Confocal Immunofluorescence Imaging Research Articles

The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

PSMD9 promotes the malignant progression of hepatocellular carcinoma by interacting with c-Cbl to activate EGFR signaling and recycling

BackgroundMounting evidences shows that the ubiquitin‒proteasome pathway plays a pivotal role in tumor progression. The expression of 26S proteasome non-ATPase regulatory subunit 9 (PSMD9) is correlated with recurrence and radiotherapy resistance in several tumor types. However, the role and mechanism of PSMD9 in hepatocellular carcinoma (HCC) progression remain largely unclear.MethodsPSMD9 was identified as a prognosis-related biomarker for HCC based on analysis of clinical characteristics and RNA-seq data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and the JP Project of the International Cancer Genome Consortium (ICGC-LIRI-JP). PSMD9 expression was analyzed in cancer tissues and adjacent noncancerous tissues via immunohistochemistry and Western blotting. Multiple in vivo and in vitro experimental techniques (such as CCK-8, colony formation, EdU, and Transwell assays; flow cytometry; Western blotting; quantitative RT-PCR; Coimmunoprecipitation assay and immunofluorescence confocal imaging) were used to assess the functions of PSMD9 in the pathogenesis of HCC.ResultsWe found that the expression of PSMD9 was upregulated and associated with a poor prognosis in HCC patients. PSMD9 promoted HCC cell proliferation, migration, invasion and metastasis. Knockdown of PSMD9 significantly inhibited HCC cell proliferation by inducing G1/S cell cycle arrest and apoptosis. Mechanistically, we demonstrated that PSMD9 promoted HCC cell proliferation and metastasis via direct interaction with the E3 ubiquitin ligase c-Cbl, suppresses EGFR ubiquitination, influenced EGFR endosomal trafficking and degradation and subsequently activated ERK1/2 and Akt signaling. In addition, we showed that PSMD9 knockdown sensitized HCC cells to the tyrosine kinase inhibitor erlotinib in vitro and in vivo.ConclusionsCollectively, our results indicate that PSMD9 drives HCC progression and erlotinib resistance by suppressing c-Cbl mediated EGFR ubiquitination and therefore can be a potential therapeutic target for HCC.

Excess Intramyocellular Lipid Does Not Affect Muscle Fiber Biophysical Properties in Mice or People With Metabolically Abnormal Obesity.

Observational studies show correlations between intramyocellular lipid (IMCL) content and muscle strength and contractile function in people with "metabolically abnormal" obesity. However, a clear physiologic mechanism for this association is lacking and causation is debated. We combined immunofluorescent confocal imaging with force measurements on permeabilized muscle fibers from metabolically normal and metabolically abnormal mice and metabolically normal (defined as normal fasting plasma glucose and glucose tolerance) and metabolically abnormal (defined as pre-diabetes and type 2 diabetes) people with overweight/obesity to evaluate relationships among myocellular lipid droplet characteristics (droplet size and density) and biophysical (active contractile and passive viscoelastic) properties. The fiber type specificity of lipid droplet parameters varied between metabolically abnormal and normal mice and among metabolically normal and metabolically abnormal people. However, despite considerable quantities of IMCL in the metabolically abnormal groups, there were no significant differences in peak active tension or passive viscoelasticity between the metabolically abnormal groups and the control group in mice or people. Additionally, there were no significant relationships among IMCL parameters and biophysical variables. Thus, we conclude that IMCL accumulation per se does not impact muscle fiber biophysical properties or physically impede contraction.

Sex-dependent polymorphism in urinary output is reversed upon deletion of exchange protein directly activated by cAMP (Epac)

