Nontransgenic Littermates Research Articles

Skeletal myotubes expressing ALS mutant SOD1 induce pathogenic changes, impair mitochondrial axonal transport, and trigger motoneuron death

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2 + transients and reactive oxygen species (i.e., H2O2). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes.

Spiral volumetric optoacoustic tomography of reduced oxygen saturation in the spinal cord of M83 mouse model of Parkinson's disease.

Metabolism and bioenergetics in the central nervous system play important roles in the pathophysiology of Parkinson's disease (PD). Here, we employed a multimodal imaging approach to assess oxygenation changes in the spinal cord of the transgenic M83 murine model of PD overexpressing the mutated A53T alpha-synuclein form in comparison with non-transgenic littermates. In vivo spiral volumetric optoacoustic tomography (SVOT) was performed to assess oxygen saturation (sO2) in the spinal cords of M83 mice and non-transgenic littermates. Ex vivo high-field T1-weighted (T1w) magnetic resonance imaging (MRI) at 9.4T was used to assess volumetric alterations in the spinal cord. 3D SVOT analysis and deep learning-based automatic segmentation of T1w MRI data for the mouse spinal cord were developed for quantification. Immunostaining for phosphorylated alpha-synuclein (pS129 α-syn), as well as vascular organization (CD31 and GLUT1), was performed after MRI scan. In vivo SVOT imaging revealed a lower sO2SVOT in the spinal cord of M83 mice compared to non-transgenic littermates at sub-100μm spatial resolution. Ex vivo MRI-assisted by in-house developed deep learning-based automatic segmentation (validated by manual analysis) revealed no volumetric atrophy in the spinal cord of M83 mice compared to non-transgenic littermates at 50μm spatial resolution. The vascular network was not impaired in the spinal cord of M83 mice in the presence of pS129 α-syn accumulation. We developed tools for deep-learning-based analysis for the segmentation of mouse spinal cord structural MRI data, and volumetric analysis of sO2SVOT data. We demonstrated non-invasive high-resolution imaging of reduced sO2SVOT in the absence of volumetric structural changes in the spinal cord of PD M83 mouse model.

Super-resolution ultrasound imaging reveals temporal cerebrovascular changes with disease progression in female 5×FAD mouse model of Alzheimer's disease: correlation with pathological impairments

Vascular dysfunction is closely associated with the progression of Alzheimer's disease (AD). A critical research gap exists that no studies have explored the invivo temporal changes of cerebrovascular alterations with AD progression in mouse models, encompassing both structure and flow dynamics at micron-scale resolution across the early, middle, and late stages of the disease. In this study, ultrasound localisation microscopy (ULM) was applied to image the cerebrovascular alterations of the transgenic female 5×FAD mouse model across different stages of disease progression: early (4 months), moderate (7 months), and late (12 months). Age-matched non-transgenic (non-Tg) littermates were used as controls. Immunohistology examinations were performed to evaluate the influence of disease progression on the β-amyloid (Aβ) load and microvascular alterations, including morphological changes and the blood-brain barrier (BBB) leakage. Our findings revealed a significant decline in both vascular density and flow velocity in the retrosplenial cortex of 5×FAD mice at an early stage, which subsequently became more pronounced in the visual cortex and hippocampus as the disease progressed. Additionally, we observed a reduction in vascular length preceding diminished flow velocities in cortical penetrating arterioles during the early stages. The quantification of vascular metrics derived from ULM imaging showed significant correlations with those obtained from vascular histological images. Immunofluorescence staining identified early vascular abnormalities in the retrosplenial cortex. As the disease advanced, there was an exacerbation of Aβ accumulation and BBB disruption in a regionally variable manner. The vascular changes observed through ULM imaging exhibited a negative correlation with amyloid load and were associated with the compromise of the BBB integrity. Through high-resolution, invivo imaging of cerebrovasculature, this study reveals significant spatiotemporal dysfunction in cerebrovascular dynamics accompanying disease progression in a mouse model of AD, enhancing our understanding of its pathophysiology. This study is supported by grants from National Key Research and Development Program of China (2020YFA0908800), National Natural Science Foundation of China (12074269, 82272014, 82327804, 62071310), Shenzhen Basic Science Research (20220808185138001, JCYJ20220818095612027, JCYJ20210324093006017), STI 2030-Major Projects (2021ZD0200500) and Guangdong Natural Science Foundation (2024A1515012591, 2024A1515011342).

