Wild-type Littermate Controls Research Articles

Targeting hypoxia-induced signaling via HIF-2 inhibition, a promising treatment strategy for intestinal fibrosis?

Abstract Background and aims With this project, we aim to evaluate if targeting hypoxia-induced signalling via hypoxia inducible factor (HIF)-2 inhibition impacts experimental intestinal fibrosis and validate our findings in stenotic fibroblasts from CD patients. Methods Colonic fibroblasts will be sorted from mice with a fibroblast-specific deletion of the oxygen sensitive enzyme prolyl hydroxylase (PHD2) (i.e. PHD2ff- Col1a2-ERT2:Cre) and their wildtype littermate controls, followed by RNA sequencing and pathway analysis. Validation of key fibrotic mediators will be performed via qPCR or immunostainings. Intestinal fibrosis will be induced in PHD2ff Col1a2-ERT2:Cre mice wildtype littermate controls by chronic administration of dextran sodium sulphate in their drinking water, followed by treatment with the oral HIF-2 inhibitor, belzutifan. Translatability of preclinical findings will be confirmed in bio-banked fibrostenotic fibroblasts from CD patients. Anticipated impact Intestinal fibrosis is a debilitating complication of IBD which cannot be fully prevented by current anti-inflammatory therapies, highlighting the unmet clinical need for anti-fibrotic treatment strategies. Our preliminary data demonstrated that genetic deletion of Phd2 in fibroblasts aggravates experimental intestinal fibrosis without affecting inflammation and is most likely mediated via HIF-2. This suggests that signaling induced by this transcriptional modulator plays a crucial role in the fibrotic process. This proposal will identify the molecular mediators driving this fibrosis-promoting effect and validate if HIF-2 targeting could represent a promising novel anti-fibrotic treatment strategy. Data resulting from this project will therefore represent a final preclinical step and provide proof-of-concept data with the perspective to initiate a clinical trial in fibrostenotic IBD with belzutifan (awaiting EMA approval, but recently FDA-approved for advanced renal cell carcinoma).

Use of a predictor cue during a speech sound discrimination task in a Cntnap2 knockout rat model of autism.

Autism is a common neurodevelopmental disorder that despite its complex etiology, is marked by deficits in prediction that manifest in a variety of domains including social interactions, communication, and movement. The tendency of individuals with autism to focus on predictable schedules and interests that contain patterns and rules highlights the likely involvement of the cerebellum in this disorder. One candidate-autism gene is contact in associated protein 2 (CNTNAP2), and variants in this gene are associated with sensory deficits and anatomical differences. It is unknown, however, whether this gene directly impacts the brain's ability to make and evaluate predictions about future events. The current study was designed to answer this question by training a genetic knockout rat on a rapid speech sound discrimination task. Rats with Cntnap2 knockout (KO) and their littermate wildtype controls (WT) were trained on a validated rapid speech sound discrimination task that contained unpredictable and predictable targets. We found that although both genotype groups learned the task in both unpredictable and predictable conditions, the KO rats responded more often to distractors during training as well as to the target sound during the predictable testing conditions compared to the WT group. There were only minor effects of sex on performance and only in the unpredictable condition. The current results provide preliminary evidence that removal of this candidate-autism gene may interfere with the learning of unpredictable scenarios and enhance reliance on predictability. Future research is needed to probe the neural anatomy and function that drives this effect.

Longevity, enhanced memory, and altered density of dendritic spines in hippocampal CA3 and dentate gyrus after hemizygous deletion of Pde2a in mice.

Studies using acute or subchronic pharmacological inhibition of phosphodiesterase 2 A (PDE2A) have led to its proposal as a target for treatment of cognitive deficits associated with neuropsychiatric and neurodegenerative disease. However, the impact of continuous inhibition of PDE2A on memory is unknown. Moreover, the neuroanatomical regions mediating memory enhancement have not been categorically identified. To address these open questions, we studied knockout mice and hippocampus restricted manipulations. Pde2a heterozygous knockout mice are viable with no gross histological abnormalities. The mice exhibit enhanced spatial and object recognition memory that is independent of anxiolytic effects and is paralleled by increased density of dendritic mushroom and thin spines in hippocampal CA3 and dentate gyrus in adult mice. In CA1, subtle alterations in spine density were seen, while theta-burst LTP and paired-pulse facilitation were normal. Spatial memory enhancement persists in aged Pde2a heterozygous knockout mice, and to our surprise these mice live significantly longer than wild-type littermate controls. In summary, we provide evidence that life-long reduction of PDE2A expression promotes spine formation and maturation, exerts beneficial effects on memory, and increases lifespan.

