD pathogenesis and test future therapies.

A humanized ACE2 mouse model recapitulating age- and sex-dependent immunopathogenesis of COVID-19.

In the ongoing battle against coronavirus disease 2019 (COVID-19), understanding its pathogenesis and developing effective treatments remain critical challenges. The creation of animal models that closely replicate human infection stands as a critical step forward in this research. Here, we present a genetically engineered mouse model with specifically-humanized knock-in ACE2 (hiACE2) receptors. This model, featuring nine specific amino acid substitutions for enhanced interaction with the viral spike protein, enables efficient severe acute respiratory syndrome coronavirus 2 replication in respiratory organs without detectable infection in the central nervous system. Moreover, it mirrors the age- and sex-specific patterns of morbidity and mortality, as well as the immunopathological features observed in human COVID-19 cases. Our findings further demonstrate that the depletion of eosinophils significantly reduces morbidity and mortality, depending on the infecting viral dose and the sex of the host. This reduction is potentially achieved by decreasing the pathogenic contribution of eosinophil-mediated inflammation, which is strongly correlated with neutrophil activity in human patients. This underscores the model's utility in studying the immunopathological aspects of COVID-19 and represents a significant advancement in COVID-19 modeling. It offers a valuable tool for testing vaccines and therapeutics, enhancing our understanding of the disease mechanisms and potentially guiding more targeted and effective treatments.

Cognitive Effects of Simulated Galactic Cosmic Radiation Are Mediated by ApoE Status, Sex, and Environment in APP Knock-In Mice.

Cosmic radiation experienced during space travel may increase the risk of cognitive impairment. While simulated galactic cosmic radiation (GCRsim) has led to memory deficits in wildtype (WT) mice, it has not been investigated whether GCRsim in combination with genetic risk factors for Alzheimer's disease (AD) worsens memory further in aging mice. Here, we investigated the central nervous system (CNS) effects of 0 Gy (sham) or 0.75 Gy five-ion GCRsim or 2 Gy gamma radiation (IRR) in 14-month-old female and male APPNL-F/NL-F knock-in (KI) mice bearing humanized ApoE3 or ApoE4 (APP;E3F and APP;E4F). As travel to a specialized facility was required for irradiation, both traveled sham-irradiated C57BL/6J WT and KI mice and non-traveled (NT) KI mice acted as controls for potential effects of travel. Mice underwent four behavioral tests at 20 months of age and were euthanized for pathological and biochemical analyses 1 month later. Fecal samples were collected pre- and post-irradiation at four different time points. GCRsim seemed to impair memory in male APP;E3F mice compared to their sham counterparts. Travel tended to improve cognition in male APP;E3F mice and lowered total Aß in female and male APP;E3F mice compared to their non-traveled counterparts. Sham-irradiated male APP;E4F mice accumulated more fibrillar amyloid than their APP;E3F counterparts. Radiation exposure had only modest effects on behavior and brain changes, but travel-, sex-, and genotype-specific effects were seen. Irradiated mice had immediate and long-term differences in their gut bacterial composition that correlated to Alzheimer's disease phenotypes.

Analysis of early effects of human APOE isoforms on Alzheimer's disease and type III hyperlipoproteinemia pathways using knock-in rat models with humanized APP and APOE.

APOE is a major genetic factor in late-onset Alzheimer's disease (LOAD), with APOE4 increasing risk, APOE3 acting as neutral, and APOE2 offering protection. APOE also plays key role in lipid metabolism, affecting both peripheral and central systems, particularly in lipoprotein metabolism in triglyceride and cholesterol regulation. APOE2 is linked to Hyperlipoproteinemia type III (HLP), characterized by mixed hypercholesterolemia and hypertriglyceridemia due to impaired binding to Low-Density Lipoproteins receptors. To explore the impact of human APOE isoforms on LOAD and lipid metabolism, we developed Long-Evans rats with human APOE2, APOE3, or APOE4 in place of rat Apoe. These rats were crossed with those carrying a humanized App allele to express human Aß, which is more aggregation-prone than rodent Aß, enabling the study of human APOE-human Aß interactions. In this study, we focused on 80-day-old adolescent rats to analyze early changes that may be associated with the later development of LOAD. We found that APOE2hAß rats had the highest levels of APOE in serum and brain, with no significant transcriptional differences among isoforms, suggesting variations in protein translation or stability. Aß43 levels were significantly higher in male APOE4hAß rats compared to APOE2hAß rats. However, no differences in Tau or phosphorylated Tau levels were observed across the APOE isoforms. Neuroinflammation analysis revealed lower levels of IL13, IL4 and IL5 in APOE2hAß males compared to APOE4hAß males. Neuronal transmission and plasticity tests using field Input-Output (I/O) and long-term potentiation (LTP) recordings showed increased excitability in all APOE-carrying rats, with LTP deficits in APOE2hAßand APOE4hAß rats compared to ApoehAß and APOE3hAß rats. Additionally, a lipidomic analysis of 222 lipid molecular species in serum samples showed that APOE2hAß rats displayed elevated triglycerides and cholesterol, making them a valuable model for studying HLP. These rats also exhibited elevated levels of phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and lysophosphatidylcholine. Minimal differences in lipid profiles between APOE3hAß and APOE4hAß rats reflect findings from mouse models. Future studies will include comprehensive lipidomic analyses in various CNS regions and at older ages to further validate these models and explore the effects of APOE isoforms on lipid metabolism in relation to AD pathology.