It is well-described that there are notable sex-dependent differences in urinary production with females having a smaller volume and greater urinary concentrating ability. Apart from contribution of the androgenic inputs, the underlying signaling intermediates remain largely unexplored. We previously showed that exchange protein directly activated by cAMP (Epac) is a critical regulator of water-solute transport in the proximal tubule and collecting duct. Deletion of either Epac1 or Epac2 isoforms increases urinary output and Na+ excretion. Here, we used metabolic cage balance studies to test whether Epac signaling mediates sex-dependent differences in urinary production. We found that concomitant deletion of Epac1&2 moderately increases 24h urinary volume by 38% (from 1.82+/-0.13 to 2.52+/-0.21 ml) in males, but drastically augment urinary production in females by 320% (from 1.39+/-0.10 to 4.45+/-0.37 ml). This was associated with only a mild though significant reduction in urinary osmolarity in males by 35%, whereas urine osmolarity was markedly decreased from 2511+/-5 to 977+/-60 mOsm in females. Consistently, plasma osmolarity was not different in EpacWT versus Epac1&2−/− males, but became significantly more hypertonic in Epac1&2−/− females. We further determine the functional consequences of impaired Epac cascade on fluid reabsorption in the proximal tubule and collecting duct in males and females. Deletion of Epac1 and Epac2 decreases NHE-3 expression in the proximal tubule in an additive manner. In addition, Na+/H+-dependent recovery after acidification was drastically reduced in freshly isolated split-opened proximal tubules from Epac1&2−/− mice when compared to EpacWT. Interestingly, the reduction of NHE-3 expression was more pronounced in Epac1&2−/− females (by 89%) than in males (by 59%). Immunofluorescence confocal images revealed much more diffuse intracellular distribution of AQP2 water channel in Epac1&2−/− females when compared to males. Furthermore, osmotically-driven water influx was reduced in principal cells within split-opened collecting ducts of Epac1&2−/− females but not males, when compared to respective EpacWT. In summary, our results reveal a substantially greater role of Epac signaling in renal water handling in females than in males. Interestingly, Epac1&2 deletion leads to a reversed pattern of sexual dimorphism in urinary production with females excreting larger volumes of hypotonic urine than males. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk). This is the full abstract presented at the American Physiology Summit 2024 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.

Abstract 4212: Mimicking HCC complex biology and diverse treatment response in a patient-derived microfluidic model

Abstract Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is closely associated with progressive stages of liver diseases. Its transformation, survival, progression and metastasis have been associated with regulation mechanisms orchestrated by the tumor stroma. Patient differences in tumor composition and cellular signaling are reflected in the diversity of therapy response. Hence, comprehensive in vitro models that capture patient-specific tumor and stromal components are necessary to elucidate and evaluate anticancer therapy. Patient-derived dissociated HCC cells from eight different patients were combined with tumor-derived fibroblasts and endothelial cells within a microfluidic setup comprising 64 parallel chips in a microtiter plate format. Cultures were challenged with an individual or combinatorial treatment from a panel of tool compounds for 72 hours in an automated setup. We used immunofluorescence and high content confocal imaging to assess the structural and biological interaction between cancer cells and the associated stroma. Viability assessment, artificial intelligence-based vascular morphology characterization and multiplexed chemokine/cytokine release measurements were used to elucidate patient and drug-dependent effects of single or combinatorial treatments. Cellular interaction within the microfluidic platform led to a vascularized tumor construct, with hepatocellular carcinoma aggregates being enveloped and traversed by a lumenized and interconnected vascular plexus, in association with tumor-derived fibroblasts. Compound treatment led to profound differences across the tested parameters, indicating different anticancer effects in conjunction with the biological diversity of the patient material. Finally, morphological assessment revealed distinct compound-induced effects on the tumor’s vascular organization. We present a vascularized patient-derived HCC model that includes relevant cellular players of the tumor microenvironment found in vivo. These co-cultures are highly suitable for studying specific cell types as well as patient-specific responses. We envision that this system has the potential to provide a platform for understanding the interplay between different cell types present in hepatocellular carcinomas, with sufficient scalability ease of use for industrial and clinical implementation. Citation Format: Orsola Mocellin, Abbie Robinson, Aleksandra Olczyk, Stephane Treillard, Thomas Olivier, Chee P. Ng, Jeroen Heijmans, Arthur Stok, Gilles van Tienderen, Monique Verstegen, Sebastian J. Trietsch, Henriëtte L. Lanz, Paul Vulto, Jos Joore, Karla Queiroz. Mimicking HCC complex biology and diverse treatment response in a patient-derived microfluidic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4212.