Abstract Or104: Targeting Tropomyosin Overlap Flexibility via Small Molecule Intervention in a Murine Dilated Cardiomyopathy Model

Familial dilated cardiomyopathy (DCM) is a genetic heart disorder characterized by enlargement of one or both ventricles, rendering the heart unable to pump blood efficiently. Mutations in cardiac thin filament (CTF) proteins have been associated with characterized clinical manifestations of DCM. Genetic segregation identified a mutation in α-Tropomyosin (Tm) at residue 230 (D230N-Tm) in two unrelated, multigenerational families linked to severe, early onset DCM. To meaningfully link genotype to phenotype our group sought to investigate the effects of D230N-Tm on the biophysical structure of Tm filament. We showed that D230N-Tm caused a compaction of the cardiac Troponin T (cTnT)–Tm overlap region accompanied by a decreased in flexibility of the Tm filament. Given the altered biophysical structure we investigated whether we could target this decrease in flexibility via small molecule intervention and improve function. Computational modeling and biophysical experiments identified small molecule 4-Hydroxy Duloxetine β-D-Glucuronide (Z06) shown to improve compaction of the cTnT-Tm overlap and restore flexibility of the Tm filament. We hypothesized that the decrease in flexibility of Tm impairs its ability to shift through its different conformational states, leading to impaired crossbridge formation. To investigate whether Z06 improves myofilament function, we performed an NADH-coupled ATPase assay using isolated myofibrils from our D230N-Tm transgenic mice and compared them to Non-Transgenic (NTg) littermates. ATPase activity was measured at saturating calcium (100uM) in the presence (250uM) and absence (0uM) of Z06. In the absence of Z06, D230N-Tm exhibited a decrease in ATPase activity (1.793±0.203 umol/min) compared to NTg littermates (5.420±0.562 umol/min), a common trend in DCM models. In the presence of 250uM Z06, D230N-Tm ATPase rates improve to levels comparable to NTg mice (4.816±0.544 umol/min). Moving forward, we will perform calcium titrations, allowing us to investigate if Z06 can restore cooperativity of myofilament activation. Additionally, we will test if the restorative effects of Z06 are present at the myocellular level, and if so, we will optimize delivery to test in vivo effects of Z06 in our D230N-Tm mouse model.

Abstract Mo028: Persistence of Fetal Cardiac Troponin T Modulates Disease Severity in Anthracycline-Induced Cardiomyopathy

Anthracyclines are a key component in the management of pediatric cancers long recognized to carry significant risk of dose-dependent cardiotoxicity. Despite advancements in clinical management, considerable variation in the incidence of cardiotoxicity persists across dosing regimens suggesting modifiers exist. Recently, changes in the splicing of TNNT2, the gene encoding cardiac troponin T (cTnT), were postulated to contribute to anthracycline cardiotoxicity, potentially providing a direct link to the myofilament. Previous studies in our lab have demonstrated that the fetal isoform, cTnT1, is a potent modifier of cardiac function contributing to disease heterogeneity in animal models of cardiomyopathies. Thus, we hypothesize that the persistent expression of cTnT1 predisposes a subset of the population to anthracycline cardiomyopathy. Using molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC), we assessed a range of anthracyclines for their potential to bind the cardiac thin filament (CTF). MD calculated Glide scores showed direct binding to the troponin complex. ITC indicated that this binding occurs at an order of magnitude higher affinity in complexes containing cTnT1. Stopped-flow kinetics of cTnT1 CTFs exposed to Doxorubicin showed an additive reduction in calcium dissociation rates, reinforcing our observations of direct binding to the CTF. Longitudinal 2D echocardiography of Doxorubicin treated mice (3mg/kg/week) indicated that both male and female cTnT1 mice exhibit an additive, dose-dependent reduction in cardiac systolic function compared to non-transgenic (NT) littermates. Consistent with clinical data, female mice exhibited an earlier reduction in % fractional shortening than males as well as delayed recovery after treatment cessation. Transcriptional profiling of mice treated with Doxorubicin revealed a 2- to 7-fold additive increase in type I and type II interferon signaling in cTnT1 mice above that observed in treated NT littermates. These data suggest that cTnT1 may increase binding of anthracyclines at the myofilament, eliciting accelerated cardiac dysfunction, and represent a unique, targetable genetic risk factor involved in anthracycline cardiotoxicity.

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo.

