Age-matched Littermate Controls Research Articles

Inferring Parameters of Pyramidal Neuron Excitability in Mouse Models of Alzheimer’s Disease Using Biophysical Modeling and Deep Learning

Alzheimer’s disease (AD) is believed to occur when abnormal amounts of the proteins amyloid beta and tau aggregate in the brain, resulting in a progressive loss of neuronal function. Hippocampal neurons in transgenic mice with amyloidopathy or tauopathy exhibit altered intrinsic excitability properties. We used deep hybrid modeling (DeepHM), a recently developed parameter inference technique that combines deep learning with biophysical modeling, to map experimental data recorded from hippocampal CA1 neurons in transgenic AD mice and age-matched wildtype littermate controls to the parameter space of a conductance-based CA1 model. Although mechanistic modeling and machine learning methods are by themselves powerful tools for approximating biological systems and making accurate predictions from data, when used in isolation these approaches suffer from distinct shortcomings: model and parameter uncertainty limit mechanistic modeling, whereas machine learning methods disregard the underlying biophysical mechanisms. DeepHM addresses these shortcomings by using conditional generative adversarial networks to provide an inverse mapping of data to mechanistic models that identifies the distributions of mechanistic modeling parameters coherent to the data. Here, we demonstrated that DeepHM accurately infers parameter distributions of the conductance-based model on several test cases using synthetic data generated with complex underlying parameter structures. We then used DeepHM to estimate parameter distributions corresponding to the experimental data and infer which ion channels are altered in the Alzheimer’s mouse models compared to their wildtype controls at 12 and 24 months. We found that the conductances most disrupted by tauopathy, amyloidopathy, and aging are delayed rectifier potassium, transient sodium, and hyperpolarization-activated potassium, respectively.

Identification of Molecular Mechanisms in Radiation Cystitis: Insights from RNA Sequencing.

Pelvic cancer survivors who were treated with radiation therapy are at risk for developing (hemorrhagic) radiation cystitis (RC) many years after completion of radiation therapy. Patients with RC suffer from lower urinary tract symptoms, including frequency, nocturia, pelvic pain, and incontinence. In advanced stages, hematuria can occur, potentially escalating to life-threatening levels. Current therapeutic options for RC are limited, partly due to ethical concerns regarding bladder biopsy in patients with fragile bladder tissue. This study aimed to leverage our established preclinical model to elucidate the molecular pathways implicated in radiation-induced tissue changes in the bladder. Female C57Bl/6 mice received a single dose of 40 Gy using CT-guided imaging and a two-beam irradiation approach using the SARRP irradiator. Bladders from irradiated and age-matched littermate controls were harvested at 1 week [n = 5/group] or 6 months [n = 5/group] after irradiation, RNA was harvested, and mRNA sequencing was performed at paired-end 150bp on the Illumina NovaSeq6000 with a target of 30 million reads per sample. Following RNA sequencing, thorough bioinformatics analysis was performed using iPathwayGuide v2012 (ADVAITA Bioinformatics). Findings of the RNA sequencing were validated using qPCR analysis. At 1 week post-irradiation, altered gene expression was detected in genes involved in DNA damage response, apoptosis, and transcriptional regulation. By 6 months post-irradiation, significant changes in gene expression were observed in inflammation, collagen catabolism, and vascular health. Affected pathways included the p53, JAK-STAT, and PI3K-Akt pathways. These findings were validated in vivo in bladder tissues from our preclinical model. This is the first study to determine the molecular changes in the bladder in response to radiation treatment. We have successfully pinpointed several pathways and specific genes that undergo modification, thereby contributing to the progression of radiation cystitis. These insights enhance our understanding of the pathophysiology of radiation cystitis and may ultimately pave the way to the identification of potential new therapeutic targets.

0258 Altered sleep regulation in a mouse model of Parkinson’s disease

Abstract Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by pathognomonic motor abnormalities that lead to significant disability and morbidity in advanced stages. PD is also associated with sleep abnormalities such as increased fragmentation, insomnia, and excessive daytime sleepiness. Little is known of the underlying mechanisms, in part due to the limited ability of existing mouse models to adequately replicate human PD patient sleep phenotypes. Here we characterize sleep-wake behavior in an innovative genetic mouse model of PD (MCI-Park) targeting a mitochondrial complex I gene (Ndufs2), which experiences progressive dopaminergic neuron loss and displays motor phenotypes resembling those seen in human PD. Methods Undisturbed sleep-wake behavior during 12 hr light: 12 hr dark cycles was evaluated in MCI-Park mice during prodromal stages (6-8 weeks of age) and later parkinsonian stages (14-18 weeks of age) as well as in age-matched wildtype littermate controls. Electroencephalographic (EEG) and electromyographic (EMG) recording electrodes were surgically implanted using sterile technique in male and female MCI-Park and wildtype mice. After one week of recovery and acclimation to recording chambers, EEG and EMG were recorded continuously and sleep phenotypes were analyzed. Results In prodromal stages, MCI-Park mice exhibited similar overall sleep state amounts compared to wildtype mice, however there were significantly more state shifts as well as longer bouts of wakefulness and shorter bouts of non-REM (NREM) sleep. As mice progressed to parkinsonian stages, these fragmentation measures worsened. In addition, parkinsonian MCI-Park mice experienced a significant increase in wakefulness, a significant reduction in NREM sleep amount, and alterations in REM sleep, with pronounced reductions in REM sleep amount and number of REM bouts. Power spectral analysis revealed consistent decreases in theta2 (also known as alpha) power across all sleep-wake states in MCI-Park mice. Conclusion These results indicate that mice harboring a genetic defect causing selective disruption of dopaminergic neurons exhibit progressive sleep abnormalities that resemble sleep disturbances in PD patients. This model thus offers an opportunity to evaluate mechanisms underlying and therapeutic strategies targeting altered sleep in PD patients. Support (if any) This work was supported by the Department of Defense award number W81XWH-21-0582 (F.W.T. and D.J.S.).

