Nontransgenic Mice Research Articles

SNCA genetic lowering reveals differential cognitive function of alpha-synuclein dependent on sex

Antisense oligonucleotide (ASO) therapy for neurological disease has been successful in clinical settings and its potential has generated hope for Alzheimer’s disease (AD). We previously described that ablating SNCA encoding for α-synuclein (αSyn) in a mouse model of AD was beneficial. Here, we sought to demonstrate whether transient reduction of αSyn expression using ASOSNCA could be therapeutic in a mouse model of AD. The efficacy of the ASOSNCA was measured via immunocytochemistry, RT-qPCR and western blotting. To assess spatial learning and memory, ASOSNCA or PBS-injected APP and non-transgenic (NTG) mice, and separate groups of SNCA-null mice, were tested on the Barnes circular maze. Hippocampal slice electrophysiology and transcriptomic profiling were used to explore synaptic function and differential gene expression between groups. Reduction of SNCA transcripts alleviated cognitive deficits in male transgenic animals, but surprisingly, not in females. To determine the functional cause of this differential effect, we assessed memory function in SNCA-null mice. Learning and memory were intact in male mice but impaired in female animals, revealing that the role of αSyn on cognitive function is sex-specific. Transcriptional analyses identified a differentially expressed gene network centered around EGR1, a central modulator of learning and memory, in the hippocampi of SNCA-null mice. Thus, these novel results demonstrate that the function of αSyn on memory differs between male and female brains.

Transcranial Nongenetic Neuromodulation via Bioinspired Vesicle-Enabled Precise NIR-II Optical Stimulation.

Regulating the activity of specific neurons is essentially important in neurocircuit dissection and neuropathy therapy. As a recently developed strategy, nanomaterial-enabled nongenetic neuromodulations that realize remote physical stimuli have made vast progress and shown great clinical potential. However, minimal invasiveness and high spatiotemporal resolution are still challenging for nongenetic neuromodulation. Herein, a second near-infrared (NIR-II)-light-induced transcranial nongenetic neurostimulation via bioinspired nanovesicles is reported. The rationally designed vesicles are obtained from vesicle-membrane-confined enzymatic reactions. This study demonstrates that the vesicle-enabled NIR-II photothermal stimuli can elicit neuronal signaling dynamics with precise spatiotemporal control and thus evoke defined neural circuits in nontransgenic mice. Moreover, the vesicle-mediated NIR-II optical stimulation can regulate mouse motor behaviors with minimal invasiveness by eliminating light-emitting implants. Furthermore, the biological modulation is integrated with photoacoustic brain imaging, realizing navigational, and efficient neuromodulation. Such transcranial and precise NIR-II optical neuromodulation mediated by bioinspired vesicles shows the potential for the optical-theranostics of neurological diseases in nontransgenic organisms.

Hydroxychloroquine Mitigates Dilated Cardiomyopathy Phenotype in Transgenic D94A Mice.

In this study, we aimed to investigate whether short-term and low-dose treatment with hydroxychloroquine (HCQ), an antimalarial drug, can modulate heart function in a preclinical model of dilated cardiomyopathy (DCM) expressing the D94A mutation in cardiac myosin regulatory light chain (RLC) compared with healthy non-transgenic (NTg) littermates. Increased interest in HCQ came with the COVID-19 pandemic, but the risk of cardiotoxic side effects of HCQ raised concerns, especially in patients with an underlying heart condition, e.g., cardiomyopathy. Effects of HCQ treatment vs. placebo (H2O), administered in Tg-D94A vs. NTg mice over one month, were studied by echocardiography and muscle contractile mechanics. Global longitudinal strain analysis showed the HCQ-mediated improvement in heart performance in DCM mice. At the molecular level, HCQ promoted the switch from myosin's super-relaxed (SRX) to disordered relaxed (DRX) state in DCM-D94A hearts. This result indicated more myosin cross-bridges exiting a hypocontractile SRX-OFF state and assuming the DRX-ON state, thus potentially enhancing myosin motor function in DCM mice. This bottom-up investigation of the pharmacological use of HCQ at the level of myosin molecules, muscle fibers, and whole hearts provides novel insights into mechanisms by which HCQ therapy mitigates some abnormal phenotypes in DCM-D94A mice and causes no harm in healthy NTg hearts.