Elevated plasma and CSF neurofilament light chain concentrations are stabilized in response to mutant huntingtin lowering in the brains of Huntington’s disease mice

BackgroundTherapeutic approaches aimed at lowering toxic mutant huntingtin (mHTT) levels in the brain can reverse disease phenotypes in animal models of Huntington's disease (HD) and are currently being evaluated in clinical trials. Sensitive and dynamic response biomarkers are needed to assess the efficacy of such candidate therapies. Neurofilament light chain (NfL) is a biomarker of neurodegeneration that increases in cerebrospinal fluid (CSF) and blood with progression of HD. However, it remains unknown whether NfL in biofluids could serve as a response biomarker for assessing the efficacy of disease-modifying therapies for HD.MethodsLongitudinal plasma and cross-sectional CSF samples were collected from the YAC128 transgenic mouse model of HD and wild-type (WT) littermate control mice throughout the natural history of disease. Additionally, biofluids were collected from YAC128 mice following intracerebroventricular administration of an antisense oligonucleotide (ASO) targeting the mutant HTT transgene (HTT ASO), at ages both before and after the onset of disease phenotypes. NfL concentrations in plasma and CSF were quantified using ultrasensitive single-molecule array technology.ResultsPlasma and CSF NfL concentrations were significantly elevated in YAC128 compared to WT littermate control mice from 9 months of age. Treatment of YAC128 mice with either 15 or 50 µg HTT ASO resulted in a dose-dependent, allele-selective reduction of mHTT throughout the brain at a 3-month interval, which was sustained with high-dose HTT ASO treatment for up to 6 months. Lowering of brain mHTT prior to the onset of regional brain atrophy and HD-like motor deficits in this model had minimal effect on plasma NfL at either dose, but led to a dose-dependent reduction of CSF NfL. In contrast, initiating mHTT lowering in the brain after the onset of neuropathological and behavioural phenotypes in YAC128 mice resulted in a dose-dependent stabilization of NfL increases in both plasma and CSF.ConclusionsOur data provide evidence that the response of NfL in biofluids is influenced by the magnitude of mHTT lowering in the brain and the timing of intervention, suggesting that NfL may serve as a promising exploratory response biomarker for HD.

NINJ1-mediated Macrophage Plasma Membrane Rupture and Neutrophil Extracellular Trap Formation Contribute to Oxalate Nephropathy.

Oxalate nephropathy is characterized by calcium oxalate crystals deposition, which triggers necrosis of renal tubular epithelial cells, initiates an inflammatory cascade characterized by neutrophil and macrophage activation within the renal microenvironment. Despite the close association of immune cells with acute oxalate nephropathy, the underlying mechanisms still remain unclear. Nerve injury-induced protein 1 (NINJ1) plays an essential role in the induction of plasma membrane rupture (PMR), leading to damage-associated molecular patterns (DAMPs) release and triggering inflammation. We hypothesize that NINJ1-mediated high mobility group box 1 (HMGB1) release from macrophage PMR and neutrophil extracellular traps (NETs) formation synergistically contribute to the progression of acute oxalate nephropathy. Using a murine model of acute oxalate nephropathy, myeloid cell-specific deletion of Ninj1 mice (Ninj1fl/flvavcre) and their wild-type littermate control mice (Ninj1wt/wtvavcre) were administered intraperitoneal injection of 100 mg/kg sodium oxalate followed by drinking water with 3% sodium oxalate. Evaluation was conducted on tubular injury and inflammatory cell infiltration. In vitro studies involved isolation and culture of renal proximal tubular epithelial cells (RTECs), bone marrow-derived macrophages, and neutrophils to investigate NETs formation and HMGB1 release. Targeted deletion of Ninj1 in myeloid cells significantly mitigated oxalate-induced acute kidney injury by suppressing both HMGB1 release and NETs formation in vivo. In vitro investigations demonstrated that HMGB1 release from macrophage PMR and NETs formation in neutrophils mediated by NINJ1 oligomerization, which consequently coordinated to enhance renal tubular epithelial cell death. Our findings elucidate the pivotal role of NINJ1-dependent macrophage PMR and NETs formation in the progression of acute oxalate nephropathy, providing novel insights for its prevention and therapeutic targets.