Aberrant adaptation of the inner medullary collecting ducts results in sodium retention and hypernatremia in a thick ascending limb specific Myh9 and Myh10 conditional knockout mouse model

Sodium reabsorption in the thick ascending limb (TAL) epithelium is essential to generate and sustain the interstitial osmotic gradient, which facilitates water reabsorption in the adjacent collecting duct system. Impaired TAL function can compromise this gradient and lead to electrolyte imbalance. Our previous work demonstrated that pan renal tubular conditional genetic inactivation of nonmuscle myosin II genes Myh9&10 in adult mice results in progressive kidney disease of TAL origin with aberrant regulation of TAL proteins U MOD and NKCC2 (Otterpohl et al; 2020). To confirm the cell-autonomous role for NM2 proteins in adult TAL epithelial cells, we generated a TAL-specific conditional knockout mouse model of Myh9&10 using the UMOD->CreERT2 mouse line (cKO). Genetic inactivation of Myh9&10 in the TAL was induced at 5 weeks of age with tamoxifen injections. Age matched control and cKO littermates were part of every cohort. Mice underwent metabolic, histologic, immunohistochemical, and biochemical analyses at varying time points until moribund. Development of progressive kidney disease in cKO mice was indicated by increasing levels of blood urea nitrogen, serum creatinine, and the tubular injury marker NGAL. Our results show that cKO females experience significant loss of weight at 12 weeks of age and are moribund between 13 – 14 weeks, while cKO males survive up to 25 weeks. Interestingly, female cKO mice exhibit hypernatremia at 12 weeks of age and male cKO mice between 16 - 20 weeks of age. We analyzed NKCC2 protein levels in whole kidney lysates and a drastic reduction was evident in both female and male cKO mice by 9 weeks of age, therefore hypernatremia was unexpected. We examined if the hypernatremia in TAL-Myh9&10 cKO mice is due to tubular adaptation in the distal nephron and/or the collecting duct segments. Systematic analysis of sodium cotransporters and channels in the distal nephron and collecting duct segments revealed an increase in NCC/pNCC levels in males and a decrease in females when compared with age matched control littermates. No apparent volume loss (increased urine output) has been observed in the female mice. Additionally, immunostaining experiments showed aberrant increase in expression of ENaC gamma in the inner medullary collecting duct cells in female and not male mice by 9-weeks of age. ENaC expression is limited to cortical collecting duct and the connecting segment under normal conditions in the mouse kidney. Consistent with this, our control mice do not have ENaC gamma expression in the inner medullary collecting ducts. Further experiments are underway to determine the cellular state/identity of these ENaC expressing inner medullary collect duct cells. These experiments confirm the critical role for Myh9&10 proteins in TAL tubule NKCC2 transport, and reveal novel, sex specific, adaptive mechanisms in distal nephron and collecting ducts in response to compromised NKCC2 transport within the TAL. NIDDK RO1 DK131020-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of metabolic substrate modification on myocardial efficiency in a rat model of obesity and diabetes

Abstract Background Congenic leptin receptor deficient rat generated by introgression of the Koletsky leptin receptor mutation into BioBreeding Diabetes Resistant rat (BBDR.lepr−/−) is a novel animal model combining obesity, systemic insulin resistance and diabetes. Systemic insulin resistance is associated with reduced myocardial glucose utilization, but its effect on myocardial external efficiency, i.e. the ability of the myocardium to convert energy into external stroke work, remains uncertain. Purpose To characterize cardiac energy metabolism and function in BBDR.lepr−/− rats and to study the effect of dipeptidyl peptidase 4 (DPP-4) inhibitor linagliptin in this model. Methods Cardiac phenotype was evaluated in six-month-old male BBDR.lepr−/− rats (n=11) and age-matched male non-diabetic lean control littermates (BBDR.lepr+/− or BBDR.lepr+/+ rats, n=14). Of these, 7 BBDR.lepr−/− rats and 6 controls underwent cardiac ultrasound, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT), and [11C]acetate PET in order to evaluate cardiac structure and function as well as glucose and oxidative metabolism. In the remaining rats, fatty acid metabolism was evaluated by [18F]fluorothia-6-heptadecanoic acid ([18F]FTHA) PET/CT. In the linagliptin intervention study, 25 BBDR.lepr−/− male rats were randomly divided into control group (n=11) that received regular chow diet and linagliptin group (n=14) that received linagliptin (10mg/kg/d) mixed in the chow diet for three months. After the intervention, the rats underwent cardiac ultrasound, [18F]FDG PET/CT, and [11C]acetate PET. Results Compared with controls, BBDR.lepr−/− rats showed increased left ventricle (LV) mass (∼40%, p>0.001) and higher systolic blood pressure (∼10%, p=0.02). However, fractional shortening and cardiac output were similar in both groups. Myocardial fractional uptake rate of glucose measured with [18F]FDG PET was significantly reduced (∼86%, p=0.004) (Fig. 1A, E), whereas myocardial fatty acid uptake measured by [18F]FTHA PET was not significantly increased (free fatty acid (FFA) corrected standardized uptake value (SUV) ∼21%, p=0.54) (Fig. 1B) in BBDR.lepr−/− compared to controls. Myocardial oxygen consumption assessed by [11C]acetate PET was similar in both groups (Fig. 1C, E), but LV work per gram of myocardium was reduced (∼28%, p=0.001) resulting in reduced myocardial external efficiency (∼21%, p=0.03) (Fig. 1D) in BBDR.lepr−/− compared to controls. Treatment with linagliptin significantly enhanced myocardial fractional uptake rate of glucose (∼166%, p=0.006) (Fig. 2A, C), but had no effect on efficiency of cardiac work (Fig. 2B). Conclusions Obese and diabetic BBDR.lepr−/− rats demonstrate LV hypertrophy and markedly reduced myocardial glucose utilization associated with impaired myocardial external efficiency despite normal LV systolic function. Enhancement of myocardial glucose uptake by linagliptin did not improve efficiency of cardiac work. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): IMI-SUMMIT

