Age-matched Wild-type Littermates Research Articles

Gait as a quantitative translational outcome measure in Angelman syndrome

Angelman syndrome (AS) is a genetic neurodevelopmental disorder characterized by developmental delay, lack of speech, seizures, intellectual disability, hypotonia, and motor coordination deficits. Motor abilities are an important outcome measure in AS as they comprise a broad repertoire of metrics including ataxia, hypotonia, delayed ambulation, crouched gait, and poor posture, and motor dysfunction affects nearly every individual with AS. Guided by collaborative work with AS clinicians studying gait, the goal of this study was to perform an in‐depth gait analysis using the automated treadmill assay, DigiGait. Our hypothesis is that gait presents a strong opportunity for a reliable, quantitative, and translational metric that can serve to evaluate novel pharmacological, dietary, and genetic therapies. In this study, we used an automated gait analysis system, in addition to standard motor behavioral assays, to evaluate components of motor, exploration, coordination, balance, and gait impairments across the lifespan in an AS mouse model. Our study demonstrated marked global motoric deficits in AS mice, corroborating previous reports. Uniquely, this is the first report of nuanced aberrations in quantitative spatial and temporal components of gait in AS mice compared to sex‐ and age‐matched wildtype littermates followed longitudinally using metrics that are analogous in AS individuals. Our findings contribute evidence toward the use of nuanced motor outcomes (i.e., gait) as valuable and translationally powerful metrics for therapeutic development for AS, as well as other genetic neurodevelopmental syndromes.Lay SummaryMovement disorders affect nearly every individual with Angelman Syndrome (AS). The most common motor problems include spasticity, ataxia of gait (observed in the majority of ambulatory individuals), tremor, and muscle weakness. This report focused on quantifying various spatial and temporal aspects of gait as a reliable, translatable outcome measure in a preclinical AS model longitudinally across development. By increasing the number of translational, reliable, functional outcome measures in our wheelhouse, we will create more opportunities for identifying and advancing successful medical interventions.

Amyloid Beta Pathology Exacerbates Weight Loss and Brain Cytokine Responses following Low-Dose Lipopolysaccharide in Aged Female Tg2576 Mice.

Systemic inflammation has been implicated in the progression of Alzheimer’s disease (AD); however, less is understood about how existing AD pathology contributes to adverse outcomes following acute inflammatory insults. In the present study, our goal was to determine how AD-associated amyloid beta (Aβ) pathology influences the acute neuroinflammatory and behavioral responses to a moderate systemic inflammatory insult. We treated 16–18-month-old female Tg2576 (Tg) mice, which overproduce human Aβ and develop plaques, and age-matched wild-type (WT) littermate mice with an intraperitoneal injection of 0.33 mg/kg lipopolysaccharide (LPS) or saline. Mice were then evaluated over the next 28 h for sickness/depressive-like behaviors (food intake, weight loss, locomotion, and sucrose preference), systemic inflammation (serum amyloid A, SAA), blood-brain barrier (BBB) disruption, astrogliosis (glial fibrillary acidic protein/GFAP), Aβ, and cytokine levels in the brain. We found that LPS caused a larger reduction in body weight in Tg vs. WT mice, but that other behavioral responses to LPS did not differ by genotype. BBB disruption was not apparent in either genotype following LPS. Concentrations of the systemic inflammatory marker, SAA, in the blood and brain were significantly increased with LPS but did not significantly differ by genotype. GFAP was increased in Tg mice vs. WT but was not significantly affected by LPS in either genotype. Finally, LPS-induced increases of eight cytokines (IL-1β, IL-6, IL-12 (p40), IL-10, IL-17A, MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5) were found to be significantly higher in Tg mice vs. WT. In summary, our data show that Aβ pathology exacerbates the neuroinflammatory response to LPS and identifies cytokines that are selectively regulated by Aβ. The association of worse neuroinflammation with greater weight loss in Tg mice suggests that Aβ pathology could contribute to poor outcomes following a systemic inflammatory insult.