4-Phenylbutyric Acid Treatment Reduces Low-Molecular-Weight Proteinuria in a Clcn5 Knock-in Mouse Model for Dent Disease-1.

Dent disease-1 (DD-1) is a rare X-linked tubular disorder characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrolithiasis and nephrocalcinosis. This disease is caused by inactivating mutations in the CLCN5 gene which encodes the voltage-gated ClC-5 chloride/proton antiporter. Currently, the treatment of DD-1 is only supportive and focused on delaying the progression of the disease. Here, we generated and characterized a Clcn5 knock-in mouse model that carries a pathogenic CLCN5 variant, c. 1566_1568delTGT; p.Val523del, which has been previously detected in several DD-1 unrelated patients, and presents the main clinical manifestations of DD-1 such as high levels of urinary b2-microglobulin, phosphate and calcium. Mutation p.Val523del causes partial ClC-5 retention in the endoplasmic reticulum. Additionally, we assessed the ability of sodium 4-phenylbutyrate, a small chemical chaperone, to ameliorate DD-1 symptoms in this mouse model. The proposed model would be of significant value in the investigation of the fundamental pathological processes underlying DD-1 and in the development of effective therapeutic strategies for this rare condition.

The human AMPKγ3 R225W mutation negatively impacts site-1 nucleotide binding and does not enhance basal AMPKγ3-associated activity nor glycogen production in human or mouse skeletal muscle.

AIM: AMP-activated protein kinase (AMPK) is activated during cellular energy perturbation. AMPK complexes are composed of three subunits and several variants of AMPK are expressed in skeletal muscle. The regulatory AMPKγ3 subunit is predominantly expressed in fast-twitch muscle fibers. A human AMPKγ3 R225W mutation has been described. The mutation increases the total pool of AMPK activity in cells cultured from R225W carrier muscle and is associated with increased glycogen levels in mature skeletal muscle. This led to the idea of AMPKγ3 being involved in the regulation of skeletal muscle glycogen levels. Evidence for this causative link remains to be provided. METHODS: We studied muscle biopsies from human carriers of the AMPKγ3 R225W mutation and we developed a novel AMPKγ3 R225W knock-in mouse model (KI HOM). Through in vitro, in situ, and ex vivo techniques, we investigated AMPK activity, AMPK function, and glycogen levels in skeletal muscle of humans and mice. RESULTS: In human carriers, the basal AMPKγ3-associated activity was reduced when assayed in the absence of exogenous AMP. No difference was observed when assayed under AMP saturation, which was supported by findings in muscle of KI HOM mice. Furthermore, effects of AICAR/muscle contraction on AMPKγ3-associated activity were absent in KI HOM muscle. Muscle glycogen levels were not affected by the mutation in human carriers or in KI HOM mice. CONCLUSIONS: The AMPKγ3 R225W mutation does not impact the AMPK-associated activity in human skeletal muscle and the mutation is not linked to glycogen accumulation. The R225W mutation ablates the AMPKγ3-associated activation by AICAR/muscle contractions, presumably due to loss of nucleotide binding in the CBS 1 domain of AMPKγ3.

KARS Mutations Impair Brain Myelination by Inducing Oligodendrocyte Deficiency: One Potential Mechanism and Improvement by Melatonin.

