Generation and Application of Inducible Chimeric RNA ASTN2-PAPPAas Knockin Mouse Model

Chimeric RNAs (chiRNAs) play many previously unrecognized roles in different diseases including cancer. They can not only be used as biomarkers for diagnosis and prognosis of various diseases but also serve as potential therapeutic targets. In order to better understand the roles of chiRNAs in pathogenesis, we inserted human sequences into mouse genome and established a knockin mouse model of the tamoxifen-inducible expression of ASTN2-PAPPA antisense chimeric RNA (A-PaschiRNA). Mice carrying the A-PaschiRNA knockin gene do not display any apparent abnormalities in growth, fertility, histological, hematopoietic, and biochemical indices. Using this model, we dissected the role of A-PaschiRNA in chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis of esophageal squamous cell carcinoma (ESCC). To our knowledge, we are the first to generate a chiRNA knockin mouse model using the Cre-loxP system. The model could be used to explore the roles of chiRNA in pathogenesis and potential targeted therapies.

Cholinergic deficits selectively boost cortical intratelencephalic control of the striatum in Huntington’s disease

Huntington’s disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in the huntingtin gene. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology remain a matter of speculation. To help fill this gap, the zQ175+/- knockin mouse model of HD was studied using approaches that allowed optogenetic interrogation of intratelencephalic (IT) and pyramidal tract (PT) connections with principal striatal spiny projection neurons (SPNs). These studies revealed that the connectivity of IT, but not PT, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced inhibitory control of IT terminals by striatal cholinergic interneurons (ChIs). Lowering mutant huntingtin selectively in ChIs with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and IT functional connectivity – revealing a novel node in the network underlying corticostriatal pathophysiology in HD.

Generation and characterization of a Myh6-driven Cre knockin mouse line

Gene deletion by the Cre-Loxp system has facilitated functional studies of many critical genes in mice, offering important insights and allowing deeper understanding on the mechanisms underlying organ development and diseases, such as heart development and diseases. In this study, we generated a Myh6-Cre knockin mouse model by inserting the IRES-Cre-wpre-polyA cassette between the translational stop codon and the 3′ untranslated region of the endogenous Myh6 gene. By crossing knockin mice with the Rosa26 reporter lines, we found that Myh6-Cre targeted cardiomyocytes at the embryonic and postnatal stages. In addition, we were able to inactivate the desmosome gene Desmoplakin (Dsp) by breeding Myh6-Cre mice with a conditional Dspflox knockout mouse line, which resulted in embryonic lethality during the mid-term pregnancy. These results suggest that the new Myh6-Cre mouse line can serve as a robust tool to dissect the distinct roles of genes involved in heart development and function.

Differential expression of striatal proteins in a mouse model of DOPA-responsive dystonia reveals shared mechanisms among dystonic disorders

Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting movements and postures. Although basal ganglia dysfunction is implicated in many forms of dystonia, the underlying mechanisms are unclear. Therefore, to reveal abnormal striatal cellular processes and pathways implicated in dystonia, we used an unbiased proteomic approach in a knockin mouse model of DOPA-responsive dystonia, a model in which the striatum is known to play a central role in the expression of dystonia. Fifty-seven of the 1805 proteins identified were differentially regulated in DOPA-responsive dystonia mice compared to control mice. Most differentially regulated proteins were associated with gene ontology terms that implicated either mitochondrial or synaptic dysfunction whereby proteins associated with mitochondrial function were generally over-represented whereas proteins associated with synaptic function were largely under-represented. Remarkably, nearly 20% of the differentially regulated proteins identified in our screen are associated with pathogenic variants that cause inherited dystonic disorders in humans suggesting shared mechanisms across many different forms of dystonia.

(D620N) VPS35 causes the impairment of Wnt/β-catenin signaling cascade and mitochondrial dysfunction in a PARK17 knockin mouse model

Patients with familial type 17 of Parkinson’s disease (PARK17) manifest autosomal dominant pattern and late-onset parkinsonian syndromes. Heterozygous (D620N) mutation of vacuolar protein sorting 35 (VPS35) is genetic cause of PARK17. We prepared heterozygous VPS35D620N/+ knockin mouse, which is an ideal animal model of (D620N) VPS35-induced autosomal dominant PARK17. Late-onset loss of substantia nigra pars compacta (SNpc) dopaminergic (DAergic) neurons and motor deficits of Parkinson’s disease were found in 16-month-old VPS35D620N/+ mice. Normal function of VPS35-containing retromer is needed for activity of Wnt/β-catenin cascade, which participates in protection and survival of SNpc DAergic neurons. It was hypothesized that (D620N) VPS35 mutation causes the malfunction of VPS35 and resulting impaired activity of Wnt/β-catenin pathway. Protein levels of Wnt1 and nuclear β-catenin were reduced in SN of 16-month-old VPS35D620N/+ knockin mice. Downregulated protein expression of survivin, which is a target gene of nuclear β-catenin, and upregulated protein levels of active caspase-8 and active caspase-9 were observed in SN of VPS35D620N/+ mice at age of 16 months. VPS35 is involved in controlling morphology and function of mitochondria. Impaired function of VPS35 caused by (D620N) mutation could lead to abnormal morphology and malfunction of mitochondria. A significant decrease in mitochondrial size and resulting mitochondrial fragmentation was found in tyrosine hydroxylase-positive and neuromelanin-positive SNpc DAergic neurons of 16-month-old VPS35D620N/+ mice. Mitochondrial complex I activity or complex IV activity was reduced in SN of 16-month-old VPS35D620N/+ mice. Increased level of mitochondrial ROS and oxidative stress were found in SN of 16-month-old VPS35D620N/+ mice. Levels of cytosolic cytochrome c and active caspase-3 were increased in SN of VPS35D620N/+ mice aged 16 months. Our results suggest that PARK17 mutant (D620N) VPS35 impairs activity of Wnt/β-catenin signaling pathway and causes abnormal morphology and dysfunction of mitochondria, which could lead to neurodegeneration of SNpc DAergic cells.

Huntingtin lowering reduces somatic instability at CAG-expanded loci

Expanded trinucleotide repeats cause many human diseases, including Huntington’s disease (HD). Recent studies indicate that somatic instability of these repeats contributes to pathogenesis in several expansion disorders. We find that lowering huntingtin protein (HTT) levels reduces somatic instability of both the Htt and Atxn2 CAG tracts in knockin mouse models, and the HTT CAG tract in human iPSC-derived neurons, revealing an unexpected role for HTT in regulating somatic instability.

In vivo mapping of a GPCR interactome using knockin mice

With over 30% of current medications targeting this family of proteins, G-protein–coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.