Hippocampal neural circuit connectivity alterations in an Alzheimer's disease mouse model revealed by monosynaptic rabies virus tracing

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with growing major health impacts, particularly in countries with aging populations. The examination of neural circuit mechanisms in AD mouse models is a recent focus for identifying new AD treatment strategies. We hypothesize that age-progressive changes of both long-range and local hippocampal neural circuit connectivity occur in AD. Recent advancements in viral-genetic technologies provide new opportunities for semi-quantitative mapping of cell-type-specific neural circuit connections in AD mouse models. We applied a recently developed monosynaptic rabies tracing method to hippocampal neural circuit mapping studies in AD model mice to determine how local and global circuit connectivity to hippocampal CA1 excitatory neurons may be altered in the single APP knock-in (APP-KI) AD mouse model. To determine age-related AD progression, we measured circuit connectivity in age-matched littermate control and AD model mice at two different ages (3–4 vs. 10–11 months old). We quantitatively mapped the connectivity strengths of neural circuit inputs to hippocampal CA1 excitatory neurons from brain regions including hippocampal subregions, medial septum, subiculum and entorhinal cortex, comparing different age groups and genotypes. We focused on hippocampal CA1 because of its clear relationship with learning and memory and that the hippocampal formation shows clear neuropathological changes in human AD. Our results reveal alterations in circuit connectivity of hippocampal CA1 in AD model mice. Overall, we find weaker extrinsic CA1 input connectivity strengths in AD model mice compared with control mice, including sex differences of reduced subiculum to CA1 inputs in aged female AD mice compared with aged male AD mice. Unexpectedly, we find a connectivity pattern shift with an increased proportion of inputs from the CA3 region to CA1 excitatory neurons when comparing young and old AD model mice, as well as old wild-type mice and old AD model mice. These unexpected shifts in CA3-CA1 input proportions in this AD mouse model suggest the possibility that compensatory circuit increases may occur in response to connectivity losses in other parts of the hippocampal circuits. We expect that this work provides new insights into the neural circuit mechanisms of AD pathogenesis.

Deficiency in FTSJ1 Affects Neuronal Plasticity in the Hippocampal Formation of Mice.

Simple SummaryNeuronal plasticity refers to the brain’s ability to adapt in response to activity-dependent changes. This process, among others, allows the brain to acquire memory or to compensate for a neurocognitive deficit. We analyzed adult FTSJ1-deficient mice in order to gain insight into the role of FTSJ1 in neuronal plasticity. These mice displayed alterations in the hippocampus (a brain structure that is involved in memory and learning, among other functions) e.g., in the form of changes in dendritic spines. Changes in dendritic spines are considered to represent a morphological hallmark of altered neuronal plasticity, and thus FTSJ1 deficiency might have a direct effect upon the capacity of the brain to adapt to plastic changes. Long-term potentiation (LTP) is an electrophysiological correlate of neuronal plasticity, and is related to learning and to processes attributed to memory. Here we show that LTP in FTSJ1-deficient mice is reduced, hinting at disturbed neuronal plasticity. These findings suggest that FTSJ1 deficiency has an impact on neuronal plasticity not only morphologically but also on the physiological level.The role of the tRNA methyltransferase FTSJ1 in the brain is largely unknown. We analyzed whether FTSJ1-deficient mice (KO) displayed altered neuronal plasticity. We explored open field behavior (10 KO mice (aged 22–25 weeks)) and 11 age-matched control littermates (WT) and examined mean layer thickness (7 KO; 6 WT) and dendritic spines (5 KO; 5 WT) in the hippocampal area CA1 and the dentate gyrus. Furthermore, long-term potentiation (LTP) within area CA1 was investigated (5 KO; 5 WT), and mass spectrometry (MS) using CA1 tissue (2 each) was performed. Compared to controls, KO mice showed a significant reduction in the mean thickness of apical CA1 layers. Dendritic spine densities were also altered in KO mice. Stable LTP could be induced in the CA1 area of KO mice and remained stable at for at least 1 h, although at a lower level as compared to WTs, while MS data indicated differential abundance of several proteins, which play a role in neuronal plasticity. FTSJ1 has an impact on neuronal plasticity in the murine hippocampal area CA1 at the morphological and physiological levels, which, in conjunction with comparable changes in other cortical areas, might accumulate in disturbed learning and memory functions.