It is very crucial to investigate key molecules that are involved in myelination to gain an understanding of brain development and injury. We have reported for the first time that pathogenic variants p.R477H and p.P505S in KARS, which encodes lysyl-tRNA synthetase (LysRS), cause leukoencephalopathy with progressive cognitive impairment in humans. The role and action mechanisms of KARS in brain myelination during development are unknown. Here, we first generated Kars knock-in mouse models through the CRISPR-Cas9 system. Kars knock-in mice displayed significant cognitive deficits. These mice also showed significantly reduced myelin density and content, as well as significantly decreased myelin thickness during development. In addition, Kars mutations significantly induced oligodendrocyte differentiation arrest and reduction in the brain white matter of mice. Mechanically, oligodendrocytes' significantly imbalanced expression of differentiation regulators and increased capase-3-mediated apoptosis were observed in the brain white matter of Kars knock-in mice. Furthermore, Kars mutations significantly reduced the aminoacylation and steady-state level of mitochondrial tRNALys and decreased the protein expression of subunits of oxidative phosphorylation complexes in the brain white matter. Kars knock-in mice showed decreased activity of complex IV and significantly reduced ATP production and increased reactive oxygen species in the brain white matter. Significantly increased percentages of abnormal mitochondria and mitochondrion area were observed in the oligodendrocytes of Kars knock-in mouse brain. Finally, melatonin (a mitochondrion protectant) significantly attenuated mitochondrion and oligodendrocyte deficiency in the brain white matter of KarsR504H/P532S mice. The mice treated with melatonin also showed significantly restored myelination and cognitive function. Our study first establishes Kars knock-in mammal models of leukoencephalopathy and cognitive impairment and indicates important roles of KARS in the regulation of mitochondria, oligodendrocyte differentiation and survival, and myelination during brain development and application prospects of melatonin in KARS (or even aaRS)-related diseases.

Convergent generation of atypical prions in knockin mouse models of genetic prion disease.

Most cases of human prion disease arise due to spontaneous misfolding of WT or mutant prion protein, yet recapitulating this event in animal models has proven challenging. It remains unclear whether spontaneous prion generation can occur within the mouse lifespan in the absence of protein overexpression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knockin mice that express the misfolding-prone bank vole prion protein (BVPrP). While mice expressing WT BVPrP (I109 variant) remained free from neurological disease, a subset of mice expressing BVPrP with mutations (D178N or E200K) causing genetic prion disease developed progressive neurological illness. Brains from spontaneously ill knockin mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant BVPrP. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP-knockin mice exhibited prion seeding activity and transmitted disease to mice expressing WT BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical before and after transmission, suggesting that both mutations guide the formation of a similar atypical prion strain. These findings imply that knockin mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action.

Candesartan restores blood-brain barrier dysfunction, mitigates aberrant gene expression, and extends lifespan in a knockin mouse model of epileptogenesis.

Blockade of Angiotensin type 1 receptor (AT1R) has potential therapeutic utility in the treatment of numerous detrimental consequences of epileptogenesis, including oxidative stress, neuroinflammation, and blood-brain barrier (BBB) dysfunction. We have recently shown that many of these pathological processes play a critical role in seizure onset and propagation in the Scn8a-N1768D mouse model. Here we investigate the efficacy and potential mechanism(s) of action of candesartan (CND), an FDA-approved angiotensin receptor blocker (ARB) indicated for hypertension, in improving outcomes in this model of pediatric epilepsy. We compared length of lifespan, seizure frequency, and BBB permeability in juvenile (D/D) and adult (D/+) mice treated with CND at times after seizure onset. We performed RNAseq on hippocampal tissue to quantify differences in genome-wide patterns of transcript abundance and inferred beneficial and detrimental effects of canonical pathways identified by enrichment methods in untreated and treated mice. Our results demonstrate that treatment with CND gives rise to increased survival, longer periods of seizure freedom, and diminished BBB permeability. CND treatment also partially reversed or 'normalized' disease-induced genome-wide gene expression profiles associated with inhibition of NF-κB, TNFα, IL-6, and TGF-ß signaling in juvenile and adult mice. Pathway analyses reveal that efficacy of CND is due to its known dual mechanism of action as both an AT1R antagonist and a PPARγ agonist. The robust effectiveness of CND across ages, sexes and mouse strains is a positive indication for its translation to humans and its suitability of use for clinical trials in children with SCN8A epilepsy.

A Chimeric IL-7Rα/IL-2Rß Receptor Promotes the Differentiation of T Cell Progenitors into B Cells and Type 2 Innate Lymphoid Cells.