Ectoine protects corneal epithelial survival and barrier from hyperosmotic stress by promoting anti-inflammatory cytokine IL-37

PurposeTo explore novel role and molecular mechanism of a natural osmoprotectant ectoine in protecting corneal epithelial cell survival and barrier from hyperosmotic stress. MethodsPrimary human corneal epithelial cells (HCECs) were established from donor limbus. The confluent cultures in isosmolar medium were switched to hyperosmotic media (400–500 mOsM), with or without ectoine or rhIL-37 for different time periods. Cell viability and proliferation were evaluated by MTT or WST assay. The integrity of barrier proteins and the expression of cytokines and cathepsin S were evaluated by RT-qPCR, ELISA, and immunostaining with confocal microscopy. ResultsHCECs survived well in 450mOsM but partially damaged in 500mOsM medium. Ectoine well protected HCEC survival and proliferation at 500mOsM. The integrity of epithelial barrier was significantly disrupted in HCECs exposed to 450mOsM, as shown by 2D and 3D confocal immunofluorescent images of tight junction proteins ZO-1 and occludin. Ectoine at 5–20 mM well protected these barrier proteins under hyperosmotic stress. The expression of TNF-α, IL-1β, IL-6 and IL-8 were dramatically stimulated by hyperosmolarity but significantly suppressed by Ectoine at 5–40 mM. Cathepsin S, which was stimulated by hyperosmolarity, directly disrupted epithelial barrier. Interestingly, anti-inflammatory cytokine IL-37 was suppressed by hyperosmolarity, but restored by ectoine at mRNA and protein levels. Furthermore, rhIL-37 suppressed cathepsin S and rescued cell survival and barrier in HCECs exposed to hyperosmolarity. ConclusionOur findings demonstrate that ectoine protects HCEC survival and barrier from hyperosmotic stress by promoting IL-37. This provides new insight into pathogenesis and therapeutic potential for dry eye disease.

Abstract C064: Chronic platinum exposure induces cell-intrinsic type1 interferon/STING pathway activation in homologous recombination-deficient pancreatic cancer to drive sensitivity to checkpoint immunotherapy

Abstract Background: Although platinum-based chemotherapy is standard treatment for pancreatic ductal adenocarcinoma (PDAC) patients with germline/somatic mutations in homologous recombination (mutHRD), a subset become platinum-resistant. We have shown that patients with mutHRD PDAC progressing on platinum chemotherapy respond to dual immune checkpoint blockade (ICB) with anti-PD1+CTLA-4. We sought to model these clinical findings in preclinical models to interrogate mechanisms in platinum-resistant mutHRD PDAC that drive ICB sensitivity. Methods: We examined the association between objective disease response following anti-PD1+CTLA-4 ICB and duration of platinum chemotherapy exposure in platinum-resistant mutHRD PDAC patients. Cisplatin- resistant and sensitive Brca2-silenced (via shRNA knockdown) KPC cancer cells were generated and characterized in vitro and in vivo. Results: In 12 mutHRD PDAC patients treated with salvage dual ICB after progression on platinum-based chemotherapy, 7 (58%) demonstrated either partial response or stable disease. Notably, duration of prior platinum exposure was associated with post-ICB disease response. To model these findings preclinically, syngeneic mice implanted with cisplatin-sensitive or resistant shBrca2 or wildtype KPC tumor cells and subsequently treated with gemcitabine+cisplatin chemotherapy demonstrated expected rapid tumor growth of cisplatin-resistant shBrca2 tumors. However, we observed a profound reduction in tumor volumes when these chronically cisplatin-resistant KPC-shBrca2 tumors were subsequently treated with anti-PD-1+CTLA4 ICB, compared to wildtype-KPC or cisplatin-sensitive KPC-shBrca2 tumors (P<0.001). To understand mechanistic underpinnings of these observations, whole transcriptome sequencing revealed significant differential upregulation of type1 interferon and cGAS-STING pathways in cisplatin-resistant KPC-shBrca2 tumor cells compared to wildtype-KPC or cisplatin-sensitive KPC-shBrca2 controls (P-adj≤0.05). Confocal immunofluorescence imaging confirmed constitutively upregulated cGAS and STING expression in cisplatin-resistant KPC-shBrca2 compared to wildtype-KPC or cisplatin-sensitive KPC-shBrca2 tumor cells. Activation of type1 interferon/STING manifested in a secretome enriched for T-cell trafficking cytokines CXCL10, CXCL9, and CCL5 from cisplatin-resistant KPC-shBrca2 cells. Intriguingly, evaluable mutHRD patient tumors responsive to dual ICB also demonstrated baseline increase in CXCL9/10 and CCL5 expression compared with ICB-resistant mutHRD tumors (n=3 each) via Nanostring analysis. As such, we observed increased transwell (in vitro) and intratumoral (in vivo) CD8+ T-cell trafficking to cisplatin-resistant KPC-shBrca2 tumors compared with cisplatin-sensitive KPC-shBrca2 controls. Conclusions: Chronicity of platinum exposure in mutHRD PDAC potentiates ICB sensitivity via induction of cell-autonomous type-1 interferon/cGAS-STING signaling and ensuing T-cell trafficking to the PDAC tumor microenvironment. Citation Format: Ifeanyichukwu Ogobuiro, Anna Bianchi, Lucas Caeiro, Haleh Amirian, Luis Nivelo, Iago De Castro Silva, Karthik Rajkumar, Andrew Adams, Vanessa Garrido, Samara Singh, Nagaraj Nagathihalli, Nipun Merchant, Ramiro Verdun, Peter Hosein, Jashodeep Datta. Chronic platinum exposure induces cell-intrinsic type1 interferon/STING pathway activation in homologous recombination-deficient pancreatic cancer to drive sensitivity to checkpoint immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C064.