A18 DEVELOPMENT OF CHRONIC ILEITIS IN THE TNFΔARE MOUSE IS ASSOCIATED WITH A LOSS OF MESENTERIC LYVE1+ MACROPHAGES

Abstract Background Crohn’s disease (CD) is a form of inflammatory bowel disease that causes transmural inflammation of any part of the gastrointestinal (GI) tract but preferentially the terminal ileum and/or the colon. This chronic inflammation is not limited to the GI tract but also affects the mesentery of inflamed regions where it induces the formation of tertiary lymphoid organs (TLOs). The homeostatic LYVE1+ macrophages (LYmΦ) have been identified in many different organs including the mesentery. They have been shown to typically align along blood vessels where they participate in the control of collagen deposition and during inflammation, regulate vascular permeability and immune cell recruitment. Here we investigate the role of the mesenteric LYmΦ in a transgenic model of CD, the TNFΔARE mouse which develops terminal ileitis over time and expresses similar mesenteric alterations (TLOs) as seen in CD. Aims To determine whether the mesenteric LYmΦ population is altered during the development of chronic inflammation in the TNFΔARE mouse and whether they participate in the development of TLOs. Methods Confocal immunofluorescent imaging of the terminal ileal mesentery of TNFΔARE mice and littermate controls were preformed to identify changes to the populations of LYmΦ during the development of chronic inflammation. Myeloperoxidase activity was used to assess terminal ileitis. Results During the initial stage of ileitis (8 weeks) LYmΦs are present across the terminal ileal mesentery in both TNFΔARE and age-matched WT littermate controls with a subpopulation aligning along the collecting lymphatic vessels of the mesentery (n=6). At 20 weeks, while the ileitis progresses, aggregates of CD45+ cells have formed within the TNFΔARE mice where LYmΦs also accumulate (n=5). At 28 weeks when the ileitis worsens, the LYmΦs are greatly reduced from the terminal ileal mesentery as well as from the CD45+ cell aggregates (n=5). WT littermate controls never develop large CD45+ cell aggregates (n=5 for each time point). Antibodies to the LYVE1 cytosolic and extracellular domains confirmed the loss of the LYVE1 receptor over receptor cleavage, however, there was no correlation of altered collagen-1 deposition within the mesentery at 28 weeks. Conclusions Here we show a spatial relationship between LYmΦs and collecting lymphatic vessels as well as a decrease in LYmΦs during the development of chronic ileitis in the TNFΔARE mouse. Whether they contribute to TLO formation, or their loss exacerbates terminal ileitis is still unknown and under current investigation. Funding Agencies CCC

Age-associated increase in anxiety-like behavior in Lpaatδ/Agpat4 knockout mice

• Rearing activity is elevated in 14- and 20-month old Lpaatδ −/− mice. • 20 month-old Lpaatδ −/− mice show increased anxiety-like behavior in the E.P.M. and O.F.T. • NMDA receptor levels are not significantly different in aged Lpaatδ −/− mice. • Brain expression of serotonin regulatory genes 5htr1a and slc6a4 is lower in 20 month-old Lpaatδ −/− mice. We have previously reported deficits in phospholipids and NMDA-receptors in brains of 2–3 month-old mice deficient in the mitochondrial enzyme lysophosphatidic acid acyltransferase delta (LPAATδ)/acylglycerophosphate acyltransferase 4 (AGPAT4), which were associated with deficits in learning and memory. An initial assessment suggested these mice may also be predisposed to an anxiogenic-like behavioral phenotype, but this was not fully evident in young mice. Since some animal and human studies report worsening anxiety from middle-to-older adulthood, we hypothesized that aging may reveal a significant anxiety-like phenotype in Lpaatδ -deficient mice. We analyzed anxiety-like behavior in 14- and 20-month old Lpaatδ deficient (Lpaat δ −/− ) mice and their wildtype age-matched littermate controls. Fourteen month-old Lpaatδ -deficient mice had ∼3-fold greater rearing activity over 24-h than controls. Aging was associated with a significant decrease in rearing activity in Lpaatδ -deficient mice, but not in wildtype control mice. However, rearing activity remained 59% higher in Lpaatδ -deficient mice than controls at 20 months of age. Behavior of Lpaatδ -deficient mice in the open field test and elevated plus maze also suggested a more anxiogenic-like phenotype, although differences between the genotypes were statistically significant only at 20 months. Brain NMDA receptor subunits were not significantly different between different ages or genotypes, which differed from prior results in younger mice. An initial screen of genes related to behavior indicated lower brain expression in Lpaatδ -deficient mice of the serotonin metabolism genes 5htr1a and slc6a4. These studies have implications for understanding associations between LPAATδ/AGPAT4 gene variants and human anxiety.

Activation of Raf signalling in NF2-null Schwann cells leads to sustained proliferation; an investigation of a new and inducible model for human schwannoma