IL-7 and IL-2 are evolutionarily related cytokines that play critical roles in the development and expansion of immune cells. Although both IL-7R and IL-2R activate similar signaling molecules, whether their signals have specific or overlapping functions during lymphocyte differentiation remains unclear. To address this question, we generated IL-7R α-chain (IL-7Rα)/IL-2R ß-chain (IL-24ß) (72R) knock-in mice expressing a chimeric receptor consisting of the extracellular domain of IL-7Rα and the intracellular domain of IL-2Rß under the control of the endogenous IL-7Rα promoter. Notably, this 72R receptor induced higher levels of STAT5 and Akt phosphorylation in T cells. In the periphery of 72R mice, the number of T cells, B cells, and type 2 innate lymphoid cells (ILC2s) was increased, whereas early T cell progenitors and double-negative 2 thymocytes were reduced in the thymus. In addition, cell proliferation and Notch signaling were impaired in the early thymocytes of 72R mice, leading to their differentiation into thymic B cells. Interestingly, ILC2s were increased in the thymus of 72R mice. Early T cell progenitors from 72R mice, but not from wild-type mice, differentiated into NK cells and ILC2-like cells when cocultured with a thymic stromal cell line. Thus, this study indicates that the chimeric 72R receptor transduces more robust signals than the authentic IL-7Rα, thereby inducing the alternative differentiation of T cell progenitors into other cell lineages. This suggests that cytokine receptors may provide instructive signals for cell fate decisions.

Ubiquitination of VE-cadherin regulates inflammation-induced vascular permeability in vivo.

VE-cadherin is a major component of the cell adhesion machinery which provides integrity and plasticity of the barrier function of endothelial junctions. Here, we analyze whether ubiquitination of VE-cadherin is involved in the regulation of the endothelial barrier in inflammation in vivo. We show that histamine and thrombin stimulate ubiquitination of VE-cadherin in HUVEC, which is completely blocked if the two lysine residues K626 and K633 are replaced by arginine. Similarly, these mutations block histamine-induced endocytosis of VE-cadherin. We describe two knock-in mouse lines with endogenous VE-cadherin being replaced by either a VE-cadherin K626/633R or a VE-cadherin KallR mutant, where all seven lysine residues are mutated. Mutant mice are viable, healthy and fertile with normal expression levels of junctional VE-cadherin. Histamine- or LPS-induced vascular permeability in the skin or lung of both of these mutant mice are clearly and similarly reduced in comparison to WT mice. Additionally, we detect a role of K626/633 for lysosomal targeting. Collectively, our findings identify ubiquitination of VE-cadherin as important for the induction of vascular permeability in the inflamed skin and lung.

Generation of green fluorescent protein reporter knock-in iPSC line at the 3'UTR region of the KLOTHO locus.

We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids.

CRISPR/Cas9 mediated targeted knock-in of eglA gene to improve endoglucanase activity of Aspergillus fumigatus LMB-35Aa.

Bioeconomy goals for using biomass feedstock for biofuels and bio-based production has arisen the demand for fungal strains and enzymes for biomass processing. Despite well-known Trichoderma and Aspergillus commercial strains, continuous bioprospecting has revealed the fungal biodiversity potential for production of biomass degrading enzymes. The strain Aspergillus fumigatus LMB-35Aa has revealed a great potential as source of lignocellulose-degrading enzymes. Nevertheless, genetic improvement should be considered to increase its biotechnological potential. Molecular manipulation based on homologous direct recombination (HDR) in filamentous fungi poses a challenge since its low recombination rate. Currently, CRISPR/Cas9-mediated mutagenesis can enable precise and efficient editing of filamentous fungi genomes. In this study, a CRISPR/Cas9-mediated gene editing strategy for improving endoglucanase activity of A. fumigatus LMB-35Aa strain was successfully used, which constitutes the first report of heterologous cellulase production in filamentous fungi using this technology. For this, eglA gene from A. niger ATCC 10,864 was integrated into conidial melanin pksP gene locus, which facilitated the selection of edited events discerned by the emergence of albino colonies. Heterologous production of the EglA enzyme in a biofilm fermentation system resulted in a 40% improvement in endoglucanase activity of the mutant strain compared to the wild type.

Tyrosine phosphorylation of both STAT5A and STAT5B is necessary for maximal IL-2 signaling and T cell proliferation.

Cytokine-mediated STAT5 protein activation is vital for lymphocyte development and function. In vitro tyrosine phosphorylation of a C-terminal tyrosine is critical for activation of STAT5A and STAT5B; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generate Stat5a and Stat5b tyrosine-to-phenylalanine mutant knockin mice and find they have greatly reduced CD8+ T-cell numbers and profoundly diminished IL-2-induced proliferation of these cells, and this correlates with reduced induction of Myc, pRB, a range of cyclins and CDKs, and a partial G1→S phase-transition block. These mutant CD8+ T cells also exhibit decreased IL-2-mediated activation of pERK and pAKT, which we attribute in part to diminished expression of IL-2Rß and IL-2Rγ. Our findings thus demonstrate that tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling. Moreover, our transcriptomic and proteomic analyses elucidate the molecular basis of the IL-2-induced proliferation of CD8+ T cells.