Immunofluorescent Staining of Human Hepatic Multicellular Spheroids: A Model for Studying Liver Diseases.

In vitro studies on liver diseases, such as non-alcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma, are traditionally performed in two-dimensional (2D) cultures of isolated primary cells or immortalized cell lines. However, this approach has limitations, as 2D cultures inadequately replicate the cell-cell and cell-extracellular matrix interactions found in three-dimensional (3D) environments. To overcome this limitation, various 3D models, such as spheroids, have been developed. These spheroids serve as simplified biomimetic in vitro models for studying liver diseases. They can be generated using a variety of cells from healthy and pathological tissues, including liver cancer. Here, we present a comprehensive protocol for performing immunofluorescent staining and confocal imaging on whole human hepatic multicellular spheroids, utilizing primary cells or cell lines. The immunofluorescence technique is a potent tool to understand the spatial distribution of different cell types within the spheroids and define the interactions that occur among these cells.

A surfactant-based dressing can reduce the appearance of Pseudomonas aeruginosa pigments and uncover the dermal extracellular matrix in an ex vivo porcine skin wound model.

From previous studies, we have shown that viable colony forming units of bacteria and bacterial biofilms are reduced after sequential treatment with a surfactant-based dressing. Here, we sought to test the impact on visible bacterial pigments and the ultrastructural impact following the sequential treatment of the same surfactant-based dressing. Mature Pseudomonas aeruginosa biofilms were grown on ex vivo porcine skin explants, and an imaging-based analysis was used to compare the skin with and without a concentrated surfactant. In explants naturally tinted by bacterial chromophores, wiping alone had no effect, while the use of a surfactant-based dressing reduced coloration. Similarly, daily wiping led to increased immunohistochemical staining for P. aeruginosa antigens, but not in the surfactant group. Confocal immunofluorescent imaging revealed limited bacterial penetration and coating of the dermis and loose pieces of sloughing material. Ultrastructural analysis confirmed that the biofilms were masking the extracellular matrix (ECM), but the surfactant could remove them, re-exposing the ECM. The masking of the ECM may provide another non-inflammatory explanation for delayed healing, as the ECM is no longer accessible for wound cell locomotion. The use of a poloxamer-based surfactant appears to be an effective way to remove bacterial chromophores and the biofilm coating the ECM fibres.