Fkbp38 deletion induces premature ovarian insufficiency in mice by activating mTOR signaling and inducing granulosa cell apoptosis

To investigate the role of tacrolimus-binding protein 38 (FKBP38) in follicle development and the mechanism by which Fkbp38 gene deletion causes premature ovarian insufficiency (POI). The Cre-loxp system was used to construct oocyte-specific Fkbp38 knockout transgenic mice. The genotype of the transgenic mice was identified using PCR, and the expression of FKBP38 in the oocytes was verified. The numbers of primordial follicles, primary follicles, secondary follicles and antral follicles in Fkbp38 knockout mice and non-transgenic littermate control mice were counted with HE staining under a microscope for analyzing the effect of Fkbp38 deletion on follicular development. The fertility and serum sex hormone levels of the mice were determined by reproduction experiments and ELISA to assess ovarian function. Ovarian granulosa cell apoptosis of the mice was assessed using TUNEL assay. The activity of the downstream target protein of phosphorylated ribosomal S6 (PS6) of mTOR signaling pathway was detected, and the expressions of BCL-2 and BAX proteins were determined using immunofluorescence assay for assessing oocyte development in the mice. The oocyte-specific Fkbp38 knockout transgenic mouse model was successfully constructed, which showed decreased fertility, disordered sex hormone levels, and significantly reduced primordial follicles, primary follicles and secondary follicles in the ovary (P < 0.05), demonstrating POI-like changes. Compared with the control mice, oocyte-specific Fkbp38 knockout caused activation of the mTOR signaling pathway, significantly increased apoptosis of the granulosa cells, and obviously increased the BAX/BCL- 2 ratio by increasing BAX expression and reducing BCL-2 expression in the oocytes (P < 0.05). FKBP38 plays an important role in follicle development, and Fkbp38 gene deletion in mice causes POI possibly by activating the mTOR signaling pathway and inducing granulosa cell apoptosis.

Sex-dependent worsening of NMDA-induced responses, anxiety, hypercortisolemia, and organometry of early peripheral immunoendocrine impairment in adult 3xTg-AD mice and their long-lasting ontogenic modulation by neonatal handling

The neuroimmunomodulation hypothesis for Alzheimer’s disease (AD) postulates that alterations in the innate immune system triggered by damage signals result in adverse effects on neuronal functions. The peripheral immune system and neuroimmunoendocrine communication are also impaired. Here we provide further evidence using a longitudinal design that also studied the long-lasting effects of an early life sensorial intervention (neonatal handling, from postnatal day 1–21) in 6-month-old (early stages of the disease) male and female 3xTg-AD mice compared to age- and sex-matched non-transgenic (NTg) mice with normal aging. The behavioral patterns elicited by the direct exposure to an open field, and the motor depression response evoked by NMDA (25 mg/kg, i.p) were found correlated to the organometry of peripheral immune-endocrine organs (thymus involution, splenomegaly, and adrenal glands’ hypertrophy) and increased corticosterone levels, suggesting their potential value for diagnostic and biomonitoring.The NMDA-induced immediate and depressant motor activity and endocrine (corticosterone) responses were sensitive to sex and AD-genotype, suggesting worse endogenous susceptibility/neuroprotective response to glutamatergic excitotoxicity in males and in the AD-genotype. 3xTg-AD females showed a reduced immediate response, whereas the NTg showed higher responsiveness to subsequent NMDA-induced depressant effect than their male counterparts. The long-lasting ontogenic modulation by handling was shown as a potentiation of NMDA-depressant effect in NTg males and females, while sex × treatment effects were found in 3xTg-AD mice. Finally, NMDA-induced corticosterone showed sex, genotype and interaction effects with sexual dimorphism enhanced in the AD-genotype, suggesting different endogenous vulnerability/neuroprotective capacities and modulation of the neuroimmunoendocrine system.

SMN Is Physiologically Downregulated at Wild-Type Motor Nerve Terminals but Aggregates Together with Neurofilaments in SMA Mouse Models.

Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle synthesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED super-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of β-actin mRNA, ribosomes, and polysomes in the presynaptic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein 1B (MAP1B) and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the presynaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high expression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal.