Resident Self-Tissue of Proinflammatory Cytokines Rather Than Their Systemic Levels Correlates with Development of Myelofibrosis in Gata1low Mice.

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-β1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakaryocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice.

Crucial role of high-mobility group box 2 in mouse ovarian follicular development through estrogen receptor beta.

High-mobility group box 2 (HMGB2) is a chromatin-associated protein that is an important regulator of gene transcription, recombination, and repair processes. The functional importance of HMGB2 has been reported in various organs, including the testis, heart, and cartilage. However, its role in the ovary is largely unknown. In this study, ovary tissues from wild-type (WT) and HMGB2-knock-out (KO) mice were examined by histopathological staining and immunohistochemistry. The ovary size and weight were significantly lower in HMGB2-KO mice than in age-matched WT littermates. Histopathological analysis revealed ovarian atrophy and progressive fibrosis in 10-month-old HMGB2-KO mouse ovaries. Compared to age-matched WT mice, the numbers of oocytes and developing follicles were significantly decreased at 2months of age and were completely depleted at 10months of age in HMGB2-KO mice. Immunohistochemistry revealed the expression of HMGB2 in the granulosa cells of developing follicles, oocytes, some corpora lutea, and stromal cells. Importantly, HMGB2-positive cells were co-localized with estrogen receptor beta (ERβ), but not ERα. Estrogen response element-binding activity was demonstrated by southwestern histochemistry, and it was decreased in HMGB2-KO mouse ovaries. Cell proliferation activity was also decreased in HMGB2-KO mouse ovaries in parallel with the decreased folliculogenesis. These results indicated that the depletion of HMGB2 induced ovarian atrophy that was characterized by a decreased ovarian size and weight, progressive fibrosis, as well as decreased oocytes and folliculogenesis. In conclusion, we demonstrated the crucial role of HMGB2 in mouse ovarian folliculogenesis through ERβ expression.

Hsp22 Deficiency Induces Age-Dependent Cardiac Dilation and Dysfunction by Impairing Autophagy, Metabolism, and Oxidative Response.

Heat shock protein 22 (Hsp22) is a small heat shock protein predominantly expressed in skeletal and cardiac muscle. Previous studies indicate that Hsp22 plays a vital role in protecting the heart against cardiac stress. However, the essential role of Hsp22 in the heart under physiological conditions remains largely unknown. In this study, we used an Hsp22 knockout (KO) mouse model to determine whether loss of Hsp22 impairs cardiac growth and function with increasing age under physiological conditions. Cardiac structural and functional alterations at baseline were measured using echocardiography and invasive catheterization in Hsp22 KO mice during aging transition compared to their age-matched wild-type (WT) littermates. Our results showed that Hsp22 deletion induced progressive cardiac dilation along with declined function during the aging transition. Mechanistically, the loss of Hsp22 impaired BCL-2–associated athanogene 3 (BAG3) expression and its associated cardiac autophagy, undermined cardiac energy metabolism homeostasis and increased oxidative damage. This study showed that Hsp22 played an essential role in the non-stressed heart during the early stage of aging, which may bring new insight into understanding the pathogenesis of age-related dilated cardiomyopathy.

Abstract P426: Inactivating Grk5 Impairs Basal Cardiac Function And Survival Via P53 Modulation