8672 Disrupted Glucocorticoid Receptor Mediated Signalling Causes a Primary Cilia Defect in the Fetal Mouse Renal Tubule

Abstract Disclosure: J. Lao: None. K.L. Short: None. J. Ng: None. D.L. Cottle: None. T.J. Cole: None. Primary cilia are microtubule-based organelles that protrude from cell membranes to mediate diverse developmental signalling pathways and senses extracellular stimuli to maintain tissue homeostasis. We show that glucocorticoid (GC) signalling via the glucocorticoid receptor (GR) is a regulator of normal primary cilia formation in kidney renal tubules. RNA sequencing of whole E18.5 fetal kidney from mice with global deletion of the GR (GR-null) compared to littermate wild-type controls identified significantly reduced mRNA levels of key ciliogenesis-related genes. Further analysis of whole fetal kidney RNA by real-time-qPCR confirmed reduced mRNA levels for the cilia-associated proteins Ccp110 (fold -2.18), Cep97 (fold -1.78), Cep290 (fold -2.91), Kif3a (fold -1.82) and Rpgr (fold -1.90). Confocal microscopy revealed abnormal, stunted primary cilia in renal proximal tubules, collecting ducts and podocytes in GR-null or in renal tubule conditional GR-deleted mice, created using the HoxB7 Cre-driver allele. Primary cilia length was significantly decreased in kidney proximal tubule cells in GR-null mice (5.01 ± 0.11μm) compared with wildtype controls (6.20 ± 0.15μm). Furthermore, scanning electron microscopy of E18.5 GR-null kidney further confirmed aberrant primary cilia morphology in the proximal renal tubules and the absence of an apical brush border. Activation of GR signalling with the synthetic GC dexamethasone in cultured mouse IMCD3 kidney tubule cells significantly increased primary cilia length (2.89 ± 0.04μm) compared to vehicle controls (2.46 ± 0.28μm), an effect blocked by the GR antagonist RU486 (vehicle + RU486: 2.06 ± 0.02μm, dexamethasone + RU486: 2.00 ± 0.02μm). Aurora kinase A (AURKA) is a known regulator of primary cilia formation via the AKT cell signalling pathway and distinct proteins at different stages of the mitotic cell cycle. AURKA protein levels were downregulated in GR-null kidney and in IMCD3 cells which suggest GR regulates AURKA to control the assembly and disassembly of primary cilia. Together, these results demonstrate that GC signalling via the GR is required for normal primary ciliogenesis in the developing renal tubule and suggests that synthetic GR agonists may provide a novel therapeutic option for human ciliopathies such as those observed in forms of polycystic kidney disease. Presentation: 6/2/2024

The role of cerebellar FOXP1 in the development of motor and communicative behaviors in mice.

The gene FOXP2 is well established for a role in human speech and language; far less is known about FOXP1. However, this related gene has also been implicated in human language development as well as disorders associated with features of autism spectrum disorder (ASD). FOXP1 protein expression has also recently been identified in the cerebellum-a neural structure previously shown to express FOXP2 protein. The current study sought to elucidate the behavioral implications of a conditional knock-out of Foxp1 using an En1-Cre driver, which is active in the entirety of the cerebellum and a subset of neurons in the midbrain and spinal cord, in mice using a test battery including motor tasks associated with cerebellar dysfunction, as well as communicative and autistic-relevant behaviors. Male and female mice with a conditional knock-out (cKO, n = 31) and wildtype littermate controls (WT, n = 34) were assessed for gross and orofacial motor control, motor-coordination learning, locomotion, social behavior, anxiety, auditory processing and expressive vocalizations. Overall results suggest Foxp1 plays a specific role in the development of communicative systems, and phenotypic expression of disruptions may interact with sex. Robust motor deficits associated with Foxp1 protein loss may particularly affect vocalizations based on significant orofacial motor deficits in cKO subjects could also contribute to vocalization anomalies. In summary, the current study provides key insights into the role of Foxp1 in cerebellar function and associated behaviors in mice, with implications for an improved understanding of communicative and motor-based neurodevelopmental disabilities in humans.

LRRK2G2019S Gene Mutation Causes Skeletal Muscle Impairment in Animal Model of Parkinson's Disease.