Sleep Alterations in a Mouse Model of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder showing progressive neuronal loss in several brain areas and a broad spectrum of motor and non-motor symptoms, including ataxia and altered sleep. While sleep disturbances are known to play pathophysiologic roles in other neurodegenerative disorders, their impact on SCA3 is unknown. Using spectrographic measurements, we sought to quantitatively characterize sleep electroencephalography (EEG) in SCA3 transgenic mice with confirmed disease phenotype. We first measured motor phenotypes in 18–31-week-old homozygous SCA3 YACMJD84.2 mice and non-transgenic wild-type littermate mice during lights-on and lights-off periods. We next implanted electrodes to obtain 12-h (zeitgeber time 0-12) EEG recordings for three consecutive days when the mice were 26–36 weeks old. EEG-based spectroscopy showed that compared to wild-type littermates, SCA3 homozygous mice display: (i) increased duration of rapid-eye movement sleep (REM) and fragmentation in all sleep and wake states; (ii) higher beta power oscillations during REM and non-REM (NREM); and (iii) additional spectral power band alterations during REM and wake. Our data show that sleep architecture and EEG spectral power are dysregulated in homozygous SCA3 mice, indicating that common sleep-related etiologic factors may underlie mouse and human SCA3 phenotypes.

Hypothalamic-pituitary-adrenal (HPA) axis activity and anxiety-like behavior during aging: A test of the glucocorticoid cascade hypothesis in amyloidogenic APPswe/PS1dE9 mice

Alzheimer’s disease (AD) is a progressive, dementing, whole-body disorder that presents with decline in cognitive, behavioral, and emotional functions, as well as endocrine dysregulation. The etiology of AD is not fully understood but stress- and anxiety-related hormones may play a role in its development and trajectory. The glucocorticoid cascade hypothesis posits that levels of glucocorticoids increase with age, leading to dysregulated negative feedback, further elevated glucocorticoids, and resulting neuropathology. We examined the impact of age (from 2 to 10 months) and stressor exposure (predator odor) on hormone levels (corticosterone and ghrelin), anxiety-like behavior (open field and light dark tests), and memory-related behavior (novel object recognition; NOR), and whether these various measures correlated with neuropathology (hippocampus and cortex amyloid beta, Aβ) in male and female APPswe/PS1dE9 transgenic and non-transgenic mice. Additionally, we performed exploratory analyses to probe if the open field and light dark test as commonly used tasks to assess anxiety levels were correlated. Consistent with the glucocorticoid cascade hypothesis, baseline corticosterone increased with age. Predator odor exposure elevated corticosterone at each age, but in contrast to the glucocorticoid cascade hypothesis, the magnitude of stressor-induced elevations in corticosterone levels did not increase with age. Overall, transgenic mice had higher post-stressor, but not baseline, corticosterone than non-transgenic mice, and across both genotypes, females consistently had higher (baseline and post-stressor) corticosterone than males. Behavior in the open field test primarily showed decreased locomotion with age, and this was pronounced in transgenic females. Anxiety-like behaviors in the light dark test were exacerbated following predator odor, and female transgenic mice were the most impacted. Compared to transgenic males, transgenic females had higher Aβ concentrations and showed more anxiety-like behavior. Performance on the NOR did not differ significantly between genotypes. Lastly, we did not find robust, statistically significant correlations among corticosterone, ghrelin, recognition memory, anxiety-like behaviors, or Aβ, suggesting outcomes are not strongly related on the individual level. Our data suggest that despite Aβ accumulation in the hippocampus and cortex, male and female APPswePS1dE9 transgenic mice do not differ robustly from their non-transgenic littermates in physiological, endocrine, and behavioral measures at the range of ages studied here.

Transgenic Expression of Nrf2 Induces a Pro-Reductive Stress and Adaptive Cardiac Remodeling in the Mouse.

Nuclear factor, erythroid 2 like 2 (Nfe2l2 or Nrf2), is a transcription factor that protects cells by maintaining a homeostatic redox state during stress. The constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling. Transgenic and non-transgenic (NTG) mice at 7–8 months of age were used to analyze the myocardial transcriptome, structure, and function. Next generation sequencing (NGS) for RNA profiling and qPCR-based validation of the NGS data, myocardial redox levels, and imaging (echocardiography) were performed. Transcriptomic analysis revealed that out of 14,665 identified mRNAs, 680 were differently expressed (DEG) in TG hearts. Of 680 DEGs, 429 were upregulated and 251 were downregulated significantly (FC > 2.0, p < 0.05). Gene set enrichment analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of the glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 µM/mg protein in mNrf2-TG and CaNrf2-TG vs. NTG). Genes involved in fetal development, hypertrophy, cytoskeletal rearrangement, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG. Non-invasive echocardiography analysis revealed an increase in systolic function (ejection fraction) in mNrf2-TG, suggesting an adaptation, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks). The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e., pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.