Introduction: G protein-coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein and depending on its localization within the cell, it has been shown to elicit either protective or deleterious effects. For instance in the heart, when anchored to the plasma membrane, this kinase can regulate specific GPCRs via canonical phosphorylation that can confer cardioprotection. However, when it accumulates in the nucleus its non-canonical activity can drive pathological hypertrophic gene transcription. Interestingly, the latter effects may not be kinase-dependent. Hypothesis: The role played by GRK5’s catalytic activity in the heart has not been fully elucidated and for that reason we sought to assess the in vivo consequences of inactivating the catalytic site of GRK5 with an initial focus at examining the basal cardiac phenotype and response to stress. Methods: We used CRISPR/Cas9 technology to generate a novel knock-in mouse model, with the ATP binding lysine (K) 215 in the catalytic cleft replaced by arginine (R) (GRK5-K215R) resulting in mice devoid of any GRK5 catalytic activity. We studies baseline cardiac function in these mutant mice compared to wild-type (WT) littermates and then stressed them via transverse aortic constriction (TAC). In vitro, we used H9c2 cardiomyocytes and various GRK5 mutants for mechanistic studies. Results: Compared to age-matched WT littermates, GRK5-K215R mice revealed marked and early (9 weeks) deterioration of cardiac function, with augmented apoptosis and fibrosis basally. Importantly, mutant knock-in mice displayed increased p53 gene expression (both at mRNA and protein levels). Moreover, TAC induced increased dysfunction and fibrosis in GRK5-K215R mice compared to WT. Mechanistically, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad-GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad-NLS) and when GRK5 was localized only outside the nucleus, there was a significant protection against apoptosis, with reduced p53 protein and mRNA levels. Conversely, when we overexpressed a mutant GRK5 without nuclear export signal (GRK5-ΔNES) to trap GRK5 within the nucleus, we found a significant increase in apoptosis, with high p53 protein expression levels. Conclusions: Inactivating GRK5’s catalytic activity impairs its nuclear regulation of p53. This can result in higher levels of p53 mRNA and protein resulting in higher rates of apoptosis in the heart leading to significant cardiac dysfunction and an intolerance to stress.

Differential expression of microRNAs associated with neurodegenerative diseases and diabetic nephropathy in protein l-isoaspartyl methyltransferase-deficient mice.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), an enzyme repairing isoaspartate residues in peptides and proteins that result from the spontaneous decomposition of normal l-aspartyl and l-asparaginyl residues during aging, has been revealed to be involved in neurodegenerative diseases (NDDs) and diabetes. However, the molecular mechanisms for a putative association of PIMT dysfunction with these diseases have not been clarified. Our study aimed to identify differentially expressed microRNAs (miRNAs) in the brain and kidneys of PIMT-deficient mice and uncover the epigenetic mechanism of PIMT-involved NDDs and diabetic nephropathy (DN). Differentially expressed miRNAs by sequencing underwent target prediction and enrichment analysis in the brain and kidney of PIMT knockout (KO) mice and age-matched wild-type (WT) littermates. Sequence analysis revealed 40 differentially expressed miRNAs in the PIMT KO mouse brain including 25 upregulated miRNAs and 15 downregulated miRNAs. In the PIMT KO mouse kidney, there were 80 differentially expressed miRNAs including 40 upregulated miRNAs and 40 downregulated miRNAs. Enrichment analysis and a systematic literature review of differentially expressed miRNAs indicated the involvement of PIMT deficiency in the pathogenesis in NDDs and DN. Some overlapped differentially expressed miRNAs between the brain and kidney were quantitatively assessed in the brain, kidney, and serum-derived exosomes, respectively. Despite being preliminary, these results may aid in investigating the pathological hallmarks and identify the potential therapeutic targets and biomarkers for PIMT dysfunction-related NDDs and DN.

BCL11B Regulates Arterial Stiffness and Related Target Organ Damage.

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Memory formation in old age requires GSK-3β.