Abnormal alpha-synuclein (αSyn) and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim to visualize αSyn inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. The fluorescent pyrimidoindole derivative THK-565 probe was characterized by means of recombinant fibrils and brains from 10- to 11-month-old M83 mice. Concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging were subsequently performed in vivo. Structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 T as well as scanning transmission x-ray microscopy (STXM) were performed to characterize the iron deposits in the perfused brains. Immunofluorescence and Prussian blue staining were further performed on brain slices to validate the detection of αSyn inclusions and iron deposition. THK-565 showed increased fluorescence upon binding to recombinant αSyn fibrils and αSyn inclusions in post-mortem brain slices from patients with PD and M83 mice. Administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 min post-intravenous injection by wide-field fluorescence compared to nontransgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. In conclusion, we demonstrated in vivo mapping of αSyn by means of noninvasive epifluorescence and vMSOT imaging and validated the results by targeting the THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice.

JOURNAL/nrgr/04.03/01300535-202508000-00025/figure1/v/2024-09-30T120553Z/r/image-tiff TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon. TAU is missorted and aggregated in an array of diseases known as tauopathies. Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications, changes of which affect microtubule stability and dynamics, microtubule interaction with other proteins and cellular structures, and mediate recruitment of microtubule-severing enzymes. As impairment of microtubule dynamics causes neuronal dysfunction, we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics. We therefore aimed to study the effects of a disease-causing mutation of TAU (P301L) on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics, to assess whether P301L-TAU causes stability-changing modifications to microtubules. To investigate TAU localization, phosphorylation, and effects on tubulin post-translational modifications, we expressed wild-type or P301L-TAU in human MAPT -KO induced pluripotent stem cell-derived neurons (iNeurons) and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU (pR5 mice). Human neurons expressing the longest TAU isoform (2N4R) with the P301L mutation showed increased TAU phosphorylation at the AT8, but not the p-Ser-262 epitope, and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons. P301L-TAU showed pronounced somatodendritic presence, but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU. P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation, but reduced acetylation, of microtubules compared with non-transgenic littermates. In sum, P301L-TAU results in changes in microtubule PTMs, suggestive of impairment of microtubule stability. This is accompanied by missorting and aggregation of TAU in mice but not in iNeurons. Microtubule PTMs/impairment may be of key importance in tauopathies.

Effect of astaxanthin in type-2 diabetes -induced APPxhQC transgenic and NTG mice

ObjectivesAggregation and misfolding of amyloid beta (Aβ) and tau proteins, suggested to arise from post-translational modification processes, are thought to be the main cause of Alzheimer's disease (AD). Additionally, a plethora of evidence exists that links metabolic dysfunctions such as obesity, type 2 diabetes (T2D), and dyslipidemia to the pathogenesis of AD. We thus investigated the combinatory effect of T2D and human glutaminyl cyclase activity (pyroglutamylation), on the pathology of AD and whether astaxanthin (ASX) treatment ameliorates accompanying pathophysiological manifestations. MethodsMale transgenic AD mice, APPxhQC, expressing human APP751 with the Swedish and the London mutation and human glutaminyl cyclase (hQC) enzyme and their non-transgenic (NTG) littermates were used. Both APPxhQC and NTG mice were allocated to 3 groups, control, T2D-control, and T2D-ASX. Mice were fed control or high fat diet ± ASX for 13 weeks starting at an age of 11–12 months. High fat diet fed mice were further treated with streptozocin for T2D induction. Effects of genotype, T2D induction, and ASX treatment were evaluated by analysing glycemic readouts, lipid concentration, Aβ deposition, hippocampus-dependent cognitive function and nutrient sensing using immunosorbent assay, ELISA-based assays, western blotting, immunofluorescence staining, and behavioral testing via Morris water maze (MWM), respectively. ResultsAPPxhQC mice presented a higher glucose sensitivity compared to NTG mice. T2D-induced brain dysfunction was more severe in NTG compared to the APPxhQC mice. T2D induction impaired memory functions while increasing hepatic LC3B, ABCA1, and p65 levels in NTG mice. T2D induction resulted in a progressive shift of Aβ from the soluble to insoluble form in APPxhQC mice. ASX treatment reversed T2D-induced memory dysfunction in NTG mice and in parallel increased hepatic pAKT while decreasing p65 and increasing cerebral p-S6rp and p65 levels. ASX treatment reduced soluble Aβ38 and Aβ40 and insoluble Aβ40 levels in T2D-induced APPxhQC mice. ConclusionsWe demonstrate that T2D induction in APPxhQC mice poses additional risk for AD pathology as seen by increased Aβ deposition. Although ASX treatment reduced Aβ expression in T2D-induced APPxhQC mice and rescued T2D-induced memory impairment in NTG mice, ASX treatment alone may not be effective in cases of T2D comorbidity and AD.