Abstract Aims The NF2 gene encodes the tumour suppressor Merlin, which is deleted in 100% of patients with the familial tumour predisposition syndrome neurofibromatosis type 2 but also in 70% of those who develop sporadic schwannomas. The Raf-TR mouse model uses a tamoxifen-inducible Raf-kinase/ oestrogen receptor fusion protein (Raf-TR) expressed in myelinating Schwann cells to mimic a nerve injury response in Schwann cell by activating Raf/MEK/ERK signalling in the absence of peripheral nerve injury. We will assess whether Raf/MEK/ERK activation on an NF2 null background leads to tumourigenesis within the vestibular nerves and dorsal root ganglia (DRGs), two tumour sites identified in the Periostin-Cre mouse model in which schwannoma formation is spontaneous, with a view to generating an inducible NF2 null schwannoma mouse model. Method Mice with a Schwann cell specific loss of Merlin were crossed with mice carrying a tamoxifen-inducible Raf-TR gene to generate Raf-TR+/-; P0-Cre+/-; NF2fl/fl (Cre+) mice which were NF2 null and compared to Raf-TR+/-; P0-Cre-/-; NF2fl/fl (Cre-) littermate controls. Mice were injected with tamoxifen or vehicle for five consecutive days and their vestibular nerves and dorsal root ganglia (DRGs) were analysed at various timepoints . An EdU proliferation assay was used to quantify the proliferation in the vestibular ganglia, as well as the DRGs. Rates of proliferation were compared to Cre- age-matched littermate controls treated with tamoxifen or vehicle. Results In the Periostin-Cre NF2 null schwannoma model, tumours form spontaneously in the DRGs and vestibular ganglia. In our new model, we see a clear increase in proliferation at 21 d post-injection in the NF2 null (Cre+) tamoxifen-treated mice compared to control (Cre-) tamoxifen-treated controls in both DRGs and vestibular ganglia. Cre- tamoxifen-treated mice do not show increased proliferation compared to Cre- vehicle controls. Taken together, this shows that activation of the Raf/MEK/ERK pathway in Schwann cells only causes a sustained proliferation response on an NF2 null background in the DRGs and vestibular ganglia. We are assessing later timepoints to further characterise tumour development in these mice. Conclusion Combining the Raf-TR mouse model to create a demyelinating phenotype with an NF2 null background leads to vastly increased rates of proliferation at the sites of schwannoma tumourigenesis within the peripheral nervous system: the DRGs and the vestibular ganglia. The high proliferation in the vestibular ganglia in particular is similar to the development of vestibular schwannomas in patients with Neurofibromatosis type 2. The new mouse model used in this study shows potential to be very useful as an inducible schwannoma tumour model, in which we can study the early events of tumour formation.

Age-Dependent Degradation of Locomotion Encoding in Huntington's Disease R6/2 Model Mice.

Background:Huntington’s disease (HD) is an inherited fatal neurodegenerative disease, leading to neocortical and striatal atrophy. The commonly studied R6/2 HD transgenic mouse model displays progressive motor and cognitive deficits in parallel to major pathological changes in corticostriatal circuitry.Objective:To study how disease progression influences striatal encoding of movement.Methods:We chronically recorded neuronal activity in the dorsal striatum of R6/2 transgenic (Tg) mice and their age-matched nontransgenic littermate controls (WTs) during novel environment exposure, a paradigm which engages locomotion to explore the novel environment.Results:Exploratory locomotion degraded with age in Tg mice as compared to WTs. We encountered fewer putative medium spiny neurons (MSNs)—striatal projection neurons, and more inhibitory interneurons—putative fast spiking interneurons (FSIs) in Tg mice as compared to WTs. MSNs from Tg mice fired less spikes in bursts without changing their firing rate, while FSIs from these mice had a lower firing rate and more of them were task-responsive as compared to WTs. Additionally, MSNs from Tg mice displayed a reduced ability to encode locomotion across age groups, likely associated with their low prevalence in Tg mice, whereas the encoding of locomotion by FSIs from Tg mice was substantially reduced solely in old Tg mice as compared to WTs.Conclusion:Our findings reveal an age-dependent decay in striatal information processing in transgenic mice. We propose that the ability of FSIs to compensate for the loss of MSNs by processes of recruitment and enhanced task-responsiveness diminishes with disease progression, possibly manifested in the displayed age-dependent degradation of exploratory locomotion.

The JAK2V617F-Bearing Vascular Niche Regulates Hematopoietic Stem/Progenitor Cell Regeneration Following Radiation Injury

IntroductionMyeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion. Endothelial cells (ECs) are an essential component of the hematopoietic niche. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. Previously, we reported that the JAK2V617F-bearing vascular niche promotes the expansion of the JAK2V617F HSPCs in preference to JAK2WT HSPCs. In this work, we test the hypothesis that the JAK2V617F-bearing vascular niche protects MPN HSPCs from radiation injury.MethodsJAK2V617F Flip-Flop (FF1) mice and Tie2-Cre mice were crossed to generate a strain in which human JAK2V617F is expressed specifically in hematopoietic cells and ECs (Tie2/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsTo investigate the effects of the JAK2V617F-bearing vascular niche on MPN hematopoiesis in vivo, WT CD45.1 marrow cells were transplanted into lethally irradiated (950cGy) Tie2/FF1 mice or age-matched littermate control mice (CD45.2) (n=12 in each group). During a 3-month follow up, while all WT control recipients displayed full donor engraftment, 7 of 12 Tie2/FF1 recipient mice displayed recovery of the JAK2V617F-mutant hematopoiesis (mixed donor/recipient chimerism) 10 weeks after transplantation. (Figure 1) In contrast to the Tie2/FF1 recipients with full WT donor engraftment, the mixed chimeric mice developed neutrophilia, thrombocytosis, splenomegaly, and significantly expanded CD45.2 JAK2V617F-mutant HSPCs during an 8 month follow up, resembling what we observed in the primary Tie2/FF1 mice in a previous study. These findings suggest that the JAK2V617F-mutant HSPCs in the Tie2/FF1 mice are relatively protected from lethal irradiation.To investigate whether this radioprotection phenotype is due to the JAK2V617F mutation in Tie2/FF1 HSPCs, we generated a chimeric murine model with JAK2V617F-mutant HSPCs and a WT vascular niche by transplanting JAK2V617F marrow cells into WT recipients. The transplantation of WT marrow cells into WT recipients served as a control. Following hematopoietic recovery, each set of mice were irradiated with 300cGy to create a radiation injury, and 2 hr later marrow Lin- HSPCs were isolated for evaluation of cellular apoptosis and cell cycle status. We found that cell apoptosis was significantly increased in the JAK2V617F-mutant HSPCs compared to JAK2WT HSPCs (14.4% vs 2.2%, P=0.035). There was no significant difference in cell cycle status between the JAK2V617F HSPCs and JAK2WT HSPCs. (Figure 2) These data suggest that, in the WT vascular niche, the JAK2V617F-mutant HSPC is more (not less) sensitive to radiation-induced apoptosis than JAK2WT HSPCs.We then investigated whether the hematopoietic radioprotection phenotype is a result of the JAK2V617F-bearing vascular niche protecting HSPCs. Marrow Lineageneg (Lin-) HSPCs were isolated from Tie2/FF1 mice and age-matched littermate controls and cultured on a feeder layer of JAK2WT or JAK2V617F ECs. After 300cGy irradiation, we observed higher numbers of cells from JAK2V617FHSPCs cultured on JAK2V617F-bearing ECs compared to their being cultured on JAK2WT ECs (1.6-fold, P= 0.026). In addition, JAK2WT ECs displayed increased cell apoptosis post irradiation compared to JAK2V617F ECs (19.5% vs 7.7%, P=0.026). qRT-PCR of irradiated murine ECs confirmed that both epidermal growth factor (EGF) (4.0-fold, P=0.011) and pleiotrophin (PTN) (11.4-fold, P=0.00001), two niche factors involved in the regulation of HSPC regeneration following radiation injury, were up-regulated in JAK2V617F-bearing ECs compared to JAK2WT ECs. (Figure 3) These results suggest that the JAK2V617F-bearing vascular niche contributes directly to HSPC radioprotection, possibly by its elaboration of soluble niche factors, such as EGF and/or PTN.ConclusionsIn summary, we report that the JAK2V617F-bearing vascular niche protects the JAK2V617F HSPCs from the otherwise lethal irradiation administered during conditioning for marrow transplantation. Together with our previous studies, our findings indicate that the mutant vascular niche could contribute to disease relapse and poor donor cell engraftment following stem cell transplantation in patients with MPNs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The JAK2V617F-Bearing Megakaryocytes Promote Hematopoietic Stem/Progenitor Cell Expansion through the Thrombopoietin/MPL Signaling

IntroductionThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for MPN HSPC expansion are not fully understood. One hallmark of MPNs is megakaryocytic (MK) hyperplasia. Very recent evidence has implicated MKs in regulating HSPC activity. Since thrombopoietin (TPO) acting through its receptor MPL are key regulators of both megakaryopoiesis and HSPC activity, we hypothesize that JAK2V617F-bearing MKs promote MPN stem cell function via altered TPO/MPL signaling.MethodsJAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice were crossed to generate a MK cell lineage-specific human JAK2V617F knock-in mouse strain (Pf4/FF1). TPO or MPL was ablated in Pf4/FF1 by crossing Pf4/FF1 mice with TPO knock-out mice (TPOnull) or MPL knockout mice (MPLnull) to generate TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice. Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsPreviously, we have demonstrated that JAK2V617F-bearing MKs cause a murine myeloproliferative syndrome with HSPC expansion in a Pf4/FF1 murine model. Here, we studied the effects of the JAK2V617F-bearing MK niche on MPN stem cell function. First, we isolated LineagenegcKit+ (Lin-cKit+) HSPCs from 28wk old Pf4/FF1 mice and their age-matched littermate controls (Pf4-Cre) (n=4 in each group) and analyzed their cell cycle status using propidium iodide staining. We found that the percentage of Lin-cKit+ cells in the G0/G1 phase was moderately increased in the Pf4/FF1 mice compared to control mice (63% vs 56%, P=0.012), suggesting that HSPCs in the JAK2V617F-bearing MK niche are more quiescent than in the WT MK niche. (Figure 1)Next, we performed a competitive repopulation assay in which both CD45.2 marrow cells from Pf4/FF1 mice or control mice were injected intravenously together with competitor CD45.1 WT marrow cells into lethally irradiated (950 cGy) CD45.1 recipients. During 16 weeks follow up post transplantation, recipients of Pf4/FF1 marrow cells displayed a higher peripheral blood donor (CD45.2) chimerism than the control mice. Marrow cell colony assays demonstrated a moderate increase of CD45.2+ hematopoietic progenitors in the Pf4/FF1 marrow recipients compared to control mice (1.5-fold, p = 0.0025). Marrow cell flow cytometry analysis revealed that, although there was no significant difference in the total CD41+ MK cell numbers between the two groups, CD45.2+ MKs were significantly increased (3.3-fold, P=0.012) while CD45.1+ MKs were significantly decreased (3.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Similarly, although the total numbers of marrow CD45+EPCR+CD48-CD150+ (E-SLAM) cells, which is a highly purified long-term repopulating HSPC population, were not changed between the two groups, CD45.2+ E-SLAM cells were significantly increased (3.5-fold, P=0.012) while CD45.1+ E-SLAM cells were significantly decreased (5.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Therefore, the JAK2V617F-bearing MK niche promoted the expansion of JAK2V617F HSPCs, probably at the expense of suppressing WT hematopoiesis. No significant difference in blood cell count, spleen weight, or total femoral cell count was observed between the Pf4/FF1 marrow recipients and control marrow recipients. (Figure 2)To determine the role(s) of TPO/MPL signaling in the development of JAK2V617F MK-induced myeloproliferation, we ablated TPO or MPL in the Pf4/FF1 mice. At 28 weeks of age, in contrast to the Pf4/FF1 mice which developed thrombocytosis, increased numbers of hematopoietic progenitors, and a substantial expansion of MKs and HSPCs compared to age-matched WT controls, TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice did not display any of these features. (Figure 3) These data suggest that TPO and MPL are required for the development of JAK2V617F MK-induced myeloproliferation.ConclusionsOur study establishes that JAK2V617F-bearing MKs form an important part of the MPN HSPC niche and TPO/MPL signaling is critical for the MK niche function. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Early manifestation of gait alterations in the Tg2576 mouse model of Alzheimer's disease.