Glycogen synthase kinase 3β (GSK-3β) is a therapeutic target for various age-related neurodegenerative diseases. It is linked to the two main pathological features of Alzheimer's disease (AD), tau and amyloid β (Aβ); GSK-3β is a major candidate to pathologically hyperphosphorylate tau and modulate Aβ production. However, inhibition of GSK-3β in clinical studies in humans has been found to not significantly improve cognitive function of AD patients, prompting us to study the physiological role of GSK-3β in old mice. Using a contextual fear-conditioning paradigm, we now report that old gsk-3β+/- mice are deficient in both short-term and long-term memory formation, suggesting that GSK-3β is required for memory formation at old age. Biochemical and immunohistochemical analyses showed that the number of synapses does not differ between gsk-3β+/- and age-matched wild-type (wt) littermate mice. Based on these observations, we propose that, GSK-3β may contribute to help maintain brain function during aging. Our results may explain the poor efficacy of GSK-3β inhibitors in preserving memory capacity in AD patients.

Abstract 15259: Breast Cancer and Heritable Pah Due to Bmpr2 Mutation: An Unexpected Discovery

Introduction: BMPR2 mutation is reported in 80% of familial PAH and 20% of idiopathic cases. Recently, a rat model of hereditary Pulmonary Arterial Hypertension with a BMPR2 loss-of-function mutation has been generated. Similarly to human, only around 20% of the rats spontaneously developed mild PAH. Unexpectedly, we observed that 20% (95%CI:12-28) of the 82 Bmpr2+/Δ71 rats (93% of females) versus 4% (95%CI:0-9)of the 84 age-matched wild type littermates spontaneously developed mammary masses requiring euthanasia. Interestingly, rats with tumor had elevated PA and clearly exhibit PAH signs. Hypothesis: We hypothesized that mammary tumor exacerbates the penetrance of PAH in the BMPR2 mutated female rats. Methods/Results: BMPR2 mutated and wild-type female young rats were treated with the breast cancer inducer 7, 12- diméthylbenz(a)anthracene (DMBA) or Vehicle. DMBA causes on average more tumors development in BMPR2 mutated rats versus WT rats (6,2 vs. 4) P=0,0275. Compare to both vehicle-treated and DMBA rats (without tumor) BMPR2 mutated rats with DMBA-induced tumors had a significant increase in mean PA pressure (p<0,0001); total pulmonary resistance (p<0,0001) and a significant decrease in cardiac output (P=0,0293). Histologically, these changes were associated with an increase of pulmonary artery remodelling assessed by immunofluorescence. Mechanistically, development of PAH in the BMPR2 mutated rats with tumor was associated with a significant upregulation of BRD4 (western blot) (p=0,0149); IL-1β (P=0,0052), BRD3 (P=0,0791) and BIRC5 (P=0,0166) mRNA expression levels. Co-culture experiments with breast cancer cells and PASMC from both WT and BMPR2 mutated rats are ongoing to identify whether cancer cells are realising factors responsible for BRD4 activation and thus PASMC proliferation and resistance to apoptosis. Conclusions: We demonstrated for the first time that breast cancer exacerbates PAH development in BMPR2 mutated rats. Increased BRDs driven pro-inflammatory signalling is associated with PAH development in BMPR2 mutated rats. We are currently exploring whether BMPR2 mutated patients are more prone to breast cancer development or not and whether PAH development occurs more frequently in breast cancer patients.

Atypical myelinogenesis and reduced axon caliber in the Scn1a variant model of Dravet syndrome: An electron microscopy pilot study of the developing and mature mouse corpus callosum