Induction of tauopathy in a mouse model of amyloidosis using intravenous administration of adeno-associated virus vectors expressing human P301L tau.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which extracellular aggregates of the amyloid beta (Aβ) peptide precede widespread intracellular inclusions of the microtubule-associated protein tau. The autosomal dominant form of AD requires mutations that increase production or aggregation of the Aβ peptide. This has led to the hypothesis that amyloid deposition initiates downstream responses that lead to the hyperphosphorylation and aggregation of tau. Here we use a novel approach, somatic gene transfer via intravenous adeno-associated virus (AAV), to further explore the effects of pre-existing amyloid deposits on tauopathy. APP+PS1 mice, which develop amyloid deposits at 3 to 6 months of age, and non-transgenic littermates were injected at 8 months of age intravenously with AAV-PHP.eB encoding P301L human tau. Tissue was collected at 13 months and tauopathy was assessed. Total human tau expression was observed to be relatively uniform throughout the brain, reflecting the vascular route of AAV administration. Phospho-tau deposition was not equal across brain regions and significantly increased in APP+PS1 mice compared to non-transgenic controls. Interestingly, the rank order of phospho-tau deposition of affected brain regions in both genotypes paralleled the rank order of amyloid plaque deposits in APP+PS1 mice. We also observed significantly increased MAPT RNA expression in APP+PS1 mice compared to non-transgenic despite equal AAV transduction efficiency between groups. This model has advantages over prior approaches with widespread uniform human tau expression throughout the brain and the ability to specify the stage of amyloidosis when the tau pathology is initiated. These data add further support to the amyloid cascade hypothesis and suggest RNA metabolism as a potential mechanism for amyloid-induced tauopathy.

Impact of Long-Term Dietary High Fat and Eicosapentaenoic Acid on Behavior and Hypothalamic-Pituitary-Adrenal Axis Activity in Amyloidogenic APPswe/PSEN1dE9 Mice

Introduction: Alzheimer’s disease (AD) alters neurocognitive and emotional function and causes dysregulation of multiple homeostatic processes. The leading AD framework pins amyloid beta plaques and tau tangles as primary drivers of dysfunction. However, many additional variables, including diet, stress, sex, age, and pain tolerance, interact in ways that are not fully understood to impact the onset and progression of AD pathophysiology. We asked: (1) does high-fat diet, compared to low-fat diet, exacerbate AD pathophysiology and behavioral decline? And, (2) can supplementation with eicosapentaenoic (EPA)-enriched fish oil prevent high-fat-diet-induced changes? Methods: Male and female APPswePSdE9 mice, and their non-transgenic littermates, were randomly assigned to a diet condition (low-fat, high-fat, high-fat with EPA) and followed from 2 to 10 months of age. We assessed baseline corticosterone concentration during aging, pain tolerance, cognitive function, stress coping, and corticosterone response to a stressor. Results: Transgenic mice were consistently more active than non-transgenic mice but did not perform worse on either cognitive task, even though we recently reported that these same transgenic mice exhibited metabolic changes and had increased amyloid beta. Mice fed high-fat diet had higher baseline and post-stressor corticosterone, but diet did not impact cognition or pain tolerance. Sex had the biggest influence, as female mice were consistently more active and had higher corticosterone than males. Conclusion: Overall, diet, genotype, and sex did not have consistent impacts on outcomes. We found little support for predicted interactions and correlations, suggesting diet impacts metabolic function and amyloid beta levels, but these outcomes do not translate to changes in behaviors measured here.

Maternal fish-oil supplementation reduces presenilin 1 level and the amyloid-beta burden in adult 5xFAD offspring without major changes in brain fatty acids

Omega-3 fatty acid interventions show potential benefits in Alzheimer?s disease (AD) when initiated during its early stages. This study investigated whether maternal diet supplemented with omega-3-rich fish oil (FO) could delay or reduce amyloid beta (A?) formation, a key feature of AD, in 5xFAD transgenic offspring. Dams received FO during mating, pregnancy, and lactation. Brain tissues from female offspring were collected at 2 and 6 months of age. The findings indicated a shift in amyloid precursor protein processing, evidenced by increased soluble amyloid precursor protein ? (sAPP?) levels, suggesting a transition from amyloidogenic to non-amyloidogenic pathway. FO influenced the expression of presenilin 1 and 2 but did not impact A? levels in 2-month-old mice. However, FO reduced the A? burden in the brains of 6-month-old animals. Lipidomic analysis revealed that 5xFAD mice have unimpaired omega-3 acquisition during gestation and lactation in comparison to non-transgenic littermates. However, a response to FO supplementation was found in non-transgenic offspring only, indicating that alterations in brain lipids are not the primary mechanism of FO-induced A? decline in 5xFAD. In conclusion, FO did not prevent or delay amyloid pathology in genetically predisposed animals but did mitigate its progression, suggesting mechanisms that warrant further investigation.