Distinct microglia alternative splicing in Alzheimer's disease.

Numerous alternative splicing (AS) events have been documented in Alzheimer's disease (AD). However, cell type-specific AS analysis is still lacking. We described AS events in the hippocampal microglia sorted by CD45 and CD11b from Aβ precursor protein (APP) and non-transgenic (Ntg) mice. GSE171195 dataset was downloaded from GEO database, aligned to GRCm39 genome. Skipped exon (SE), alternative 3’SS (A3SS), retained intron (RI), alternative 5’SS (A5SS), and mutually exclusive exons (MXE) were evaluated using rMATS and maser. Differential expressed genes or transcripts were analyzed via limma. Gene ontology and correlation analyses were performed with clusterProfiler and ggcorrplot R packages. 36,340 raw counts of AS were identified, and 95 significant AS events were eventually selected with strict criteria: (1) average coverage >5; (2) delta percent spliced in >0.1. SE was the most common AS events (68.42%), followed by A3SS and RI. Autophagy genes were mainly spliced in SE events, actin depolymerization genes spliced in A3SS events, while synaptic plasticity related genes were mainly spliced in RI pattern. These significant AS events may be regulated by dysregulated splicing factors in AD. In conclusion, we revealed microglia specific AS events in AD, and our study provides novel pathological mechanisms in the pathogenesis of AD.

Stress granule assembly in vivo is deficient in the CNS of mutant TDP-43 ALS mice.

Responding effectively to external stress is crucial for neurons. Defective stress granule dynamics has been hypothesized as one of the pathways that renders motor neurons in amyotrophic lateral sclerosis (ALS) more prone to early death. Specifically, it is thought that stress granules seed the cytoplasmic TDP-43 inclusions that are observed in the neurons of most ALS patients, as well as ~50% of all frontotemporal dementia (FTD) patients. In this study, we tested this hypothesis in an intact mammalian nervous system. We established an in vivo heat stress paradigm in mice that effectively triggers the eIF2α pathway and the formation of stress granules in the CNS. In non-transgenic mice, we report an age-dependent decline in the formation of heat-induced stress granules, with 18-month-old animals showing a significant impairment. Furthermore, although neuronal stress granules were robustly observed in non-transgenic mice and SOD1G93A mice, they were largely absent in age-matched TDP-43M337V animals. The observed defect in stress granule formation in TDP-43M337V mice correlated with deficits in expression of key protein components typically required for phase separation. Lastly, while TDP-43 was not localized to stress granules, we observed complete nuclear depletion of TDP-43 in a subset of neurons, with the highest proportion being in the TDP-43M337V mice. Overall, our results indicate that mutant TDP-43 expression is associated with defective stress granule assembly and increased TDP-43 nuclear depletion in the mammalian nervous system, which could be relevant to ALS/FTD pathogenesis.

Abstract P2045: Transcriptome Versus Proteomic Discrepancy Is Associated With Structural-functional Dys-synchrony In Reductive Stress Myocardium