While the gradually aggravated motor and non-motor disorders of Parkinson's disease (PD) lead to progressive disability and frequent falling, skeletal muscle impairment may contribute to this condition. The leucine-rich repeat kinase2 (LRRK2) is a common disease-causing gene in PD. Little is known about its role in skeletal muscle impairment and its underlying mechanisms. To investigate whether the mutation in LRRK2 causes skeletal muscle impairment, we used 3-month-old (3mo) and 14-month-old (14mo) LRRK2G2019S transgenic (TG) mice as a model of PD, compared with the age-matched littermate wild-type (WT) controls. We measured the muscle mass and strength, ultrastructure, inflammatory infiltration, mitochondrial morphology and dynamics dysfunction through behavioural analysis, electromyography (EMG), immunostaining, transmission electron microscopy (TEM) and other molecular biology techniques. The 3mo-TG mice display mild skeletal muscle impairment with spontaneous potentials in EMG (increased by 130%, p < 0.05), myofibre necrosis(p < 0.05) and myosin heavy chain-II changes (reduced by 19%, p < 0.01). The inflammatory cells and macrophage infiltration are significantly increased (CD8a+ and CD68+ cells up 1060% and 579%, respectively, both p < 0.0001) compared with the WT mice. All of the above pathogenic processes are aggravated by aging. The 14mo-TG mice EMG examinations show a reduced duration (by 31%, p < 0.01) and increased polyphasic waves of motor unit action potentials (by 28%, p < 0.05). The 14mo-TG mice present motor behavioural deficits (p < 0.05), muscle strength and mass reduction by 37% and 8% (p < 0.05 and p < 0.01, respectively). A remarkable increase in inflammatory infiltration is accompanied by pro-inflammatory cytokines in the skeletal muscles. TEM analysis shows muscle fibre regeneration with the reduced length of sarcomeres (by 6%;p < 0.05). The muscle regeneration is activated as Pax7+ cells increased by 106% (p < 0.0001), andmyoblast determination protein elevated by 71% (p < 0.01). We also document the morphological changes and dynamics dysfunction of mitochondria with the increase of mitofusin1 by 43% (p < 0.05)and voltage-dependent anion channel 1 by 115% (p < 0.001) in the skeletal muscles of 14mo-TG mice. Taken together, these findings may provide new insights into the clinical and pathogenic involvement of LRRK2G2019 mutation in muscles, suggesting that the diseases may affect not only midbrain dopaminergic neurons, but also other tissues, and it may help overall clinical management of this devastating disease.

Abstract P353: Sexual dimorphism of blood pressure responses to angiotensin II in rats subjected to early life circadian stress

Various forms of early life stress (ELS) lead to a greater risk of hypertension, but no studies have examined stress produced by circadian disruption. We hypothesize that circadian stress during adolescence will increase sensitivity to a hypertensive stimulus in adulthood recapitulating other models of early life stress (ELS). We exposed male and female rats 1-2 weeks post-weaning to 6-hour phase advances (shifts) in the light/dark cycle each week for 4 weeks. To assess whether the molecular clock contributes to the response to ELS, we performed these studies in both Bmal1 knockout (KO) rats and littermate wildtype (WT) controls, Bmal1 is a core circadian clock transcription factor. At 10-12 weeks of age, baseline mean arterial pressure (MAP) was measured (telemetry) followed by a 2-wk angiotensin II (AngII) infusion (250 ng/kg/min, s.c.). At baseline, non-ELS KO rats of both sexes had lower MAP compared to WT littermates, but this genotype effect was lost in ELS rats. Thus, exposure to circadian stress early in life has only a modest effect on baseline blood pressure in rats lacking Bmal1. AngII increased MAP in all groups but was significantly attenuated in male ELS rats compared to non-ELS controls of both genotypes (p stress = 0.039, p genotype =0.979, p SxG =0.917, 2-way ANOVA), but this attenuation was not evident in female rats (p stress = 0.497, p genotype =0.041, p SxG =0.772). We also observed a significant phase advance in peak MAP in male non-ELS rats of both genotypes that was absent in ELS males (p Genotype =0.030, p AngII &lt;0.001, p AngIIxGT =0.025). In contrast, there were no significant differences in peak MAP time in any female rats. The response to AngII was attenuated in ELS male, but not female rats. These data do not support our original hypothesis that exposure to circadian stress during adolescence induces hypersensitization to Ang II in rats but does reveal an impact on the timing of peak MAP in male rats. We propose that the attenuated MAP response to AngII seen in male rats following exposure to specific types of circadian stress during the post-weaning period may pre-condition rats against a subsequent hypertensive stimulus.