Age‐ and Sex‐ dependent changes in Translocator Protein 18 kDa (TSPO) expression, cellular sources, and association with amyloid‐β plaques in the 5XFAD murine model of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder that affects cognition, memory, and social abilities with devastating effects on individuals and families. The hallmark AD pathology is the formation of amyloid‐β‐(Aβ)‐plaques and Tau neurofibrillary tangles leading to neurodegeneration. Neuroinflammation, and microglia activation, also play an important role in the initiation and progression of AD, a process that begins earlier than cognitive decline. TSPO is a well‐validated and widely used biomarker of neuroinflammation that is expressed in glial cells, and it is markedly increased in the brain of AD subjects and in AD animal models. Previous studies have not assessed a detailed trajectory of TSPO in the progression of AD. In the present study, we determined the age‐and sex‐dependent increase in brain TSPO levels, cellular sources, and association with Aβ‐plaques in parallel with behavior.MethodBehavioral performance was measured using the Barnes Maze (cognition) in male and female wild‐type (WT) and 5XFAD mice at 3 months (3M), and 7 months (7M). To evaluate pathological endpoints, we measured Aβ‐load (number, size, and % area covered), TSPO quantitative autoradiography using the TSPO‐specific radioligand [3H]‐DPA‐713, and quadruple‐label immunofluorescent confocal imaging to determine the cellular sources of the TSPO response and its association with Aβ‐plaques.ResultAt 3M, we observed no significant differences in behavioral performance between WT and 5XFAD in either sex. At 7M, 5XFAD males showed no cognitive dysfunction, however, there was a significant cognitive impairment in 5XFAD females. We found an increase of Aβ‐plaques in an age‐and sex‐dependent manner. TSPO autoradiography showed a significant increase in TSPO levels in 5XFAD mice in both sexes and ages with a higher increase in females. Furthermore, we show that TSPO expression is colocalized with plaque‐associated microglia and not astrocytes.ConclusionThese results indicate that brain TSPO is an early biomarker of neuroinflammation and TSPO levels increase at the same time that Aβ‐aggregation and much earlier than cognitive decline. Furthermore, the increase in TSPO levels was associated with microglia surrounding or embedded with Aβ‐plaques suggesting a role in plaque formation.

Actin cytoskeleton remodeling disrupts physical barriers to infection and presents entry receptors to respiratory syncytial virus.

RSV is the leading cause of infant hospitalizations and a significant cause of paediatric and geriatric morbidity worldwide. Recently, we reported that insulin-like growth factor 1 receptor (IGF1R) was a receptor for respiratory syncytial virus (RSV) in airway epithelial cells and that activation of IGF1R recruited the coreceptor, nucleolin (NCL), to the cell surface. Cilia and mucus that line the airways pose a significant barrier to viral and bacterial infection. The cortical actin cytoskeleton has been shown by others to mediate RSV entry, so we studied the roles of the RSV receptors and actin remodelling during virus entry. We found that IGF1R expression and phosphorylation were associated with the ability of RSV to infect cells. Confocal immunofluorescence imaging showed that actin projections, a hallmark of macropinocytosis, formed around viral particles 30 min after infection. Consistent with prior reports we also found that virus particles were internalized into early endosome antigen-1 positive endosomes within 90 min. Inhibiting actin polymerization significantly reduced viral titre by approximately ten-fold. Inhibiting PI3 kinase and PKCζ in stratified air-liquid interface (ALI) models of the airway epithelium had similar effects on reducing the actin remodelling observed during infection and attenuating viral entry. Actin projections were associated with NCL interacting with RSV particles resting on apical cilia of the ALIs. We conclude that macropinocytosis-like actin projections protrude through normally protective cilia and mucus layers of stratified airway epithelium that helps present the IGF1R receptor and the NCL coreceptor to RSV particles waiting at the surface.

PMPCA-Related Encephalopathy: Novel Variants, Phenotype Extension, and Mitochondrial Morphology.

The PMPCA gene encodes the α-subunit of mitochondrial processing peptidase (α-MPP), an enzyme responsible for cleavage of nuclear-encoded mitochondrial precursor proteins after their import into mitochondria. Mutations in this gene have been described in patients with nonprogressive or slow progressive cerebellar ataxia, with variable age at onset and severity. Cerebellar atrophy and striatum changes were found in severe cases. The patient was diagnosed using whole exome sequencing. Skin fibroblasts were used for confirmation of α-MPP levels using western blot and mitochondrial morphology assessment of immunofluorescent confocal microscopy images. Two novel compound heterozygous variants in the PMPCA gene (p.Tyr241Ser and p.Met251Val) were identified in an 8-year-old proband with progressive spastic quadriparesis, delayed psychomotor development, and intellectual disability, with onset at 13 months. The brain imaging showed cortical and cerebellar atrophy, reduced volume of basal ganglia with striatum hyperintensity, and periventricular white matter changes. The patient's fibroblasts showed a decreased α-MPP level and reduced and fragmented mitochondria. The described case contributes to the number of patients with progressive PMPCA-related disease with a severe intermediate phenotype. Moreover, we extend the phenotype to Leigh-like white matter changes that have not been described in previously reported cases.