Background: A real-time action of Nrf2 in response to a stress is vital to preserve the redox homeostasis, but its activation under unstressed settings tilt the redox balance towards the reductive arm, leading to reductive stress (RS). In this study, we identified that &gt;50% mismatch between mRNA and protein levels in RS myocardium undergoing structural/functional dys-synchrony. Methods: NGS-RNA Seq (for RNA), Tandem Mass Tagging (TMT) Mass spectrometry (for proteins) and speckle tracking strain echocardiography (STE) using Vevo2100 system was performed in the cardiac-specific constitutively active Nrf2 transgenic (CaNrf2-TG/TG) and non-transgenic (NTG) mice (n=4/group) at ~6-7 months of age. Results: Comparison of NGS and TMT data in TG vs. NTG hearts showed surprising results. Out of 104 commonly found gene products in NGS and proteins at a FC≥1.5, the levels of 50 proteins are not consistent with their RNA expression. The proteins that are steadily expressed according to their respective mRNA levels are classified as “transcription-sync proteins (T-SP)” and the ones that do not match are termed as “transcription-nonsync proteins. While changes in Nrf2-driven antioxidant genes ( Nqo1, Gsr, Gsta1-4, and Gstm1 ) were grouped under “T-SP”, other transcripts including Atp5pb, Ces1d, Cmbl, Cox7a1, Cpt1b, Fah and Gstk1 did not quantitatively match with their translated products in TG hearts. Further, STE demonstrated an impaired wall motion (diastolic and systolic velocity), increased fractional shortening (20% vs 50%; p&lt;0.0001), decreased global longitudinal strain (-10% vs -25%, p&lt;0.0002), increased IVRT (6.46 vs 13.8 ms; p&lt;0.0001) and IVCT (8.06 vs 12.5 ms; p&lt;0.0005) in TG vs. NTG mice. Changes in the systolic and diastolic functions with structural abnormalities demonstrate cardiac dys-synchronicity in TG hearts with chronic RS exhibiting impaired transcriptome and proteome profiles. Conclusions: Our comparative analysis of RNA and protein in RS hearts demonstrated that 50% of the proteins do not match with the quantitative changes of their transcripts. We highlight that the RS could be a novel mechanism for disproportionate transcription vs translation, which leads to structural and functional dys-synchrony in the TG hearts.

Abstract P3091: Transgenic Expression Of Nrf2 Induces A Pro-reductive Stress And Adaptive Cardiac Remodeling In Mice

Background: NFE2L2, nuclear factor, erythroid 2 like 2 (Nrf2), is a transcription factor that protects cells through maintaining a homeostatic redox state during stress. Constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling. Methods: Cardiac-specific full-length Nrf2 transgenic (mNrf2-TG) and non-transgenic (NTG) mice hearts on the C57/BL6 background at 7-8 months of age were used to analyze the transcriptome and cardiac structure/function. Next generation sequencing (NGS) for RNA profiling (n=3/group) and qPCR-based validation (n=5/group) of the NGS data were accomplished. Myocardial redox levels and imaging (echocardiography) were performed. Results: Transcriptomic analysis revealed that out of 14665 identified mRNAs, 679 were differently expressed (DEG) in TG hearts. Of 679 DEGs, 429 were upregulated and 251 were downregulated significantly (FC&gt;2, P&lt;0.05). Gene Set Enrichment Analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 μM/mg protein in mNrf2-TG and CaNrf2-TG vs NTG). Expression of fetal genes and hypertrophic biomarkers, cytoskeletal genes, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG hearts. Non-invasive echocardiography analysis revealed an increase in systolic function (Ejection Fraction) in mNrf2-TG, suggesting an adaption, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks). Conclusion: The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e. pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.

The BMP and FGF pathways reciprocally regulate odontoblast differentiation

ABSTRACT Purpose Previous studies demonstrated that the exposure of primary dental pulp (DP) cultures to fibroblast growth factor 2 (FGF2) between days 3–7 exerted significant and long-lasting stimulatory effects on odontoblast differentiation and Dspp expression. These effects involved the increased expression of components of bone morphogenetic protein (BMP) signaling and were reverted by a BMP inhibitor noggin. FGF2 also transiently stimulated osteoblast differentiation and the expression of Ibsp and Dmp1. The present study aimed to further explore interactions between BMP and FGF signaling during odontoblast and osteoblast differentiation in DP cultures. Materials and Methods Cultures were established using DP tissue isolated from non-transgenic and fluorescent reporter (DSPP-Cerulean, BSP-GFP, and DMP1-mCherry) transgenic mice and exposed to BMP2, FGF2, SU5402 (an FGF receptor inhibitor), and noggin between days 3–7. Mineralization, gene expression, fluorescent protein expression, and odontoblast formation were examined using xylenol orange, quantitative PCR, fluorometric analysis, and immunocytochemistry, respectively. Results BMP2 activated SMAD1/5/8 but not ERK1/2 signaling, whereas FGF2 exerted opposite effects. BMP2 did not affect mineralization, the expression of Ibsp and Dmp1, and the percentage of DSPP-Cerulean+ odontoblasts but significantly increased Dspp and DSPP-Cerulean. In cultures exposed to BMP2 and FGF2, respectively, both SU5402 and noggin led to long-lasting decreases in Dspp and DSPP-Cerulean and transient decreases in Dmp1 and DMP1-mCherry without affecting Ibsp and BSP-GFP. Conclusion BMP2 and FGF2 exerted reciprocal stimulatory effects on odontoblast differentiation, whereas their effects on osteoblast differentiation were mediated independently. These data will further elucidate the perspectives of using BMP2 and FGF2 for dentin regeneration/repair.