Dravet Syndrome (DS) is a genetic neurodevelopmental disease. Recurrent severe seizures begin in infancy and co-morbidities follow, including developmental delay, cognitive and behavioral dysfunction. A majority of DS patients have an SCN1A heterozygous gene mutation. This mutation causes a loss-of-function in inhibitory neurons, initiating seizure onset. We have investigated whether the sodium channelopathy may result in structural changes in the DS model independent of seizures. Morphometric analyses of axons within the corpus callosum were completed at P16 and P50 in Scn1a heterozygote KO male mice and their age-matched wild-type littermates. Trainable machine learning algorithms were used to examine electron microscopy images of ~400 myelinated axons per animal, per genotype, including myelinated axon cross-section area, frequency distribution and g-ratios. Pilot data for Scn1a heterozygote KO mice demonstrate the average axon caliber was reduced in developing and adult mice. Qualitative analysis also shows micro-features marking altered myelination at P16 in the DS model, with myelin out-folding and myelin debris within phagocytic cells. The data has indicated, in the absence of behavioral seizures, factors that governed a shift toward small calibre axons at P16 have persisted in adult Scn1a heterozygote KO corpus callosum. The pilot study provides a basis for future meta-analysis that will enable robust estimates of the effects of the sodium channelopathy on axon architecture. We propose that early therapeutic strategies in DS could help minimize the effect of sodium channelopathies, beyond the impact of overt seizures, and therefore achieve better long-term treatment outcomes.

Electrical and structural remodeling contribute to atrial fibrillation in type 2 diabetic db/db mice

Atrial fibrillation (AF) is highly prevalent in diabetes mellitus (DM), yet the basis for this finding is poorly understood. Type 2 DM may be associated with unique patterns of atrial electrical and structural remodeling; however, this has not been investigated in detail. The purpose of this study was to investigate AF susceptibility and atrial electrical and structural remodeling in type 2 diabetic db/db mice. AF susceptibility and atrial function were assessed in male and female db/db mice and age-matched wildtype littermates. Electrophysiological studies were conducted invivo using intracardiac electrophysiology and programmed stimulation. Atrial electrophysiology was also investigated in isolated atrial preparations using high-resolution optical mapping and in isolated atrial myocytes using patch-clamping. Molecular biology studies were performed using quantitative polymerase chain reaction and western blotting. Atrial fibrosis was assessed using histology. db/db mice were highly susceptible to AF in association with reduced atrial conduction velocity, action potential duration prolongation, and increased heterogeneity in repolarization in left and right atria. In db/db mice, atrial K+ currents, including the transient outward current (Ito) and the ultrarapid delayed rectifier current (IKur), were reduced. The reduction in Ito occurred in association with reductions in Kcnd2 mRNA expression and KV4.2 protein levels. The reduction in IKur was not related to gene or protein expression changes. Interstitial atrial fibrosis was increased in db/db mice. Our study demonstrates that increased susceptibility to AF in db/db mice occurs in association with impaired electrical conduction as well as electrical and structural remodeling of the atria.

Effects of suppressing bioavailability of insulin-like growth factor on age-associated intervertebral disc degeneration.

Suppression of the insulin‐like growth factor‐1 (IGF‐1) signaling pathway reduces age‐related disorders and increases lifespan across species, making the IGF‐1 pathway a key regulator of aging. Previous in vitro intervertebral disc cell studies have reported the pro‐anabolic effect of exogenously adding IGF‐1 on matrix production. However, the overall effects of suppressing IGF‐1 signaling on age‐related intervertebral disc degeneration (IDD) is not known. Here, the effects of suppressing IGF‐1 signaling on age‐related IDD in vivo were examined using PAPPA −/− mice. These are animals with targeted deletion of pregnancy‐associated plasma protein A (PAPPA), the major protease that cleaves inhibitory IGF binding proteins that control bioavailability of IGF‐1 for cell signaling. Compared to age‐matched wild‐type (Wt) littermates, reduced levels of matrix proteoglycan (PG) and aggrecan were seen in discs of 23‐month old PAPPA −/− mice. Decreased aggrecanolysis and expression of two key catabolic markers, matrix metalloproteinase‐3 and a disintegrin and metalloproteinase with thrombospondin motifs‐4, were also observed in discs of old PAPPA −/− mice compared to Wt littermates. Suppressing IGF‐1 signaling has been implicated to shift cellular metabolism toward maintenance rather than growth and decreasing cellular senescence. Along this line, discs of old PAPPA −/− mice also exhibited lower cellular senescence, assessed by p53 and lamin B1 markers. Collectively, the data reveal complex regulation of disc matrix homeostasis by PAPPA/IGF‐1 signaling during chronologic aging, that is, reduced IGF‐1 bioavailability confers the benefit of decreasing disc cellular senescence and matrix catabolism but also the disadvantage of decreasing disc PG matrix anabolism. This pathway requires further mechanistic elucidation before IGF‐1 could be considered as a therapeutic growth factor for treating IDD.