Characterization of Anxiety-Like Behaviors and Neural Circuitry following Chronic Moderate Noise Exposure in Mice.

Commonly encountered nontraumatic, moderate noise is increasingly implicated in anxiety; however, the neural substrates underlying this process remain unclear. We investigated the neural circuit mechanism through which chronic exposure to moderate-level noise causes anxiety-like behaviors. Mice were exposed to chronic, moderate white noise [85 decibel (dB) sound pressure level (SPL)], 4 h/d for 4 wk to induce anxiety-like behaviors, which were assessed by open field, elevated plus maze, light-dark box, and social interaction tests. Viral tracing, immunofluorescence confocal imaging, and brain slice patch-clamp recordings were used to characterize projections from auditory brain regions to the lateral amygdala. Neuronal activities were characterized by invivo multielectrode and fiber photometry recordings in awake mice. Optogenetics and chemogenetics were used to manipulate specific neural circuitry. Mice chronically (4 wk) exposed to moderate noise (85 dB SPL, 4 h/d) demonstrated greater neuronal activity in the lateral amygdala (LA), and the LA played a critical role in noise-induced anxiety-like behavior in these model mice. Viral tracing showed that the LA received monosynaptic projections from the medial geniculate body (MG) and auditory cortex (ACx). Optogenetic excitation of the or circuits acutely evoked anxiety-like behaviors, whereas their chemogenetic inactivation abolished noise-induced anxiety-like behavior. Moreover, mice chronically exposed to moderate noise were more susceptible to acute stress, with more neuronal firing in the LA, even after noise withdrawal. Mice exposed to 4 wk of moderate noise (85 dB SPL, 4 h/d) demonstrated behavioral and physiological differences compared to controls. The neural circuit mechanisms involved greater excitation from glutamatergic neurons of the MG and ACx to LA neurons under chronic, moderate noise exposure, which ultimately promoted anxiety-like behaviors. Our findings support the hypothesis that nontraumatic noise pollution is a potentially serious but unrecognized public health concern. https://doi.org/10.1289/EHP12532.

Garlic extract enhances bioceramic bone scaffolds through upregulating ALP & BGLAP expression in hMSC-monocyte co-culture

Bone homeostasis is predicated by osteoblast and osteoclast cell cycles where gene expressions are responsible for their differentiation from human mesenchymal stem cells (hMSC) and monocytes, respectively. The pro-osteogenic potential of an hMSC-monocyte co-culture can be measured through complementary DNA (mRNA synthesis) within the nucleus, known as quantitative polymerase chain reaction (qPCR). Through this technique, the effects of garlic extract (allicin) release from calcium phosphate bone scaffolds on gene expression of bone forming and bone remodeling cells was explored. Results show this complex biomaterial system enhances hMSC differentiation through the upregulation of bone-forming proteins. Osteoblastic gene markers alkaline phosphatase (ALP) and osteocalcin (BGLAP), are respectively upregulated by 3-fold and 1.6-fold by day 14. These mature osteoblasts then upregulate the receptor activator of nuclear factor-kB ligand (RANKL) which recruits osteoclast cells, as captured by a nearly 2-fold higher osteoclast expression of tartrate-resistance acid-phosphatase (ACP5). This also activates antagonist osteoprotegerin (OPG) expression in osteoblasts, decreasing osteoclast resorption potential and ACP5 expression by day 21. The pro-osteogenic environment with garlic extract release is further quantified by a 4× increase in phosphatase activity and visibly captured in immunofluorescent tagged confocal images. Also corroborated by enhanced collagen formation in a preliminary in vivo rat distal femur model, this work collectively reveals how garlic extract can enhance bioceramic scaffolds for bone tissue regenerative applications.