A quantitative in vivo imaging platform for tracking pathological tau depositions and resultant neuronal death in a mouse model.

Depositions of tau fibrils are implicated in diverse neurodegenerative disorders, including Alzheimer's disease, and precise assessments of tau pathologies and their impacts on neuronal survival are crucial for pursuing the neurodegenerative tau pathogenesis with and without potential therapies. We aimed to establish an in vivo imaging system to quantify tau accumulations with positron emission tomography (PET) and brain atrophy with volumetric MRI in rTg4510 transgenic mice modeling neurodegenerative tauopathies. A total of 91 rTg4510 and non-transgenic control mice underwent PET with a tau radiotracer, 18F-PM-PBB3, and MRI at various ages (1.8-12.3 months). Using the cerebellum as reference, the radiotracer binding in target regions was estimated as standardized uptake value ratio (SUVR) and distribution volume ratio (DVR). Histopathological staining of brain sections derived from scanned animals was also conducted to investigate the imaging-neuropathology correlations. 18F-PM-PBB3 SUVR at 40-60 min in the neocortex, hippocampus, and striatum of rTg4510 mice agreed with DVR, became significantly different from control values around 4-5 months of age, and progressively and negatively correlated with age and local volumes, respectively. Neocortical SUVR also correlated with the abundance of tau inclusions labeled with PM-PBB3 fluorescence, Gallyas-Braak silver impregnation, and anti-phospho-tau antibodies in postmortem assays. The in vivo and ex vivo 18F-PM-PBB3 binding was blocked by non-radioactive PM-PBB3. 18F-PM-PBB3 yielded a 1.6-fold greater dynamic range for tau imaging than its ancestor, 11C-PBB3. Our imaging platform has enabled the quantification of tau depositions and consequent neuronal loss and is potentially applicable to the evaluation of candidate anti-tau and neuroprotective drugs.

Abstract 1348: LMW-E induction and crosstalk with immune cells potentiates local immune responses leading to an immunosuppressive microenvironment at the early stages of breast tumorigenesis in mouse models

Abstract Introduction: Cyclin E is an independent predictor of poor outcomes and response to treatment in breast cancer (BrCa). Expression of low-molecular-weight cyclin E (LMW-E) is associated with more aggressive disease in all BrCa subtypes. While tumor infiltrating lymphocytes (TILs) are more abundant in LMW-E+ tumors, high-TIL/LMW-E+ tumors have lower probability of pathological complete response (pCR) to neoadjuvant chemotherapy. We hypothesized that LMW-E induces immune changes that create a permissive microenvironment in the mammary gland for promoting tumor initiation and subsequent growth. We aimed to evaluate the role of mammary epithelial expression of LMW-E in the temporal induction of systemic and local immune responses that ultimately prime the mammary gland for tumor development. Methods: We generated a tri-transgenic mouse model capable of conditionally expressing human LMW-E under the control of the MMTV promoter in a p53 heterozygous background (MPT) upon doxycycline (Dox) administration. Female MPT mice were treated with Dox for 3, 6 and 9 months and age-matched untreated controls were sacrificed at each time point. An independent group of non-transgenic mice in a p53 heterozygous background were maintained +/- Dox as positive and negative controls. Mammary glands and peripheral organs (spleen, lung, bone marrow) were harvested for immune profiling by flow cytometry and multiplex immunofluorescence microscopy (mIF). Serum was also collected for cytokine/chemokine assessment. Immune profiling via flow cytometry was performed using two multi-color panels to assess basic immune and more specialized T-cell subsets. For the mIF experiments, two 5-marker panels were applied to mammary tissue. Results: Histological examination of MPT mammary glands over time showed a temporal increase in acinar proliferation and mitotic figures, which was further confirmed by the co-localization of panCK+ and Ki-67+ markers. We report that although immune cell frequencies changed with age, these specific changes were dependent on LMW-E induction as compared to the non-transgenic cohort of mice. LMW-E+ mammary glands showed a temporal enrichment in B cells, macrophages, T cells (CD4+, PD1+, CD4+Ki67+, and Tregs), cDCs, and panCK+, panCK+vimentin+ populations over time. By contrast, pDCs increased from 3 to 6 months but decreased in the pre-tumorigenic mammary gland of the 9-month old LMW-E+ mice. Conclusions: LMW-E induction mediates an increase in epithelial cell proliferation and epithelial-to-mesenchymal transition events that result in local immune alterations, specifically affecting T-cell subsets. Our findings suggest that the immunological changes driven by LMW-E lead to an immunosuppressive microenvironment that may promote tumor formation at the early stages of breast tumorigenesis. Citation Format: Sofia Mastoraki, Amriti R. Lulla, Sarah Schneider, Karen Clise-Dwyer, Morgan M. Green, Natalie W. Fowlkes, Kelly K. Hunt, Stephanie S. Watowich, Khandan Keyomarsi. LMW-E induction and crosstalk with immune cells potentiates local immune responses leading to an immunosuppressive microenvironment at the early stages of breast tumorigenesis in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1348.