Effect of salbutamol on neuromuscular junction function and structure in a mouse model of DOK7 congenital myasthenia.

Congenital myasthenic syndromes (CMS) are characterized by fatigable muscle weakness resulting from impaired neuromuscular transmission. β2-adrenergic agonists are an effective treatment for DOK7-CMS. DOK7 is a component within the AGRN-LRP4-MUSK-DOK7 signalling pathway that is key for the formation and maintenance of the synaptic structure of the neuromuscular junction (NMJ). The precise mechanism of action of β2-adrenergic agonists at the NMJ is not fully understood. In this study, we investigated whether β2-adrenergic agonists improve both neurotransmission and structural integrity of the NMJ in a mouse model of DOK7-CMS. Ex-vivo electrophysiological techniques and microscopy of the NMJ were used to study the effect of salbutamol, a β2-adrenergic agonist, on synaptic structure and function. DOK7-CMS model mice displayed a severe phenotype with reduced weight gain and perinatal lethality. Salbutamol treatment improved weight gain and survival in DOK7 myasthenic mice. Model animals had fewer active NMJs, detectable by endplate recordings, compared with age-matched wild-type littermates. Salbutamol treatment increased the number of detectable NMJs during endplate recording. Correspondingly, model mice had fewer acetylcholine receptor-stained NMJs detected by fluorescent labelling, but following salbutamol treatment an increased number were detectable. The data demonstrate that salbutamol can prolong survival and increase NMJ number in a severe model of DOK7-CMS.

Effects of Neurod1 Expression on Mouse and Human Schwannoma Cells.

The objective was to explore the effect of the proneuronal transcription factor neurogenic differentiation 1 (Neurod1, ND1) on Schwann cells (SC) and schwannoma cell proliferation. Using a variety of transgenic mouse lines, we investigated how expression of Neurod1 effects medulloblastoma (MB) growth, schwannoma tumor progression, vestibular function, and SC cell proliferation. Primary human vestibular schwannoma (VS) cell cultures were transduced with adenoviral vectors expressing Neurod1. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) uptake. Basic science investigation. Expression of Neurod1 reduced the growth of slow-growing but not fast-growing MB models. Gene transfer of Neurod1 in human schwannoma cultures significantly reduced cell proliferation in dose-dependent way. Deletion of the neurofibromatosis type 2 (Nf2) tumor-suppressor gene via Cre expression in SCs led to increased intraganglionic SC proliferation and mildly reduced vestibular sensory-evoked potentials (VsEP) responses compared to age-matched wild-type littermates. The effect of Neurod1-induced expression on intraganglionic SC proliferation in animals lacking Nf2 was mild and highly variable. Sciatic nerve axotomy significantly increased SC proliferation in wild-type and Nf2-null animals, and expression of Neurod1 reduced the proliferative capacity of both wild-type and Nf2-null SCs following nerve injury. Expression of Neurod1 reduces slow-growing MB progression and reduces human SC proliferation in primary VS cultures. In a genetic mouse model of schwannomas, we find some effects of Neurod1 expression; however, the high variability indicates that more tightly regulated Neurod1 expression levels that mimic our in vitro data are needed to fully validate Neurod1 effects on schwannoma progression. NA Laryngoscope, 131:E259-E270, 2021.