Aging Associated Changes in Autophagy Markers in Mouse Lumbar Motor Neurons

Autophagy is a process through which damaged cytoplasmic structures are recycled and degraded within the cell. Aging can affect autophagy regulation in different steps and lead to the accumulation of altered proteins, leading to cell damage and death. Motor neuron (MN) loss is an important contributor to sarcopenia age-dependent motor dysfunction. Understanding the regulatory mechanisms of autophagy can help elucidate the motor dysfunction that occurs with aging and provide a target for developing future therapeutic agents. Previous immunofluorescence imaging studies in phrenic MNs showed an age-dependent increase in key autophagy proteins LC3 and p62, reflecting autophagosome accumulation and autophagy impairment. Accordingly, we hypothesized that aging in lumbar MNs leads to autophagy impairment reflected in regulatory protein expression. The current study assessed autophagy in choline acetyltransferase-positive MNs from spinal levels L1-L6, focused on crural muscles (extensor digitorum longus and soleus), using confocal immunofluorescence imaging. Expression of LC3 and p62 proteins in MNs were determined in male and female C57BL/6 mice at 6-, 18- and 24- months of age. Increased expression of LC3 in lumbar MNs was observed with aging across all three age groups and p62 expression in lumbar MNs was significantly increased by 24- and 18-months compared to 6-months (p<0.001). There was no sex effect observed in LC3, but p62 was significantly higher in lumbar MNs of female mice (p=0.007). Expression of both LC3 and p62 increased diffusely in ventral lumbar gray matter regions with age (p<0.001), an effect that was predominant in male and not female mice. Taken together, the results of this study provide evidence for that age-dependent changes in autophagy markers expression occurs in regions of the spinal cord (motor neurons and ventral horn gray matter). The neuronal mechanisms of aging effects include dysregulation of autophagy. Supported by NIH R01 AG057052. 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.

ALS Causative Mutations in KIF5A Disrupt Autoinhibition Leading to Toxic Gain of Function (P8-8.002)

<h3>Objective:</h3> The objective of this study was to elucidate the pathogenic mechanisms of ALS causative mutations in motor protein KIF5A. <h3>Background:</h3> KIF5A is a neuronal specific subunit of Kinesin-1, which is a microtubule motor protein that plays roles in axonal transport and cytoskeletal regulation. Mutations in distinct regions of Kinesin-1 lead to a broad range of neurologic diseases. KIF5A enables Kinesin-1 to walk along microtubules and transport various cargos such as mitochondria and RNA granules into the distal axon. ALS-causative mutations in KIF5A affect the structure of the domain of the protein that functions in cargo binding and autoregulation. However, the consequences of ALS-causative mutations on disease pathogenesis are not fully understood. Our central hypothesis was that KIF5A ALS mutations disrupt cargo binding and/or autoregulation leading to neurodegeneration. <h3>Design/Methods:</h3> We generated <i>in vitro</i> models of KIF5A ALS using transient transfection of disease causative variants into neuroblastoma cell lines. We performed immunofluorescence staining and confocal imaging to visualize the distribution of motor proteins KIF5A, KIF5B, and Dynein. We utilized Drosophila S2 cell lines to study mitochondrial motility using live-imaging. <h3>Results:</h3> Whereas wild type (WT) KIF5A displayed relatively homogeneous distribution, ALS mutant KIF5A developed dramatic accumulation within distal neurites. Co-expression of the ALS mutant protein with the WT caused it to accumulate as well. Mitochondria displayed similar mislocalization to distal neurites. No change was seen in the distribution of KIF5B or Dynein. <h3>Conclusions:</h3> We found that ALS mutant KIF5A caused dramatic accumulation of mutant and WT protein as well as mitochondrial within distal neurite tips. These findings suggest that ALS mutation disrupts its autoinhibition causing mislocalization of its cargos through gain of function. These changes in distribution are specific to KIF5A containing motors. Our findings establish dysregulation of KIF5A activity as the underlying pathogenic mechanism for KIF5A ALS. <b>Disclosure:</b> Dr. Brent has received research support from The American Academy of Neurology. Mr. Sterling-Angus has nothing to disclose. Dr. Deng has nothing to disclose.

Charged multivesicular body protein 4b forms complexes with gap junction proteins during lens fiber cell differentiation.