Abstract 37: Targeted Deletion of NKCC1 in Vascular Smooth Muscle Cells Lowers Blood Pressure in Normotensive and Hypertensive Mice

Background: Salt plays a major role in blood pressure homeostasis and the pathogenesis of arterial hypertension. While numerous mechanisms have been described by which Na + can influence arterial blood pressure, the role of Cl - in blood pressure regulation is still largely unexplored. The Na + -K + -2Cl - cotransporter NKCC1 is an important Cl - import pathway in vascular smooth muscle cells (VSMC). To investigate the role of NKCC1 in blood pressure regulation, we generated mice with an inducible VSMC-specific disruption of Slc12a2 . Methods: Mean arterial blood pressure and heart rate were measured by radiotelemetry in unrestrained mice lacking vascular NKCC1 (KO) and wildtype littermates (WT). Cardiac function was assessed by echocardiography. Plasma renin activity and aldosterone concentrations were quantified by radioimmunoassay. Protein expression of renal sodium transporters and channels was analyzed by Western blotting. Results: Mean arterial blood pressure was markedly decreased in Nkcc1 -deficient mice 6 weeks after induction of gene disruption (from 116±2 mm Hg to 103±4 mm Hg, n=18, p&lt;0.001, 2-way ANOVA and Bonferroni post-test), while it did not change in WT control animals (n=17). Heart rate, stroke volume, fractional shortening, and ejection fraction remained unchanged compared to non-induced controls. We did not observe a compensatory increase in plasma renin activity or aldosterone levels, and the blood pressure response to ANG II receptor 1 antagonism by losartan treatment for 7 days was similar in both groups (ΔMAP KO -8.8±3.0 mm Hg, n=8, ΔMAP WT -12.4±1.9 mm Hg, n=7; p&gt;0.05, 2-way ANOVA). In the kidney, the relative expression of total NCC (1.5- fold , n=7-8; p=&lt;0.05, Student’s t-test) and pNCCThr53 (1.9- fold , n=7-8; p=&lt;0.05, Student’s t-test) were modestly increased. The hypotensive effect of Nkcc1 disruption was maintained after induction of hypertension by either infusion of ANG II for 10 days (ΔMAP KO 43.8±6.5 mm Hg, n=6, ΔMAP WT 50.2±7.2 mm Hg, n=4; p&gt;0.05, 2-way ANOVA) or a high salt diet for 10 days (ΔMAP KO 21.7±7.0 mm Hg, n=8, ΔMAP WT 34.0±7.3 mm Hg, n=5; p&gt;0.05, 2-way ANOVA). Conclusions: Our findings indicate that NKCC1 in vascular smooth muscle contributes significantly to the maintenance of arterial blood pressure. Inhibition of NKCC1 in VSMC may provide additional blood pressure reduction in resistant hypertension.

Age-related effects of optineurin deficiency in the mouse eye

Optineurin (OPTN) is a gene associated with familial normal tension glaucoma (NTG). While NTG involves intraocular pressure (IOP)-independent neurodegeneration of the visual pathway that progresses with age, how OPTN dysfunction leads to NTG remains unclear. Here, we generated an OPTN knockout mouse (Optn−/−) model to test the hypothesis that a loss-of-function mechanism induces structural and functional eye deterioration with aging. Eye anatomy, visual function, IOP, retinal histology, and retinal ganglion cell survival were compared to littermate wild-type (WT) control mice. Consistent with OPTN’s role in NTG, loss of OPTN did not increase IOP or alter gross eye anatomy in young (2–3 months) or aged (12 months) mice. When retinal layers were quantitated, young Optn−/− mice had thinner retina in the peripheral regions than young WT mice, primarily due to thinner ganglion cell-inner plexiform layers. Despite this, visual function in Optn−/− mice was not severely impaired, even with aging. We also assessed relative abundance of retinal cell subtypes, including amacrine cells, bipolar cells, cone photoreceptors, microglia, and astrocytes. While many of these cellular subtypes were unaffected by Optn deletion, more dopaminergic amacrine cells were observed in aged Optn−/− mice. Taken together, our findings showed that complete loss of Optn resulted in mild retinal changes and less visual function impairment, supporting the possibility that OPTN-associated glaucoma does not result from a loss-of-function disease mechanism. Further research using these Optn mice will elucidate detailed molecular pathways involved in NTG and identify clinical or environmental risk factors that can be targeted for glaucoma treatment.

Exon 1-targeting miRNA reduces the pathogenic exon 1 HTT protein in Huntington's disease models.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) that results in toxic gain of function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex, and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and might contribute to HD pathology. This finding suggests that reducing the expression of HTT1a might achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT might not completely prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes and have been the rationale for phase I/II clinical studies now ongoing in the USA and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at 2 months postinjection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in mice at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit in comparison to other HTT-targeting modalities.