Loss of mural cell-derived laminin aggravates hemorrhagic brain injury

BackgroundMural cells synthesize and deposit laminin to the basement membrane. To investigate the function of mural cell-derived laminin, we generated a mutant mouse line lacking mural cell-derived laminin (termed PKO). In a previous study, we showed that the PKO mice were grossly normal under homeostatic condition, but developed blood-brain barrier (BBB) breakdown with advanced age (> 8 months), suggesting that these mutants are intrinsically weak. Based on these findings, we hypothesized that PKO mice have exacerbated injuries in pathological conditions.MethodsUsing collagenase-induced intracerebral hemorrhage (ICH) as an injury model, we examined various stroke outcomes, including hematoma volume, neurological function, neuronal death, BBB integrity, paracellular/transcellular transport, inflammatory cell infiltration, and brain water content, in PKO mice and their wildtype littermates at young age (6–8 weeks). In addition, transmission electron microscopy (TEM) analysis and an in vitro ICH model were used to investigate the underlying molecular mechanisms.ResultsCompared to age-matched wildtype littermates, PKO mice display aggravated stroke outcomes, including larger hematoma size, worse neurological function, increased neuronal cell death, enhanced BBB permeability, increased transcytosis, and elevated inflammatory cell infiltration. These mutants also exhibit high baseline brain water content independent of aquaporin-4 (AQP4). In addition, mural cell-derived laminin significantly reduced caveolin-1 without affecting tight junction proteins in the in vitro ICH model.ConclusionsThese results suggest that mural cell-derived laminin attenuates BBB damage in ICH via decreasing caveolin-1 and thus transcytosis, regulates brain water homeostasis, and plays a beneficial role in ICH.

Microglial physiological properties and interactions with synapses are altered at presymptomatic stages in a mouse model of Huntington\u2019s disease pathology

BackgroundHuntington’s disease (HD) is a dominantly inherited neurodegenerative disorder that affects cognitive and motor abilities by primarily targeting the striatum and cerebral cortex. HD is caused by a mutation elongating the CAG repeats within the Huntingtin gene, resulting in HTT protein misfolding. Although the genetic cause of HD has been established, the specific susceptibility of neurons within various brain structures has remained elusive. Microglia, which are the brain’s resident macrophages, have emerged as important players in neurodegeneration. Nevertheless, few studies have examined their implication in HD.MethodsTo provide novel insights, we investigated the maturation and dysfunction of striatal microglia using the R6/2 mouse model of HD. This transgenic model, which presents with 120+/-5 CAG repeats, displays progressive motor deficits beginning at 6 weeks of age, with full incapacitation by 13 weeks. We studied microglial morphology, phagocytic capacity, and synaptic contacts in the striatum of R6/2 versus wild-type (WT) littermates at 3, 10, and 13 weeks of age, using a combination of light and transmission electron microscopy. We also reconstructed dendrites and determined synaptic density within the striatum of R6/2 and WT littermates, at nanoscale resolution using focused ion beam scanning electron microscopy.ResultsAt 3 weeks of age, prior to any known motor deficits, microglia in R6/2 animals displayed a more mature morphological phenotype than WT animals. Microglia from R6/2 mice across all ages also demonstrated increased phagocytosis, as revealed by light microscopy and transmission electron microscopy. Furthermore, microglial processes from 10-week-old R6/2 mice made fewer contacts with synaptic structures than microglial processes in 3-week-old R6/2 mice and age-matched WT littermates. Synaptic density was not affected by genotype at 3 weeks of age but increased with maturation in WT mice. The location of synapses was lastly modified in R6/2 mice compared with WT controls, from targeting dendritic spines to dendritic trunks at both 3 and 10 weeks of age.ConclusionsThese findings suggest that microglia may play an intimate role in synaptic alteration and loss during HD pathogenesis.

Retinal analysis of a mouse model of Alzheimer's disease with multicontrast optical coherence tomography.