Charged multivesicular body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap junction alpha-3 protein (GJA3) or connexin 46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens-where large gap junction plaques begin to form-particularly, on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses, CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma membrane complexes, either directly and/or indirectly, with gap junction proteins Cx46 and Cx50 that are often associated with "ball-and-socket" double-membrane junctions during lens fiber cell differentiation.

Erratum: Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse.

An erratum was issued for:Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse. The Authors section was updated from: Ruibing Xia1,2,3 Julia Vlcek1,2 Julia Bauer1,2,3 Stefan Kääb1,3 Hellen Ishikawa-Ankerhold1,2 Dominic Adam van den Heuvel1,2 Christian Schulz1,2,3 Steffen Massberg1,2,3 Sebastian Clauss1,2,3 1University Hospital Munich, Department of Medicine I, Ludwig Maximilian University Munich 2Walter Brendel Center of Experimental Medicine, Ludwig Maximilian University Munich 3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance to: Ruibing Xia1,2,3 Julia Vlcek1,2 Julia Bauer1,2,3 Stefan Kääb1,3 Hellen Ishikawa-Ankerhold1,2 Dominic Adam van den Heuvel1,2 Christian Schulz1,2,3 Steffen Massberg1,2,3 Sebastian Clauss1,2,3 1University Hospital Munich, Department of Medicine I, Ludwig Maximilians University (LMU) Munich 2Institute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig Maximilians University (LMU) Munich 3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance.

An Innovative Toolkit to Investigate the Complex Mechanisms of Cardiac Arrhythmias

ARTICLES DISCUSSED: Tomsits, P., Schuttler, D., Kaab, S., Clauss, S., Voigt, N. Isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of Ca2+ transients and L-Type calcium current. Journal of Visualized Experiments. (165), 10.3791/61964 (2020). Seibertz, F., Reynolds, M., Voigt, N. Single-Cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (166), 10.3791/61890 (2020). Xia, R. et al. Isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node. Journal of Visualized Experiments. (171), 10.3791/62236 (2021). Xia, R. et al. Whole-Mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse. Journal of Visualized Experiments. (166), 10.3791/62058 (2020). Kumar, P., Si, M., Paulhus, K., Glasscock, E. Microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice. Journal of Visualized Experiments. (173), 10.3791/62735 (2021). Scherschel, K. et al. Location, dissection, and analysis of the murine stellate ganglion. Journal of Visualized Experiments. (166), 10.3791/62026 (2020). Shimura, D., Hunter, J., Katsumata, M., Shaw, R. M. Removal of an internal translational start site from mRNA while retaining expression of the full-length protein. Journal of Visualized Experiments. (181), 10.3791/63405 (2022). Rotzer, R. D. et al. Implantation of combined telemetric ECG and blood pressure transmitters to determine spontaneous baroreflex sensitivity in conscious mice. Journal of Visualized Experiments. (168), 10.3791/62101 (2021). Tomsits, P. et al. Analyzing long-term electrocardiography recordings to detect arrhythmias in mice. Journal of Visualized Experiments. (171), 10.3791/62386 (2021). Grab, M. et al. Development and evaluation of 3D-printed cardiovascular phantoms for interventional planning and training. Journal of Visualized Experiments. (167), 10.3791/62063 (2021).

Junctophilin-2 allosterically interacts with ryanodine receptor type 2 to regulate calcium release units in mouse cardiomyocytes.

The interaction between junctophilin-2 (JPH2) and ryanodine receptor type 2 (RyR2) regulated Ca2+ signaling in mouse cardiomyocytes. However, their exact interaction remains unclear. This study elucidates the interaction between JPH2 with RyR2 using co-immunoprecipitation of cardiac sarcoplasmic reticulum vesicles. Additionally, a glutathione S-transferase (GST) pull-down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. JPH2 interacted with RyR2 and the C terminus of the JPH2 protein can pull-down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines in isolated mouse cardiomyocytes. Knockdown of JPH2 reduced the amplitude of Ca2+ transients and disrupted its interaction with RyR2. Therefore, the C-terminus domain of JPH2 is required for interactions with RyR2 in mouse cardiomyocytes, which provides a molecular mechanism for seeking Ca2+-related disease prevention strategies.