Tartrate-resistant acid phosphatase (TRAP/ACP5) promotes bone length, regulates cortical and trabecular bone mass, and maintains growth plate architecture and width in a sex- and site-specific manner in mice

Tartrate-resistant acid phosphatase (TRAP) serum levels reflect osteoclast number, bone remodeling activity, and fracture risk. Deletion or loss of function of TRAP results in short stature in mice and man. Yet, the impact and mechanisms of TRAP for the site- and sex-specific development of bone and cartilage is not well understood. Here, we use a global TRAP knockout (TRAPKO) and wildtype littermate control (WT) mice of both sexes to investigate TRAP as a possible sex- and site-specific regulator of bone and growth plate development. TRAPKO mice of both sexes weighed less and had shorter tibial length than their WT, features that were more accentuated in male than female TRAPKO mice. These changes were not associated with a general reduction in growth as not all organs displayed a proportionally lower mass, and serum IGF-1 was unchanged. Using μCT and site-specificity analysis of the cortical bone revealed wider proximal tibia, a higher trabecular thickness, and lower trabecular separation in male TRAPKO compared to WT mice, an effect not seen in female mice. Histomorphometric analysis revealed that the growth plate height as well as height of terminal hypertrophic chondrocytes were markedly increased, and the number of columns was decreased in TRAPKO mice of both sexes. These effects were more accentuated in female mice. Proliferation and differentiation of bone marrow derived macrophages into osteoclasts, as well as C-terminal cross links were normal in TRAPKO mice of both sexes. Collectively, our results show that TRAP regulates bone and cartilage development in a sex-and site-specific manner in mice.

Abstract Mo062: Cpne5 is Necessary for Normal Sinoatrial Node Function

Background: Cpne5 (Copine 5) is a Ca2+-dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing (scRNA-seq) of the developing mouse heart, we found Cpne5 gene expression to be highly enriched throughout the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesize that Cpne5 is necessary for normal CCS function. Objective: Determine whether Cpne5 is necessary for the normal development and/or function of the CCS. Methods: To assess the necessity of Cpne5, homozygous systemic knockout (KO) mice were generated using CRISPR/Cas9. Cpne5KO and littermate wild-type (WT) control mice were characterized using surface electrocardiogram (ECG), continuous telemetry and echocardiogram. Protein expression of Cpne5 and CCS-related ion channels were assessed by immunofluorescence of tissue sections, isolated adult murine SAN cells and whole mount immunostaining with 3D volumetric imaging (iDISCO+). Further, CRISPR-mediated deletion of CPNE5 was performed in human induced pluripotent stem cells (hiPSC). Differentiated SAN-like cells were phenotyped via calcium transient (CaT) dynamics and microelectrode array. Results: Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, sinus pauses, and increased heart rate variability compared to WT controls on long term telemetry. Additionally, CPNE5KO hiPSC-derived SAN-like cells exhibited irregular CaT as well as significant beat period variability compared to isogenic WT controls. Conclusion: Systemic loss-of-function of Cpne5 in mice resulted in sinus node dysfunction, consistent with an hiPSC-derived SAN model and human GWAS data, implicating a novel, cell-intrinsic role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies are ongoing to further elucidate the underlying mechanism.

Sleep EEG signatures in mouse models of 15q11.2-13.1 duplication (Dup15q) syndrome

BackgroundSleep disturbances are a prevalent and complex comorbidity in neurodevelopmental disorders (NDDs). Dup15q syndrome (duplications of 15q11.2-13.1) is a genetic disorder highly penetrant for NDDs such as autism and intellectual disability and it is frequently accompanied by significant disruptions in sleep patterns. The 15q critical region harbors genes crucial for brain development, notably UBE3A and a cluster of gamma-aminobutyric acid type A receptor (GABAAR) genes. We previously described an electrophysiological biomarker of the syndrome, marked by heightened beta oscillations (12-30 Hz) in individuals with Dup15q syndrome, akin to electroencephalogram (EEG) alterations induced by allosteric modulation of GABAARs. Those with Dup15q syndrome exhibited increased beta oscillations during the awake resting state and during sleep, and they showed profoundly abnormal NREM sleep. This study aims to assess the translational validity of these EEG signatures and to delve into their neurobiological underpinnings by quantifying sleep physiology in chromosome-engineered mice with maternal (matDp/ + mice) or paternal (patDp/ + mice) inheritance of the full 15q11.2-13.1-equivalent duplication, and mice with duplication of just the UBE3A gene (Ube3a overexpression mice; Ube3a OE mice) and comparing the sleep metrics with their respective wildtype (WT) littermate controls.MethodsWe collected 48-h EEG/EMG recordings from 35 (23 male, 12 female) 12–24-week-old matDp/ + , patDp/ + , Ube3a OE mice, and their WT littermate controls. We quantified baseline sleep, sleep fragmentation, spectral power dynamics during sleep states, and recovery following sleep deprivation. Within each group, distinctions between Dup15q mutant mice and WT littermate controls were evaluated using analysis of variance (ANOVA) and student’s t-test. The impact of genotype and time was discerned through repeated measures ANOVA, and significance was established at p < 0.05.ResultsOur study revealed that across brain states, matDp/ + mice mirrored the elevated beta oscillation phenotype observed in clinical EEGs from individuals with Dup15q syndrome. Time to sleep onset after light onset was significantly reduced in matDp/ + and Ube3a OE mice. However, NREM sleep between Dup15q mutant and WT littermate mice remained unaltered, suggesting a divergence from the clinical presentation in humans. Additionally, while increased beta oscillations persisted in matDp/ + mice after 6-h of sleep deprivation, recovery NREM sleep remained unaltered in all groups, thus suggesting that these mice exhibit resilience in the fundamental processes governing sleep-wake regulation.ConclusionsQuantification of mechanistic and translatable EEG biomarkers is essential for advancing our understanding of NDDs and their underlying pathophysiology. Our study of sleep physiology in the Dup15q mice underscores that the beta EEG biomarker has strong translational validity, thus opening the door for pre-clinical studies of putative drug targets, using the biomarker as a translational measure of drug-target engagement. The unaltered NREM sleep may be due to inherent differences in neurobiology between mice and humans. These nuanced distinctions highlight the complexity of sleep disruptions in Dup15q syndrome and emphasize the need for a comprehensive understanding that encompasses both shared and distinct features between murine models and clinical populations.