Fragmentation of functional resting state brain networks in a transgenic mouse model of tau pathology: A metabolic connectivity study using [18F]FDG-PET

In a previous study, regional reductions in cerebral glucose metabolism have been demonstrated in the tauopathy mouse model rTg4510 (Endepols et al., 2022). Notably, glucose hypometabolism was present in some brain regions without co-localized synaptic degeneration measured with [18F]UCB-H. We hypothesized that in those regions hypometabolism may reflect reduced functional connectivity rather than synaptic damage. To test this hypothesis, we performed seed-based metabolic connectivity analyses using [18F]FDG-PET data in this mouse model. Eight rTg4510 mice at the age of seven months and 8 non-transgenic littermates were injected intraperitoneally with 11.1 ± 0.8 MBq [18F]FDG and spent a 35-min uptake period awake in single cages. Subsequently, they were anesthetized and measured in a small animal PET scanner for 30 min. Three seed-based connectivity analyses were performed per group. Seeds were selected for apparent mismatch between [18F]FDG and [18F]UCB-H. A seed was placed either in the medial orbitofrontal cortex, dorsal hippocampus or dorsal thalamus, and correlated with all other voxels of the brain across animals. In the control group, the emerging correlative pattern was strongly overlapping for all three seed locations, indicating a uniform fronto-thalamo-hippocampal resting state network. In contrast, rTg4510 mice showed three distinct networks with minimal overlap. Frontal and thalamic networks were greatly diminished. The hippocampus, however, formed a new network with the whole parietal cortex. We conclude that resting-state functional networks are fragmented in the brain of rTg4510 mice. Thus, hypometabolism can be explained by reduced functional connectivity of brain areas devoid of tau-related pathology, such as the thalamus.

MRI reveals cerebral iron, calcification and spinal cord atrophy in M83 mouse model of Parkinson’s disease

AbstractBackgroundRodent models of Parkinson’s disease have been crucial tools for understanding the pathogenic mechanisms of alpha‐synuclein‐associated alterations. Here, we employed multiparametric high‐field magnetic resonance imaging (MRI) to examine the molecular alterations and neurodegeneration in the brain and spinal cord of transgenic M83 (A53T) murine model of Parkinson’s disease.MethodTen nontransgenic littermates and twelve M83 mice at 10‐11 months of age were assessed. Gradient echo MR for susceptibility‐weighted imaging (SWI) and T1 MR scans were acquired ex vivo at 9.4 T in the brain and spinal cord of mice. Histology and immunofluorescence staining for alpha‐synuclein inclusions (phospho‐S129), NeuN, GFAP and Iba‐1 for gliosis were used to investigate the nature of the imaging observations.ResultFrom T1 MR, a reduced area of white matter in the thoracic spinal cord (T1) and a reduced area of gray matter in the cervical (C6‐C8) and thoracic (T1) spinal cord were observed in the M83 mice compared to nontransgenic mice. In addition, lower left‐right length measurements in the cervical (C8) spinal cord were observed in M83 mice than in nontransgenic mice. SW images revealed regional hypointensities in the hippocampus, cortex, striatum, midbrain and thalamus of M83 mice, which in corresponding phase images indicated both diamagnetic (calcification) and paramagnetic (iron) lesions in the brains of M83 mice. Immunofluorescence staining demonstrated an increased Iba‐1 level in the brains of M83 mice, indicating microgliosis.ConclusionWe found cerebral iron, calcification, microgliosis and spinal cord atrophy in M83 mice.

Cooperative Somatic Alterations in XPO1 and TNFAIP3 (A20) Promote Immune Modulation and Tissue Infiltration in Primary Mediastinal B Cell Lymphoma

