The effect of obesity and subsequent weight reduction on cardiac morphology and function in cats.

BACKGROUND: In people, obesity is a risk factor for cardiovascular disease, associated with systemic hypertension, cardiac remodelling and systolic and diastolic dysfunction. Weight reduction can reverse myocardial remodelling and reduce risk of subsequent cardiovascular disease. In cats, far less is known regarding the effects of obesity and subsequent weight reduction on cardiovascular morphology and function. This prospective study aimed to assess cardiac morphology and function, heart rate variability, cardiac biomarkers and body composition before and after controlled weight reduction in cats with obesity. Body composition analysis (by dual energy x-ray absorptiometry, DEXA) and cardiovascular assessment (echocardiography, systemic arterial systolic blood pressure, electrocardiography, plasma cardiac biomarkers) were performed prior to weight management in twenty cats with obesity. These investigations were repeated in eleven cats that reached target weight. RESULTS: At baseline, systemic hypertension was not documented, but the majority of cats with obesity (15 out of 19) showed echocardiographic evidence of diastolic dysfunction. Eleven of 20 cats had increased maximal end-diastolic septal or left ventricular free wall thickness (≥ 6.0 mm) at baseline. Median (interquartile range) percentage of weight lost in the cats reaching target weight was 26% (17-29%), with a median reduction in body fat mass of 45% (26-64%). Both the end-diastolic left ventricular free wall (median magnitude of change -0.85 mm, IQR -0.05 mm to -1.55 mm, P = 0.019; median percentage reduction 14.0%) and end-diastolic interventricular septum (median magnitude of change -0.5 mm, IQR -0.2 mm to -1.225 mm, P = 0.047; median percentage reduction 7.9%) thickness decreased after weight reduction. Following weight reduction, pulsed wave tissue Doppler imaging of the left ventricular free wall was consistent with improved diastolic function in 4 out of 8 cats, however there was no significant difference in overall diastolic function class. Further, there was no change in heart rate variability or cardiac biomarkers with weight reduction. CONCLUSION: An increase in left ventricular wall thickness and diastolic dysfunction were common echocardiographic features in cats with obesity within our study and may be reversible with successful weight and fat mass loss. Further studies are required to clarify the clinical consequences of these findings.

An aco-2::gfp knock-in enables the monitoring of mitochondrial morphology throughout C. elegans lifespan.

We used CRISPR/Cas9 gene editing in C. elegans in order to fluorescently tag endogenous aconitase-2 (ACO-2). ACO-2 is a mitochondrially localized protein, and the aco-2::gfp strain enabled the examination of native mitochondrial morphology in live animals. Here we validate that the aco-2::gfp strain displays the prototypic changes in mitochondrial morphology known to occur during aging and upon paraquat (PQ) induced mitochondrial stress. We also provide evidence that the ACO-2::GFP reporter can serve as a superior means for tracking mitochondrial morphology than conventional MitoTracker dyes-especially in aged-worms.

Screening Method for Identifying Toxicants Capable of Inducing Astrocyte Senescence.

Cellular senescence is a tumor-suppressive mechanism which leads to near irreversible proliferative arrest. However, senescent cells can cause tissue dysfunction, in large part because they express a senescence-associated secretory phenotype (SASP) involving secretion of, amongst other factors, proinflammatory cytokines known to compromise neuronal health. Therefore, established neurotoxicants may cause neurotoxicity in vivo, in part by triggering mitotic cells in the brain to undergo senescence and adopt an inflammatory SASP which in turn could cause deleterious effects to surrounding neurons. To begin to address this hypothesis, we examined whether we could screen known neurotoxicants for their ability to cause astrocytes (a mitotic cell type especially important for maintaining neuronal health) to undergo senescence. For this purpose, we utilized inducible pluripotent stem cell-derived human astrocytes and screened an 80 compound neurotoxicant library provided by the Biomolecular Screening Branch of the NIEHS National Toxicology Program. Here we present a screening method based on induction of the senescent marker, senescent-associated beta-galactosidase (SA-ß-gal). We describe in detail an automated method for the unbiased quantitation of percentage of SA-ß-gal + astrocytes. Although our results suggest that conducting an SA-ß-gal senescence screen using human inducible pluripotent stem cell-derived astrocytes may be feasible, they also highlight challenges that likely preclude its adaptation to high-throughput. We also explore the possibility of using primary mouse astrocytes for this purpose and explain why this platform is problematic and very unlikely to yield meaningful results, even in small screens with compound replicates.

Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease.

Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.

Cellular senescence and the aging brain.

Cellular senescence is a potent anti-cancer mechanism that arrests the proliferation of mitotically competent cells to prevent malignant transformation. Senescent cells accumulate with age in a variety of human and mouse tissues where they express a complex 'senescence-associated secretory phenotype' (SASP). The SASP includes many pro-inflammatory cytokines, chemokines, growth factors and proteases that have the potential to cause or exacerbate age-related pathology, both degenerative and hyperplastic. While cellular senescence in peripheral tissues has recently been linked to a number of age-related pathologies, its involvement in brain aging is just beginning to be explored. Recent data generated by several laboratories suggest that both aging and age-related neurodegenerative diseases are accompanied by an increase in SASP-expressing senescent cells of non-neuronal origin in the brain. Moreover, this increase correlates with neurodegeneration. Senescent cells in the brain could therefore constitute novel therapeutic targets for treating age-related neuropathologies.

Loss of PSD-95 enrichment is not a prerequisite for spine retraction.

Changes in neuronal structure are thought to underlie long-term behavioral modifications associated with learning and memory. In particular, considerable evidence implicates the destabilization and retraction of dendritic spines along with the loss of spine synapses as an important cellular mechanism for refining brain circuits, yet the molecular mechanisms regulating spine elimination remain ill-defined. The postsynaptic density protein, PSD-95, is highly enriched in dendritic spines and has been associated with spine stability. Because spines with low levels of PSD-95 are more dynamic, and the recruitment of PSD-95 to nascent spines has been associated with spine stabilization, we hypothesized that loss of PSD-95 enrichment would be a prerequisite for spine retraction. To test this hypothesis, we used dual-color time-lapse two-photon microscopy to monitor rat hippocampal pyramidal neurons cotransfected with PSD-95-GFP and DsRed-Express, and we analyzed the relationship between PSD-95-GFP enrichment and spine morphological changes. Consistent with our hypothesis, we found that the majority of spines that retracted were relatively unenriched for PSD-95-GFP. However, in the subset of PSD-95-GFP-enriched spines that retracted, spine shrinkage and loss of PSD-95-GFP were tightly coupled, suggesting that loss of PSD-95-GFP enrichment did not precede spine retraction. Moreover, we found that, in some instances, spine retraction resulted in a significant enrichment of PSD-95-GFP on the dendritic shaft. Our data support a model of spine retraction in which loss of PSD-95 enrichment is not required prior to the destabilization of spines.

Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.

Ephrin-as guide the formation of functional maps in the visual cortex.

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. Here, we map the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally, using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps.

Ephrin-As and neural activity are required for eye-specific patterning during retinogeniculate mapping.

In mammals, retinal ganglion cell (RGC) projections initially intermingle and then segregate into a stereotyped pattern of eye-specific layers in the dorsal lateral geniculate nucleus (dLGN). Here we found that in mice deficient for ephrin-A2, ephrin-A3 and ephrin-A5, eye-specific inputs segregated but the shape and location of eye-specific layers were profoundly disrupted. In contrast, mice that lacked correlated retinal activity did not segregate eye-specific inputs. Inhibition of correlated neural activity in ephrin mutants led to overlapping retinal projections that were located in inappropriate regions of the dLGN. Thus, ephrin-As and neural activity act together to control patterning of eye-specific retinogeniculate layers.