Dopamine production in neurotensin receptor 1 neurons is required for diet-induced obesity and increased day eating on a high-fat diet.

This study aimed to determine a dopaminergic circuit required for diet-induced obesity in mice. We created conditional deletion mutants for tyrosine hydroxylase (TH) using neurotensin receptor 1 (Ntsr1) Cre and other Cre drivers and measured feeding and body weight on standard and high-fat diets. We then used an adeno-associated virus to selectively restore TH to the ventral tegmental area (VTA) Ntsr1 neurons in conditional knockout (cKO) mice. Mice with cKO of Th using Vglut2-Cre, Cck-Cre, Calb1-Cre, and Bdnf-Cre were susceptible to obesity on a high-fat diet; however, Ntsr1-Cre Th cKO mice resisted weight gain on a high-fat diet and did not experience an increase in day eating unlike their wild-type littermate controls. Restoration of TH to the VTA Ntsr1 neurons of the Ntsr1-Cre Th cKO mice using an adeno-associated virus resulted in an increase in weight gain and day eating on a high-fat diet. Ntsr1-Cre Th cKO mice failed to increase day eating on a high-fat diet, offering a possible explanation for their resistance to diet-induced obesity. These results implicate VTA Ntsr1 dopamine neurons as promoting out-of-phase feeding behavior on a high-fat diet that could be an important contributor to diet-induced obesity in humans.

Multi-omics analysis of diabetic pig lungs reveals molecular derangements underlying pulmonary complications of diabetes mellitus.

Growing evidence shows that the lung is an organ prone to injury by diabetes mellitus. However, the molecular mechanisms of these pulmonary complications have not yet been characterized comprehensively. To systematically study the effects of insulin deficiency and hyperglycaemia on the lung, we combined proteomics and lipidomics with quantitative histomorphological analyses to compare lung tissue samples from a clinically relevant pig model for mutant INS gene-induced diabetes of youth (MIDY) with samples from wild-type littermate controls. Among others, the level of pulmonary surfactant-associated protein A (SFTPA1), a biomarker of lung injury, was moderately elevated. Furthermore, key proteins related to humoral immune response and extracellular matrix organization were significantly altered in abundance. Importantly, a lipoxygenase pathway was dysregulated as indicated by 2.5-fold reduction of polyunsaturated fatty acid lipoxygenase ALOX15 levels, associated with corresponding changes in the levels of lipids influenced by this enzyme. Our multi-omics study points to an involvement of reduced ALOX15 levels and an associated lack of eicosanoid switching as mechanisms contributing to a proinflammatory milieu in the lungs of subjects with diabetes mellitus.

Adult Inception of Ketogenic Diet Therapy Increases Sleep during the Dark Cycle in C57BL/6J Wild Type and Fragile X Mice.