.Significance. Recent Alzheimer’s disease (AD) patient studies have focused on retinal analysis, as the retina is the only part of the central nervous system that can be imaged noninvasively by optical methods. However, as this is a relatively new approach, the occurrence and role of retinal pathological features are still debated.Aim. The retina of an APP/PS1 mouse model was investigated using multicontrast optical coherence tomography (OCT) in order to provide a documentation of what was observed in both transgenic and wild-type mice.Approach. Both eyes of 24 APP/PS1 transgenic mice (age: 45 to 104 weeks) and 15 age-matched wild-type littermates were imaged by the custom-built OCT system. At the end of the experiment, retinas and brains were harvested from a subset of the mice (14 transgenic, 7 age-matched control) in order to compare the in vivo results to histological analysis and to quantify the cortical amyloid beta plaque load.Results. The system provided a combination of standard reflectivity data, polarization-sensitive data, and OCT angiograms. Qualitative and quantitative information from the resultant OCT images was extracted on retinal layer thickness and structure, presence of hyper-reflective foci, phase retardation abnormalities, and retinal vasculature.Conclusions. Although multicontrast OCT revealed abnormal structural properties and phase retardation signals in the retina of this APP/PS1 mouse model, the observations were very similar in transgenic and control mice.

Abstract P157: Role of P47 phox in Angiotensin II Induced Cardiac Hypertrophy and Fibrosis in Mice

NADPH oxidase mediated ROS production has been recognized to be an important contributory mechanism for hypertension and left ventricular hypertrophy (LVH). However, the regulatory role of NADPH oxidase mediated superoxide production in Angiotensin II (AngII) induced cardiac fibrosis and macrophage infiltration is still elusive. In the present study, we investigated the role of phagocytic NADPH oxidase enzyme system in cardiac remodelling induced by AngII in young adult (2-3 months old) male mice deficient in p47 phox , a cytosolic subunit of the NADPH oxidase (KO, n=13) and age-matched wild-type littermates (WT, n=9). Animals were infused with AngII pressor dose (1500 ng/kg/min, s.c) or saline via osmotic minipump (Alzet 2002, USA) for 14 consecutive days and then, subjected to echocardiography (VEVO 1100, Canada). Further animals were sacrificed and hearts was harvested for histological studies and gene expression analysis. AngII treatment in WT and KO mice showed prominent alterations in echocardiographic measurements with no changes in E/A, EF and FS. However, KO mice showed aggravated cardiac hypertrophic response (LVAWd: WT, 1.20±0.09 vs. KO, 1.42±0.04, p<0.05; LVIDd: WT, 3.02±0.20 vs. KO, 2.60±0.07, p<0.05; LVPWd: WT, 1.49±0.06 vs. KO, 1.78±0.06, p<0.05). Histologically, AngII infused KO mice also showed increased cardiomyocyte diameter compared to AngII infused WT mice (WT, 388±16.37 vs. KO, 560.3±41.28 μm 2 ; p<0.01 respectively). Further, AngII infused KO mice showed elevated immune cell infiltration, which were positive for Mac-3 staining compared to AngII infused WT mice (WT, 2.61±0.32 vs. KO, 4.09±0.44 %; p<0.05). Besides, AngII infused KO mice showed augmented interstitial fibrosis (WT, 11.11±0 0.67 vs. KO, 20.85± 3.63 %; p<0.05), thick collagen fibres deposition (WT, 28.93± 0.94 vs. KO, 37.60± 2.49 %; p<0.05) compared to AngII infused WT mice. Moreover, AngII infused KO mice also showed upregulated gene expression of hypertrophic and fibrotic markers, including Nppa, Nppb, Acta1, Myh6, TGF-β1, collagen (I and III) and α-sma compared to AngII infused WT mice. Together, these data suggest that NADPH oxidase and its subunit p47 phox play a pivotal role in AngII induced LVH by regulating fibrotic machinery and macrophage infiltration.

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of Tcell receptor excision circles (TRECs) and Tcell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent Tcell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cellcounts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of Tcell lymphopenia at birth.