The significance of electrical signals in maturing spermatozoa for phosphoinositide regulation through voltage-sensing phosphatase.

Voltage-sensing phosphatase (VSP) exhibits voltage-dependent phosphatase activity toward phosphoinositides. VSP generates a specialized phosphoinositide environment in mammalian sperm flagellum. However, the voltage-sensing mechanism of VSP in spermatozoa is not yet characterized. Here, we found that VSP is activated during sperm maturation, indicating that electric signals in immature spermatozoa are essential. Using a heterologous expression system, we show the voltage-sensing property of mouse VSP (mVSP). The voltage-sensing threshold of mVSP is approximately -30 mV, which is sensitive enough to activate mVSP in immature spermatozoa. We also report several knock-in mice in which we manipulate the voltage-sensitivity or electrochemical coupling of mVSP. Notably, the V312R mutant, with a minor voltage-sensitivity change, exhibits abnormal sperm motility after, but not before, capacitation. Additionally, the V312R mutant shows a significant change in the acyl-chain profile of phosphoinositide. Our findings suggest that electrical signals during sperm maturation are crucial for establishing the optimal phosphoinositide environment in spermatozoa.

Impact of OATP2B1 on Pharmacokinetics of Atorvastatin Investigated in rSlco2b1-Knockout and SLCO2B1-Knockin Rats.

The organic anion transporting polypeptide (OATP) 2B1 is considered an emerging drug transporter that is found expressed in pharmacokinetically relevant organs such as the liver, small intestine, and kidney. Despite its interaction with various substrate drugs, the understanding of its in vivo relevance is still limited. In this study, we first validated the interaction of atorvastatin with rat OATP2B1 using transiently transfected HeLa cells. Moreover, we characterized our rSlco2b1-knockout and SLCO2B1-knockin rats for mRNA, protein expression, and localization of OATP2B1 in the liver, small intestine, and kidney. The transporter showed the highest expression in the liver followed by the small intestine. In humanized rats, human OATP2B1 is localized on the sinusoidal membrane of hepatocytes. In enterocytes of wild-type and humanized rats, the transporter was detected in the luminal membrane with the vast majority being localized subapical. Subsequently, we assessed atorvastatin pharmacokinetics in male wild-type, rSlco2b1-knockout, and SLCO2B1-knockin rats after a single-dose administration (orally and intravenously). Investigating the contribution of rat OATP2B1 or human OATP2B1 to oral atorvastatin pharmacokinetics revealed no differences in concentration-time profiles or pharmacokinetic parameters. However, when comparing the pharmacokinetics of atorvastatin after intravenous administration in SLCO2B1-humanized rats and knockout animals, notable differences were observed. In particular, the systemic exposure (area under the curve) decreased by approximately 40% in humanized animals, whereas the clearance was 57% higher in animals expressing human OATP2B1. These findings indicate that human OATP2B1 influences pharmacokinetics of atorvastatin after intravenous administration, most likely by contributing to the hepatic uptake. SIGNIFICANCE STATEMENT: Wild-type, rSlco2b1-knockout, and SLCO2B1-humanized Wistar rats were characterized for the expression of rat and human SLCO2B1/OATP2B1. Pharmacokinetic studies of atorvastatin over 24 hours were conducted in male wild-type, rSlco2b1-knockout, and SLCO2B1-humanized rats. After a single-dose intravenous administration, a lower systemic exposure and an increase in clearance were observed in SLCO2B1-humanized rats compared with knockout animals indicating a contribution of OATP2B1 to the hepatic clearance.

Selective suppression of oligodendrocyte-derived amyloid beta rescues neuronal dysfunction in Alzheimer's disease.

Reduction of amyloid beta (Aß) has been shown to be effective in treating Alzheimer's disease (AD), but the underlying assumption that neurons are the main source of pathogenic Aß is untested. Here, we challenge this prevailing belief by demonstrating that oligodendrocytes are an important source of Aß in the human brain and play a key role in promoting abnormal neuronal hyperactivity in an AD knock-in mouse model. We show that selectively suppressing oligodendrocyte Aß production improves AD brain pathology and restores neuronal function in the mouse model in vivo. Our findings suggest that targeting oligodendrocyte Aß production could be a promising therapeutic strategy for treating AD.