AT1R‐mediated neurodegeneration is part of the etiology of hypertension‐associated cognitive impairment

Hypertension has now been closely associated to cognitive impairment, due to its contribution to cerebrovascular dysfunction. To investigate whether there is neuronal mechanism, hypertension was induced in C57BL/6j male mice (6 months) by DOCA‐salt treatment (1.5 mg/g, s.c., 28 days). Firstly, behavioral study, which contains object location behavioral task, novel object recognition, Barnes maze, and nesting score test, was performed to assess cognitive function, meanwhile, blood pressure (BP) was monitored by tail‐cuff device. In NT (non‐transgenic) mice, DOCA+1%NaCl increases BP significantly, before vs. after DOCA (systolic BP): 114.8 ±0.94 vs. 133.7 ±3.3 mmHg. In whole, compared to sham controls, the treated NT mice performed worse in all four behavioral tests, indicating cognition decline after DOCA‐salt treatment. Later, brain tissues were collected to evaluate possible degenerative alterations in neurons by transmission electron microscope (TEM) imaging, Golgi‐Cox staining, fluorescence‐activated cell sorting (FACS) and qRT‐PCR. It was shown via the TEM that DOCA‐salt hypertension induces mitochondrial damage and morphological changes in prefrontal cortical neurons, meanwhile, Golgi‐Cox staining revealed decreased numbers of dendritic spines, suggesting the onset of neurodegeneration in DOCA‐salt hypertension. In both prefrontal cortex and hippocampus, the mRNA levels of BDNF (brain‐derived neurotrophic factor) and PI4KIIIβ (phosphatidylinositol 4‐kinase IIIβ) were found to be significantly lower in hypertensive NT mice. Using FACS, neurons were sorted from those regions, then analyzed via qRT‐PCR, which identified that the downregulated expression of PI4KIIIβ takes place in neurons. Using laser speckle contrast imaging system, we detected only decreased functional hyperemia, but no impairment in basal cerebral blood flow of the hypertensive mice, suggesting that the neurodegeneration is not caused by cerebral ischemia. We previously demonstrated that neuronal angiotensin type 1 receptors (AT1R) plays a pivotal role in the maintenance of neurogenic hypertension, and here we hypothesized that activation of AT1R could also exacerbate hypertension‐induced reduction in neuronal function. Particularly, in the hippocampal CA1 of adult mice, treatment of angiotensin II (Ang‐II, 500 nM, bath application) induces a significant inhibition in the induction of long‐term potentiation (LTP), while pre‐treatment with losartan (5 μM, 15 min), the AT1R antagonist, can efficiently block this Ang‐II‐induced inhibition, supporting the involvement of neuronal AT1R. Indeed, in mice with selective deletion of neuronal AT1aR, DOCA‐salt‐induced neurodegeneration was attenuated, as evidenced by significantly higher mRNA levels of cortical BDNF and PI4KIIIβ, compared with the NT. In summary, our data suggest that neuron‐expressing AT1R participates in the development of hypertension‐associated cognitive impairment, independently of vascular AT1R.

Mitochondrial Sirtuin-3 (SIRT3) Prevents Doxorubicin-Induced Dilated Cardiomyopathy by Modulating Protein Acetylation and Oxidative Stress.