There is strong clinical evidence that multifaceted gait abnormalities may be manifested at early stages of Alzheimer's disease (AD), are related to cognitive decline, and can be used as an early biomarker to identify patients at risk of progressing to full-blown dementia. Despite their importance, gait abnormalities have not been investigated in mouse models of AD, which replicate important aspects of the human disease. The Tg2576 is frequently used in AD research to test therapeutic interventions targeting cellular mechanisms contributing to the genesis of AD. This transgenic mouse strain overexpresses a mutant form of the 695 amino acid isoform of human amyloid precursor protein with K670N and M671L mutations (APPK670/671L) linked to early-onset familial AD. Tg2576 mice exhibit impaired cognitive functions and increased cortical and hippocampal soluble β-amyloid levels starting from 5months of age and increased insoluble β-amyloid levels and amyloid plaques that resemble senile plaques associated with human AD by 13months of age. To demonstrate early manifestations of gait dysfunction in this relevant preclinical model, we characterized gait and motor performance in 10-month-old Tg2576 mice and age-matched littermate controls using the semi-automated, highly sensitive, Catwalk XT system. We found that Tg2576 mice at the pre-plaque stage exhibited significantly altered duty cycle and step patterns and decreased stride length and stride time. Base-of-support, stride time variability, stride length variability, cadence, phase dispersions and gait symmetry indices were unaltered. The presence of measurable early gait abnormalities during the pre-plaque stages of AD in this relevant preclinical mouse model has direct translational relevance and supports the view that longitudinal monitoring of gait performance could be used in addition to behavioral testing to evaluate progression of the disease and to assess treatment efficacy.

IL-23 receptor deficiency results in lower bone mass via indirect regulation of bone formation

The IL-23 receptor (IL-23R) signaling pathway has pleiotropic effects on the differentiation of osteoclasts and osteoblasts, since it can inhibit or stimulate these processes via different pathways. However, the potential role of this pathway in the regulation of bone homeostasis remains elusive. Therefore, we studied the role of IL-23R signaling in physiological bone remodeling using IL-23R deficient mice. Using µCT, we demonstrate that 7-week-old IL-23R−/− mice have similar bone mass as age matched littermate control mice. In contrast, 12-week-old IL-23R−/− mice have significantly lower trabecular and cortical bone mass, shorter femurs and more fragile bones. At the age of 26 weeks, there were no differences in trabecular bone mass and femur length, but most of cortical bone mass parameters remain significantly lower in IL-23R−/− mice. In vitro osteoclast differentiation and resorption capacity of 7- and 12-week-old IL-23R−/− mice are similar to WT. However, serum levels of the bone formation marker, PINP, are significantly lower in 12-week-old IL-23R−/− mice, but similar to WT at 7 and 26 weeks. Interestingly, Il23r gene expression was not detected in in vitro cultured osteoblasts, suggesting an indirect effect of IL-23R. In conclusion, IL-23R deficiency results in temporal and long-term changes in bone growth via regulation of bone formation.

Characterization of nonmotor behavioral impairments and their neurochemical mechanisms in the MitoPark mouse model of progressive neurodegeneration in Parkinson's disease

Mitochondrial dysfunction has been implicated as a key player in the pathogenesis of Parkinson's disease (PD). The MitoPark mouse, a transgenic mitochondrial impairment model developed by specific inactivation of TFAM in dopaminergic neurons, spontaneously exhibits progressive motor deficits and neurodegeneration, recapitulating several features of PD. Since nonmotor symptoms are now recognized as important features of the prodromal stage of PD, we comprehensively assessed the clinically relevant motor and nonmotor deficiencies from ages 8–24 wk in both male and female MitoPark mice and their littermate controls. As expected, motor deficits in MitoPark mice began around 12–14 wk and became severe by 16–24 wk. Interestingly, MitoPark mice exhibited olfactory deficits in the novel and social scent tests as early as 10–12 wk as compared to age-matched littermate controls. Additionally, male MitoPark mice showed spatial memory deficits before female mice, beginning at 8 wk and becoming most severe at 16 wk, as determined by the Morris water maze. MitoPark mice between 16 and 24 wk spent more time immobile in forced swim and tail suspension tests, and made fewer entries into open arms of the elevated plus maze, indicating a depressive and anxiety-like phenotype, respectively. Importantly, depressive behavior as determined by immobility in forced swim test was reversible by antidepressant treatment with desipramine. Neurochemical and mechanistic studies revealed significant changes in CREB phosphorylation, BDNF, and catecholamine levels as well as neurogenesis in key brain regions. Collectively, our results indicate that MitoPark mice progressively exhibit deficits in olfactory discrimination, cognitive learning and memory, and anxiety- and depression-like behaviors as well as key neurochemical signaling associated with nonmotor deficits in PD. Thus, MitoPark mice can serve as an invaluable model for studying nonmotor deficits in addition to studying the motor deficits related to pathology in PD.

AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1KOCrb2LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in Crb1KOCrb2LowMGC retinas but not in wild-type or Crb1-deficient retinas. Crb1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Müller cells of Crb1KOCrb2LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.

Microglia Demonstrate Local Mixed Inflammation and a Defined Morphological Shift in an APP/PS1 Mouse Model

Microglia are traditionally described as the immune cells of the brain and have an inflammatory role in Alzheimer's disease (AD). Microglial morphological and phenotypic shifts in AD have not been fully characterized; however, microglia are often described as either pro- or anti-inflammatory. To determine microglial if microglial morphology and phenotype changes with disease status. This study observed morphology through Iba1 immunohistochemistry on tissue sections encompassing the primary motor cortex and somatosensory barrel fields. Immunohistochemistry for pro-inflammatory markers: CD14 and CD40; and anti-inflammatory markers: CD16 and TREM2, was performed at 3, 6, and 12 months of age which correlated with pre-plaque, onset, and significant plaque load in APP/PS1 brains (n = 6) and compared to age-matched littermate controls (n = 6). Microglia demonstrated a defined morphological shift with time. Deramified morphologies increased in the APP/PS1, at both 6 months (p < 0.0001) and 12 months (p < 0.0001). At 12 months, there were significantly lower numbers of ramified microglia (p < 0.001). Results indicated that microglia have a heterogenic marker immunoreactivity as CD16, TREM2, and CD40 were associated with an activated morphology at the same time points. All inflammatory markers were significantly upregulated at 12 months in the APP/PS1 mice (TREM2 (F(2,30) = 10.75, p = 0.0003), CD40 (F(2,30) = 15.86, p < 0.0001), CD14 (F(2,30) = 6.84, p = 0.0036), and CD16 (F(2,30) = 3.026, p = 0.0635)). Our data indicate that pro- and anti-inflammatory factors of microglia occur in APP/PS1 mice.

MitoPark transgenic mouse model recapitulates the gastrointestinal dysfunction and gut-microbiome changes of Parkinson’s disease

Gastrointestinal (GI) disturbances are one of the earliest symptoms affecting most patients with Parkinson’s disease (PD). In many cases, these symptoms are observed years before motor impairments become apparent. Hence, the molecular and cellular underpinnings that contribute to this early GI dysfunction in PD have actively been explored using a relevant animal model. The MitoPark model is a chronic, progressive mouse model recapitulating several key pathophysiological aspects of PD. However, GI dysfunction and gut microbiome changes have not been categorized in this model. Herein, we show that decreased GI motility was one of the first non-motor symptoms to develop, evident as early as 8 weeks with significantly different transit times from 12 weeks onwards. These symptoms were observed well before motor symptoms developed, thereby paralleling PD progression in humans. At age 24 weeks, we observed increased colon transit time and reduced fecal water content, indicative of constipation. Intestinal inflammation was evidenced with increased expression of iNOS and TNFα in the small and large intestine. Specifically, iNOS was observed mainly in the enteric plexi, indicating enteric glial cell activation. A pronounced loss of tyrosine hydroxylase-positive neurons occurred at 24 weeks both in the mid-brain region as well as the gut, leading to a corresponding decrease in dopamine (DA) production. We also observed decreased DARPP-32 expression in the colon, validating the loss of DAergic neurons in the gut. However, the total number of enteric neurons did not significantly differ between the two groups. Metabolomic gas chromatography-mass spectrometry analysis of fecal samples showed increased sterol, glycerol, and tocopherol production in MitoPark mice compared to age-matched littermate controls at 20 weeks of age while 16 s microbiome sequencing showed a transient temporal increase in the genus Prevotella. Altogether, the data shed more light on the role of the gut dopaminergic system in maintaining intestinal health. Importantly, this model recapitulates the chronology and development of GI dysfunction along with other non-motor symptoms and can become an attractive translational animal model for pre-clinical assessment of the efficacy of new anti-Parkinsonian drugs that can alleviate GI dysfunction in PD.

Stat3 in osteocytes mediates osteogenic response to loading

Signal transducer and activator of transcription 3 (Stat3) is a member of the Stat family of proteins involved in signaling in many different cell types, including osteocytes. Osteocytes are considered major mechanosensing cells in bone due to their intricate dendritic networks able to sense changes in physical force and to orchestrate the response of osteoclasts and osteoblasts. We examined the role of Stat3 in osteocytes by generating mice lacking Stat3 in these cells using the Dmp-1(8kb)-Cre promoter (Stat3cKO mice). Compared to age-matched littermate controls, Stat3cKO mice of either sex (18 weeks old) exhibit reduced bone formation indices, decreased osteoblasts and increased osteoclasts, and altered material properties, without detectable changes in bone mineral density (BMD) or content of either trabecular or cortical bone. In addition, Stat3cKO mice of either sex show significantly decreased load-induced bone formation. Furthermore, pharmacologic inhibition of Stat3 in osteocytes in vitro with WP1066 blocked the increase in cytosolic calcium induced by ATP, a mediator of the cellular responses to sheer stress. WP1066 also increased reactive oxygen species (ROS) production in cultured MLO-Y4 osteocytes. These data demonstrate that Stat3 is a critical mediator of mechanical signals received by osteocytes and suggest that osteocytic Stat3 is a potential therapeutic target to stimulate bone anabolism.