Background: Primary Mediastinal B cell lymphoma (PMBCL) is an aggressive large B cell non-Hodgkin lymphoma typically presenting as a mass in the anterior mediastinum in early adulthood, disproportionately affecting females (2:1). Previous studies have identified constitutive activation of the NF-κB pathway and immune evasion as hallmark features of PMBCL. The master negative regulator of canonical NF-κB, TNFAIP3 (A20), is inactivated in ~25-60% of PMBCL cases via disruptive mutations or deletions. Gains of the 2p16.1-15 locus ( REL, XPO1, BCL11A) are seen in ~40-75% of PMBCL cases. Interestingly, PMBCL cases with 2p16.1-15 gain/amplification display concomitant increases in XPO1 expression but lack correlative increases in REL or BCL11A. In addition, XPO1 mutations are seen in ~15-33% of PMBCL cases, with the most prevalent mutation (E571K) implicated in NF-KB activation. Despite the established occurrence of genetic events impacting XPO1 and A20 in PMBCL, the cooperative potential of these alterations remains unexplored. Consequently, we conducted an extensive review of literature describing the PMBCL genomic landscape and utilized in vivo models to examine the combined potential of XPO1 and A20 alterations to promote lymphomagenesis. Methods: Genomic data was obtained from NGS analyses from a total of 233 PMBCL patients across 6 independent published research studies and analyzed for cooccurrence of A20 & XPO1 alterations (Camus et al. 2022; Chapuy et al. 2019; Lacy et al. 2020; Mareschal et al. 2015; Mottock et al. 2019; Tuveri et al 2022). For in vivo analysis, we developed a murine model of B cell specific overexpression of wildtype (WT) or E571K- XPO1 with CD19.cre driven depletion of A20 (Eµ-XPO1xA20 KO). Whole body histopathologic evaluation of FFPE tissue with H&E staining was performed on 6-month-old, age and sex matched littermate mice. Results: We found co-occurrence of inactivating events in A20 and XPO1 missense mutations impacting 15% of PMBCL evaluated cases, with consistent results seen across all studies (range: 13.8-20%). As expected, A20 mutations/del were more prevalent than XPO1 mutations across all studies (OR 2.46, 95% CI 0.84-7.18, p = 0.02). XPO1 mutated cases trended towards a greater likelihood of having co-occurring A20 mutations (OR 8.51, 95% CI 0.84-86.47, p = 0.08). XPO1 mutations and gain (2p16) were found to be mutually exclusive events with gains co-occurring with A20 mutations/del in ~17% of PMBCL cases evaluated. Analysis of survival of PMBCL patients with cooccurring A20 inactivating alterations and XPO1 mutations is ongoing although preliminarily trend towards reduced mean 2-year survival (p=0.056) compared to all others are observed. Taken together, co-occurring genetic alterations effecting A20 and XPO1 are prevalent in PMBCL the alternative biology stemming from potential cooperative mechanisms may represent an aggressive subgroup of PMBCL. To evaluate concurrent B cell-specific alterations of A20 and XPO1 in vivo we utilized Eµ-XPO1xA20 KO mouse models. Performing histopathology in 6-month-old mice we observed enlarged lymph nodes due to marked increases in lymphocytes within the cortex, paracortex, medulla, and sinuses in mice with loss of functional A20, particularly in Eµ-XPO1xA20 KO mice with overexpression of WT XPO1. Increased lymphocyte populations drove overall enlargement of the node (mandibular, mesenteric, pancreatic, and mediastinal) and distorted normal architecture, featuring small lymphocytes populating sinus regions and larger lymphocytes populating areas resembling follicles. Eµ-XPO1(WT & E571K)xA20 KO mice additionally featured mild mononuclear infiltrates in the lung and salivary glands; all of which were observed with increased severity compared to A20KO, Eµ-XPO1, or non-transgenic littermates. Significance: We identified co-occurring genetic lesions impacting A20 and XPO1 in PMBCL tumors and show that these alterations synergize in vivo leading to nodal/extranodal lymphocyte infiltration. The immunomodulatory effects appear to be driven by the cooperation of XPO1 overexpression, more so than mutation, and A20 inactivation in young mice. Further investigation of these models will allow elucidation of underlying cooperative mechanisms and reveal targetable pathways in PMBCL.

Increased Heat Pain Tolerance but Hyperalgesia to Tonic Inflammatory Pain in the CRND8 Mouse Model of Alzheimer's Disease.

The effects of Alzheimer's disease (AD) pathology on the experience of pain are poorly understood. To understand the pathophysiological mechanisms underlying pain sensory transmission in the transgenic mouse model of AD, CRND8. We explored AD-related pathology in the spinal cord and dorsal root ganglia of 18-week-old female CRND8 mice. We assessed nociceptive responses to both acute heat stimuli and persistent inflammatory pain in CRND8 mice and non-transgenic (non-Tg) littermates. In addition, we searched for differences in biochemical correlates of inflammatory pain between CRND8 and non-Tg mice. Finally, we investigated the excitability of dorsal horn noc iceptive neurons in spinal cord slices from CRND8 and non-Tg mice. We demonstrated the presence of intracellular AD-like pathology in the spinal cord and in the dorsal root ganglia nociceptive sensory neurons of CRND8 mice. We found that CRND8 mice had a reduced susceptibility to acute noxious heat stimuli and an increased sensitivity to tonic inflammatory pain. Tonic inflammatory pain correlated with a lack of induction of pro-opiomelanocortin in the spinal cord of CRND8 mice as compared to non-Tg mice. Electrophysiological recording in acute spinal cord slice preparations indicated an increased probability of glutamate release at the membrane of dorsal horn nociceptive neurons in CRND8 mice. This study suggests that an increased thermal tolerance and a facilitation of nociception by peripheral inflammation can coexist in AD.