High doses of doxorubicin put cancer patients at risk for developing dilated cardiomyopathy. Previously, we showed that doxorubicin treatment decreases SIRT3 (sirtuin 3), the main mitochondrial deacetylase and increases protein acetylation in rat cardiomyocytes. Here, we hypothesize that SIRT3 expression can attenuate doxorubicin induced dilated cardiomyopathy in vivo by preventing the acetylation of mitochondrial proteins. Nontransgenic, M3-SIRT3 (truncated SIRT3; short isoform), and M1-SIRT3 (full-length SIRT3; mitochondrial localized) transgenic mice were treated with doxorubicin for 4 weeks (8 mg/kg body weight per week). Echocardiography was performed to assess cardiac structure and function and validated by immunohistochemistry and immunofluorescence (n=4-10). Mass spectrometry was performed on cardiac mitochondrial peptides in saline (n=6) and doxorubicin (n=5) treated hearts. Validation was performed in doxorubicin treated primary rat and human induced stem cell derived cardiomyocytes transduced with adenoviruses for M3-SIRT3 and M1-SIRT3 and deacetylase deficient mutants (n=4-10). Echocardiography revealed that M3-SIRT3 transgenic mice were partially resistant to doxorubicin induced changes to cardiac structure and function whereas M1-SIRT3 expression prevented cardiac remodeling and dysfunction. In doxorubicin hearts, 37 unique acetylation sites on mitochondrial proteins were altered. Pathway analysis revealed these proteins are involved in energy production, fatty acid metabolism, and oxidative stress resistance. Increased M1-SIRT3 expression in primary rat and human cardiomyocytes attenuated doxorubicin-induced superoxide formation, whereas deacetylase deficient mutants were unable to prevent oxidative stress. Doxorubicin reduced SIRT3 expression and markedly affected the cardiac mitochondrial acetylome. Increased M1-SIRT3 expression in vivo prevented doxorubicin-induced cardiac dysfunction, suggesting that SIRT3 could be a potential therapeutic target for mitigating doxorubicin-induced dilated cardiomyopathy.

Overexpression of lncRNA-Gm2044 in spermatogonia impairs spermatogenesis in partial seminiferous tubules

Long noncoding RNAs (lncRNAs) have been demonstrated to regulate reproduction in mammals. Our previous study revealed that the expression level of lncRNA-Gm2044 was obviously elevated in nonobstructive azoospermia with spermatogonial arrest. Here, a transgenic mouse model of lncRNA-Gm2044 in spermatogonia using the Stra8 promoter was constructed to explore the roles of upregulated lncRNA-Gm2044 in male fertility. Testicular morphology and fertility weren't affected in transgenic mice expressing lncRNA-Gm2044. However, overexpression of lncRNA-Gm2044 in spermatogonia partially impaired spermatogenesis in the transgenic mice. Then, transcriptome sequencing was executed to find the potential signaling pathway repressing spermatogenesis in germ cells of lncRNA-Gm2044 transgenic mice. Through quantitative analysis of differentially expressed genes, 442 upregulated mRNAs and 147 downregulated mRNAs were displayed in male germ cells of Gm2044-transgenic mice (Gm2044-Tg) compared with non-transgenic mice (Non-Tg). Using gene ontology (GO) analysis, differentially expressed genes were shown to play vital roles in RNA_metabolic_process, Central_element, Enzyme_binding, and Intracellular_bridge. Using Kyoto encyclopedia of genes and genomes (KEGG) analysis, differentially expressed genes were shown to participate in RNA_transport, Cell_cycle, Renin-angiotensin_system, and Chemokine_signaling_pathway. Gene Set Enrichment Analysis (GSEA) revealed that Acrosome_assembly and Sperm_plasma_membrane were involved in the overexpression of lncRNA-Gm2044 blocking spermatogenesis. Furthermore, some of the most differentially expressed mRNAs were verified by RT-qPCR. In addition, we determined that the lncRNA-Gm2044 has no ability to translate into peptides by the bioinformatics method and molecular experiment. Thus, lncRNA-Gm2044 is a novel molecular target for the diagnosis and treatment of male infertility.

Hebbian plasticity: the elusive missing link at the heart of Alzheimer's disease pathogenesis?

Hebbian plasticity: the elusive missing link at the heart of Alzheimer's disease pathogenesis?