Importance of the Q/N-rich Segment for Protein Stability and Activity of Endogenous Mouse TDP-43

TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). TDP-43 aggregation and translocation out of the nucleus are crucial factors in ALS. TDP-43 aggregation results from its resistance to degradation, to which the long-disordered C-terminal region (CTR) is thought to contribute. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the protein stability, activity, and subcellular localization of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of Q/N-rich segment greatly restored the protein stability and activity of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development.

Broad spectrum of CRISPR-induced edits in an embryonic lethal gene

Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. While CRISPR-based gene editing of multiple genes at once offers an attractive alternative strategy, subsequent breeding or establishment of permanent mouse lines will rapidly segregate the different mutant loci again. Direct phenotypic analysis of genomic edits in an embryonic lethal gene in F0 generation mice, or F0 mouse embryos, circumvents the need for breeding or establishment of mutant mouse lines. In the course of genotyping a large cohort of F0 CRISPants, where the embryonic lethal gene T/brachyury was targeted, we noted the presence of multiple CRISPR-induced modifications in individual embryos. Using long-read single-molecule Nanopore sequencing, we identified a wide variety of deletions, ranging up to 3 kb, that would not have been detected or scored as wildtype with commonly used genotyping methods that rely on subcloning and short-read or Sanger sequencing. Long-read sequencing results were crucial for accurate genotype–phenotype correlation in our F0 CRISPants. We thus demonstrate feasibility of screening manipulated F0 embryos for mid-gestation phenotypic consequences of CRISPR-induced mutations without requiring derivation of permanent mouse lines.

Pathogenic in-Frame Variants in SCN8A: Expanding the Genetic Landscape of SCN8A-Associated Disease

Numerous SCN8A mutations have been identified, of which, the majority are de novo missense variants. Most mutations result in epileptic encephalopathy; however, some are associated with less severe phenotypes. Mouse models generated by knock-in of human missense SCN8A mutations exhibit seizures and a range of behavioral abnormalities. To date, there are only a few Scn8a mouse models with in-frame deletions or insertions, and notably, none of these mouse lines exhibit increased seizure susceptibility. In the current study, we report the generation and characterization of two Scn8a mouse models (ΔIRL/+ and ΔVIR/+) carrying overlapping in-frame deletions within the voltage sensor of domain 4 (DIVS4). Both mouse lines show increased seizure susceptibility and infrequent spontaneous seizures. We also describe two unrelated patients with the same in-frame SCN8A deletion in the DIV S5-S6 pore region, highlighting the clinical relevance of this class of mutations.

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease

MYH9-related disease patients with mutations in the contractile protein non-muscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild to moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in MYH9-related disease patients, and treatment with tranexamic acid can improve the hemostatic function.

Not All That Is Gold Glitters: PV-IRES-Cre Mouse Line Shows Low Efficiency of Labeling of Parvalbumin Interneurons in the Perirhinal Cortex

The wide diversity of cortical inhibitory neuron types populating the cortex allows the assembly of diverse microcircuits and endows these circuits with different computational properties. Thus, characterizing neuronal diversity is fundamental to describe the building blocks of cortical microcircuits and probe their function. To this purpose, the mouse has emerged as a powerful tool to genetically label and manipulate specific inhibitory cell-types in the mammalian brain. Among these cell-types, the parvalbumin-expressing interneuron type (PV-INs) is perhaps the most characterized. Several mouse lines have been generated to target PV-INs. Among these mouse lines, the PV-IRES-Cre lines is the most widely used and demonstrated a high specificity and efficiency in targeting PV-INs in different cortical areas. However, a characterization of the performance across cortical regions is still missing. Here we show that the PV-IRES-Cre mouse line labels only a fraction of PV immunoreactive neurons in perirhinal cortex and other association areas. Our results point to a yet uncharacterized diversity within the PV-INs and emphasize the need to characterize these tools in specific cortical areas.

Effect of Central Administration of Brain-Derived Neurotrophic Factor (BDNF) on Behavior and Brain Monoamine Metabolism in New Recombinant Mouse Lines Differing by 5-HT1A Receptor Functionality

Experiments were carried out on recombinant B6.CBA-D13Mit76C (B6-M76C) and B6.CBA-D13Mit76B (B6-M76B) mouse lines created by transferring a 102.73–118.83 Mbp fragment of chromosome 13, containing the 5-HT1A receptor gene, from CBA or C57BL/6 strains to a C57BL/6 genetic background, correspondingly. We have recently shown different levels of 5-HT1A receptor functionality in these mouse lines. The administration of BDNF (300 ng/mouse, i.c.v.) increased the levels of exploratory activity and intermale aggression only in B6-M76B mice, without affecting depressive-like behavior in both lines. In B6-M76B mice the behavioral alterations were accompanied by a decrease in the 5-HT2A receptor functional activity and the augmentation of levels of serotonin and its main metabolite, 5-HIAA (5-hydroxyindoleacetic acid), in the midbrain. Moreover, the levels of dopamine and its main metabolites, HVA (homovanillic acid) and DOPAC (3,4-dihydroxyphenylacetic acid), were also elevated in the striatum of B6-M76B mice after BDNF treatment. In B6-M76C mice, central BDNF administration led only to a reduction in the functional activity of the 5-HT1A receptor and a rise in DOPAC levels in the midbrain. The obtained data suggest the importance of the 102.73–118.83 Mbp fragment of mouse chromosome 13, which contains the 5-HT1A receptor gene, for BDNF-induced alterations in behavior and the brain monoamine system.

Optimized immunoglobulin knock-ins using Cas9 reveal peritoneal B cell lineage relationships in vivo

Immunoglobulin (Ig) knock-in mice are valuable tools in basic and translational immunological research. Here we present Speed-Ig, a rapid Cas9-based method for generating Ig knock-in mouse lines with high on-target integration rates at both heavy and light chain alleles. With standardized target sites and promoter regions, Speed-Ig mice can be used for comparative studies of B cell biology and vaccine optimization in vivo. We used Speed-Ig to create panels of mice with Ig pairs derived from B-1a, B-1b, and B-2 cells. Surprisingly, B-1b and B-2 Ig pairs drove both B-1b and B-2 phenotypes, suggesting a previously unknown lineage relationship between these subsets. We then confirmed the B-1:B-2 relationship with transcription factor reporter lines and through adoptive cell transfer experiments. In summary, our Ig knock-in approach facilitated the discovery of previously unappreciated aspect of innate-like B cell biology.

Toward Understanding the Diverse Roles of Perisomatic Interneurons in Epilepsy

Epileptic seizures are associated with excessive neuronal spiking. Perisomatic γ-aminobutyric acid (GABA)ergic interneurons specifically innervate the subcellular domains of postsynaptic excitatory cells that are critical for spike generation. With a revolution in transcriptomics-based cell taxonomy driving the development of novel transgenic mouse lines, selectively monitoring and modulating previously elusive interneuron types is becoming increasingly feasible. Emerging evidence suggests that the three types of hippocampal perisomatic interneurons, axo-axonic cells, along with parvalbumin- and cholecystokinin-expressing basket cells, each follow unique activity patterns in vivo, suggesting distinctive roles in regulating epileptic networks.