S14-Phosphorylated RPN6 Mediates Proteasome Activation by PKA and Alleviates Proteinopathy.

A better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that PKA (cAMP-dependent protein kinase or protein kinase A) activates the 26S proteasome by pS14-Rpn6 (serine14-phosphorylated Rpn6), but this discovery and its physiological significance remain to be established in vivo. Male and female mice with Ser14 of Rpn6 (regulatory particle non-ATPase 6) mutated to Ala (S14A [Rpn6/Psmd11S14A]) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system functioning was evaluated with the GFPdgn (green fluorescence protein with carboxyl fusion of the CL1 degron) reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G [arginine120 to glycine missense mutant alpha B-crystallin]). PKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type but not S14A embryonic fibroblasts (mouse embryonic fibroblasts), adult cardiomyocytes, and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and adult mouse cardiomyocytes than in wild-type counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with nontransgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than wild-type neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates, and of aberrant CryAB (alpha B-crystallin) protein aggregates, less fetal gene reactivation, and cardiac hypertrophy, and delays in cardiac malfunction. This study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifying a new therapeutic target to reduce cardiac proteotoxicity.

Abstract P2073: Hyperbaric Oxygen Therapy Protects The Myocardium From Reductive Stress-induced Proteotoxic Remodeling

Background: Oxygen is the prime source of reactive oxygen species (ROS) in cells. We hypothesize that hyperbaric oxygen therapy (HBOT) can be used as an effective intervention to maintain redox homeostasis in the myocardium. Here, we assessed whether HBOT could prevent RS through enhanced ROS signaling, thereby protecting the hearts from proteotoxic stress. Methods: Cardiac-specific human Nrf2 transgenic (hNrf2-TG) and their non-transgenic littermates (NTG) were used in this study. Mice at the age of 8 weeks were exposed to hyperbaric oxygen (2.5 Bars, 2 hr/day) for 8 weeks. The TG and NTG (n=6) mice without HBOT served as controls. Pre- and post- echocardiography (Vevo 2100) analysis were carried out to assess the structural and functional changes over time in response to HBOT exposure. The influence of HBOT on matrixins (MMP2/9), redox (GSH & NQO1), and cardiac hypertrophy markers (ANF, BNF) were examined using zymography, immunoblotting, and qPCR analysis. The aggregation of proteins in myocardial tissues was detected by Proteostat staining. Results: While the expression of hNrf2 induces RS in the mouse heart, we noticed that HBOT ameliorates the RS through enhanced ROS signaling. A decrease in systemic GSH levels along with the downregulation of ANF and BNF mRNA levels observed in the HBOT group indicates the restoration of the redox homeostasis as well as reduced remodeling of the myocardium. Interestingly, hNRF2-induced RS was suppressed by HBOT as supported by changes in the protein levels for NQO1, GCLC, and GCLM. The degree of protein aggregation in RS hearts was significantly reduced by HBOT. Vevo strain analysis reveals that 8 weeks of HBOT restored the diastolic stretch seen in the RS hearts to a normal diastole-systole velocity equal to NTG mice. A significant decrease in the ejection fraction (80% vs. 55% in TG vs. TG-HBOT), was evident in the TG mice that received HBOT. Repression of matrixins, observed in the hearts of HBOT-TG mice signifies the benefits of HBOT in RS hearts. Conclusion: HBOT directly improves myocardial structure at the tisuue level; maintain the redox status at systemic level, and improves the myocardial performance at functional level. Our study established that HBOT can rescue the myocardium from RS-induced proteotoxic insults.

Detection of proteomic alterations at different stages in a Huntington's disease mouse model via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging.

Huntington's disease (HD) is a progressive and irreversible neurodegenerative disease leading to the inability to carry out daily activities and for which no cure exists. The underlying mechanisms of the disease have not been fully elucidated yet. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) allows the spatial information of proteins to be obtained upon the tissue sections without homogenisation. In this study, we aimed to examine proteomic alterations in the brain tissue of an HD mouse model with MALDI-MSI coupled to LC-MS/MS system. We used 3-, 6- and 12-month-old YAC128 mice representing pre-stage, mild stage and pathological stage of the HD and their non-transgenic littermates, respectively. The intensity levels of 89 proteins were found to be significantly different in YAC128 in comparison to their control mice in the pre-stage, 83 proteins in the mild stage, and 82 proteins in the pathological stage. Among them, Tau, EF2, HSP70, and NogoA proteins were validated with western blot analysis. In conclusion, the results of this study have provided remarkable new information about the spatial proteomic alterations in the HD mouse model, and we suggest that MALDI-MSI is an excellent technique for identifying such regional proteomic changes and could offer new perspectives in examining complex diseases.