Sleep problems are a significant phenotype in children with fragile X syndrome. Our prior work assessed sleep-wake cycles in Fmr1KO male mice and wild type (WT) littermate controls in response to ketogenic diet therapy where mice were treated from weaning (postnatal day 18) through study completion (5-6 months of age). A potentially confounding issue with commencing treatment during an active period of growth is the significant reduction in weight gain in response to the ketogenic diet. The aim here was to employ sleep electroencephalography (EEG) to assess sleep-wake cycles in mice in response to the Fmr1 genotype and a ketogenic diet, with treatment starting at postnatal day 95. EEG results were compared with prior sleep outcomes to determine if the later intervention was efficacious, as well as with published rest-activity patterns to determine if actigraphy is a viable surrogate for sleep EEG. The data replicated findings that Fmr1KO mice exhibit sleep-wake patterns similar to wild type littermates during the dark cycle when maintained on a control purified-ingredient diet but revealed a genotype-specific difference during hours 4-6 of the light cycle of the increased wake (decreased sleep and NREM) state in Fmr1KO mice. Treatment with a high-fat, low-carbohydrate ketogenic diet increased the percentage of NREM sleep in both wild type and Fmr1KO mice during the dark cycle. Differences in sleep microstructure (length of wake bouts) supported the altered sleep states in response to ketogenic diet. Commencing ketogenic diet treatment in adulthood resulted in a 15% (WT) and 8.6% (Fmr1KO) decrease in body weight after 28 days of treatment, but not the severe reduction in body weight associated with starting treatment at weaning. We conclude that the lack of evidence for improved sleep during the light cycle (mouse sleep time) in Fmr1KO mice in response to ketogenic diet therapy in two studies suggests that ketogenic diet may not be beneficial in treating sleep problems associated with fragile X and that actigraphy is not a reliable surrogate for sleep EEG in mice.

Hepatic ketogenesis is not required for starvation adaptation in mice

ObjectiveIn response to bacterial inflammation, anorexia of acute illness is protective and is associated with the induction of fasting metabolic programs such as ketogenesis. Forced feeding during the anorectic period induced by bacterial inflammation is associated with suppressed ketogenesis and increased mortality. As ketogenesis is considered essential in fasting adaptation, we sought to determine the role of ketogenesis in illness-induced anorexia. MethodsA mouse model of inducible hepatic specific deletion of the rate limiting enzyme for ketogenesis (HMG-CoA synthase 2, Hmgcs2) was used to investigate the role of ketogenesis in endotoxemia, a model of bacterial inflammation, and in prolonged starvation. ResultsMice deficient of hepatic Hmgcs2 failed to develop ketosis during endotoxemia and during prolonged fasting. Surprisingly, hepatic HMGCS2 deficiency and the lack of ketosis did not affect survival, glycemia, or body temperature in response to endotoxemia. Mice with hepatic ketogenic deficiency also did not exhibit any defects in starvation adaptation and were able to maintain blood glucose, body temperature, and lean mass compared to littermate wild-type controls. Mice with hepatic HMGCS2 deficiency exhibited higher levels of plasma acetate levels in response to fasting. ConclusionsCirculating hepatic-derived ketones do not provide protection against endotoxemia, suggesting that alternative mechanisms drive the increased mortality from forced feeding during illness-induced anorexia. Hepatic ketones are also dispensable for surviving prolonged starvation in the absence of inflammation. Our study challenges the notion that hepatic ketogenesis is required to maintain blood glucose and preserve lean mass during starvation, raising the possibility of extrahepatic ketogenesis and use of alternative fuels as potential means of metabolic compensation.

Cage effects on synaptic plasticity and its modulation in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is characterized by impairments in executive function including different types of learning and memory. Long-term potentiation (LTP), thought to underlie the formation of memories, has been studied in the Fmr1 mouse model of FXS. However, there have been many discrepancies in the literature with inconsistent use of littermate and non-littermate Fmr1 knockout (KO) and wild-type (WT) control mice. Here, the influence of the breeding strategy (cage effect) on short-term potentiation (STP), LTP, contextual fear conditioning (CFC), expression of N-methyl-d-aspartate receptor (NMDAR) subunits and the modulation of NMDARs, were examined. The largest deficits in STP, LTP and CFC were found in KO mice compared with non-littermate WT. However, the expression of NMDAR subunits was unchanged in this comparison. Rather, NMDAR subunit (GluN1, 2A, 2B) expression was sensitive to the cage effect, with decreased expression in both WT and KO littermates compared with non-littermates. Interestingly, an NMDAR-positive allosteric modulator, UBP714, was only effective in potentiating the induction of LTP in non-littermate KO mice and not the littermate KO mice. These results suggest that commonly studied phenotypes in Fmr1 KOs are sensitive to the cage effect and therefore the breeding strategy may contribute to discrepancies in the literature.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.