Early synaptic gene expression downregulation in the frontal cortex distinguishes Alzheimer’s disease from normal aging in a genetically diverse mouse population

AbstractBackgroundThe frontal cortex is critical to many cognitive processes and is vulnerable to pathology and neurodegeneration in Alzheimer’s disease. Cortical dysfunction and impaired connectivity in the frontal cortex have also been observed in “normal” age‐related cognitive decline, suggesting common mechanisms contributing to both age‐related and AD‐related cognitive decline. The divergence that may lead to AD versus normal aging, however, is unclear.MethodHere, we have used genetically diverse mouse populations modeling either AD or normal aging, the AD‐BXDs and their nontransgenic littermates (Neuner et al, Neuron, 2019). We measured cognitive function from early (6mo) to middle (14mo) adulthood to capture presymptomatic and advanced AD stages and early aging processes. We performed bulk RNAseq from the frontal cortex, followed by age‐ and AD‐related differential gene expression analysis and Weighted Gene Coexpression Network Analysis (WGCNA) to identify genes and gene networks whose expression is associated with normal and pathological aging.ResultIn both differential gene expression analysis and WGCNA, we found common mechanisms underlying normal aging and AD (e.g., upregulation of inflammation‐related gene pathways). We also found that downregulation of genes and gene networks associated with synaptic function specifically occurred in AD and not in normal aging, suggesting that early synaptic dysfunction and/or synaptic loss is a unique feature of AD that distinguishes it from normal aging processes. Based on differential gene expression, network analyses, presence of high‐impact variants across the BXD panel, and association with cognitive function, we identified one gene candidate, B230209E15Rik, a lncRNA, as a potential regulator of synaptic gene networks in the frontal cortex that promotes resilience to cognitive decline in AD.ConclusionOverall, our data indicate that cognitive aging and AD may share common neuroimmune activation and dysregulation of neuronal maintenance, but that AD, and particularly late AD, is uniquely characterized by synaptic dysfunction. We nominate an intriguing candidate, B230209E15Rik, as a potentially key driver of synaptic gene regulation in the frontal cortex. In order to elucidate the mechanism by which this gene may promote cognitive resilience in AD, we are currently validating this finding in vivo.

Aquaporin 4 is associated with cerebral amyloid‐beta and tau deposits in mouse models

AbstractBackgroundEmerging evidence indicates that the glymphatic system plays an important role in Alzheimer’s disease. Aquaporin 4 (AQP4) constitutes a critical part of the glymphatic system in the clearance of accumulated amyloid‐beta and tau from the brain. Here, we aimed to establish the association between AQP4 and tau as well as amyloid‐beta (Aβ).MethodPerfusion and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI, at 7 or 9.4 T) were performed in arcAβ mouse model of amyloidosis (aged 16‐18 months), P301L mouse model of 4‐repeat tau (pR5 line) and respective nontransgenic littermates (aged 10‐12 months). Immunofluorescence staining was performed using antibodies against AQP4, CD31, astrogliosis (GFAP, s100β), microgliosis (Iba1, CD68), Aβ (6E10), and tau (AT‐8 and AT‐100) in brain tissue slices from transgenic arcAβ mice and wild‐type littermates.ResultCerebral microbleeds were detected in the cortex and subcortical regions in arcAβ mice. Cerebral blood flow was reduced in the cortex and hippocampus of arcAβ compared to nontransgenic mice but preserved in P301L mice. Increased AQP4 levels were observed surrounding tau deposits in the forebrain of P301L mice and parenchymal amyloid plaques/cerebral amyloid angiopathy in the cortex and hippocampus of arcAβ mice. Depolarization of AQP4 was detected in both arcAβ and P301L mice compared to their respective nontransgenic littermates. Increased s100b‐GFAP and CD68‐Iba1 fluorescence intensities indicative of atrogliosis and microgliosis were detected around tau deposits in P301L mice and amyloid plaques in arcAβ mice.ConclusionWe observed impaired perfusion in arcAβ mice but not in P301L mice, and a spatial association of AQP4 with both tau and Aβ in the brain.