Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.

Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz syndrome patients provides insights into genotype-phenotype relation.

RATIONALE: Pathogenic (P)/likely pathogenic (LP) SMAD3 variants cause Loeys-Dietz syndrome type 3 (LDS3), which is characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with osteoarthritis. OBJECTIVES: Investigate the impact of P/LP SMAD3 variants with functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs), to optimize interpretation of SMAD3 variants. METHODS: A retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and functional analyses on SMAD3 patient-derived VSMCs and SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts. RESULTS: Individuals with dominant negative (DN) SMAD3 variant in the MH2 domain exhibited more major events (66.7% vs. 44.0%, P = 0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. The age at first major event was 35.0 years [IQR 29.0-47.0] in individuals with DN variants in MH2, compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (P = 0.065). Fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential in HI SMAD3 variant fibroblasts. HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix formation compared to control cell lines. CONCLUSION: Distinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and SMA and MYH11 expression. The differences between DN and HI SMAD3 variant fibroblasts and VSMCs potentially contribute to the differences in disease manifestation. Notably, myofibroblast differentiation seems a suitable alternative in vitro test system compared to VSMCs.

Molecular phenotyping and functional assessment of smooth muscle-like cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.

Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.

Model Systems to Study the Mechanism of Vascular Aging.

Cardiovascular diseases are the leading cause of death globally. Within cardiovascular aging, arterial aging holds significant importance, as it involves structural and functional alterations in arteries that contribute substantially to the overall decline in cardiovascular health during the aging process. As arteries age, their ability to respond to stress and injury diminishes, while their luminal diameter increases. Moreover, they experience intimal and medial thickening, endothelial dysfunction, loss of vascular smooth muscle cells, cellular senescence, extracellular matrix remodeling, and deposition of collagen and calcium. This aging process also leads to overall arterial stiffening and cellular remodeling. The process of genomic instability plays a vital role in accelerating vascular aging. Progeria syndromes, rare genetic disorders causing premature aging, exemplify the impact of genomic instability. Throughout life, our DNA faces constant challenges from environmental radiation, chemicals, and endogenous metabolic products, leading to DNA damage and genome instability as we age. The accumulation of unrepaired damages over time manifests as an aging phenotype. To study vascular aging, various models are available, ranging from in vivo mouse studies to cell culture options, and there are also microfluidic in vitro model systems known as vessels-on-a-chip. Together, these models offer valuable insights into the aging process of blood vessels.

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

Selective Phosphodiesterase 1 Inhibition Ameliorates Vascular Function, Reduces Inflammatory Response, and Lowers Blood Pressure in Aging Animals.

Diminished nitric oxide-cGMP-mediated relaxation plays a crucial role in cardiovascular aging, leading to decreased vasodilation, vascular hypertrophy and stiffening, and ultimately, cardiovascular dysfunction. Aging is the time-related worsening of physiologic function due to complex cellular and molecular interactions, and it is at least partly driven by DNA damage. Genetic deletion of the DNA repair enzyme ERCC1 endonuclease in Ercc1Δ/- mice provides us an efficient tool to accelerate vascular aging, explore mechanisms, and test potential treatments. Previously, we identified the cGMP-degrading enzyme phosphodiesterase 1 as a potential treatment target in vascular aging. In the present study, we studied the effect of acute and chronic treatment with ITI-214, a selective phosphodiesterase 1 inhibitor on vascular aging features in Ercc1Δ/- mice. Compared with wild-type mice, Ercc1Δ/- mice at the age of 14 weeks showed decreased reactive hyperemia, diminished endothelium-dependent and -independent responses of arteries in organ baths, carotid wall hypertrophy, and elevated circulating levels of inflammatory cytokines. Acute ITI-214 treatment in organ baths restored the arterial endothelium-independent vasodilation in Ercc1Δ/- mice. An 8-week treatment with 100 mg/kg per day ITI-214 improved endothelium-independent relaxation in both aorta and coronary arteries, at least partly restored the diminished reactive hyperemia, lowered the systolic and diastolic blood pressure, normalized the carotid hypertrophy, and ameliorated inflammatory responses exclusively in Ercc1Δ/- mice. These findings suggest phosphodiesterase 1 inhibition would provide a powerful tool for nitric oxide-cGMP augmentation and have significant therapeutic potential to battle arteriopathy related to aging. SIGNIFICANCE STATEMENT: The findings implicate the key role of phosphodiesterase 1 in vascular function and might be of clinical importance for the prevention of mortalities and morbidities related to vascular complications during aging, as well as for patients with progeria that show a high risk of cardiovascular disease.

Transforming Growth Factor-ß and the Renin-Angiotensin System in Syndromic Thoracic Aortic Aneurysms: Implications for Treatment.

Thoracic aortic aneurysms (TAAs) are permanent pathological dilatations of the thoracic aorta, which can lead to life-threatening complications, such as aortic dissection and rupture. TAAs frequently occur in a syndromic form in individuals with an underlying genetic predisposition, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). Increasing evidence supports an important role for transforming growth factor-ß (TGF-ß) and the renin-angiotensin system (RAS) in TAA pathology. Eventually, most patients with syndromic TAAs require surgical intervention, as the ability of present medical treatment to attenuate aneurysm growth is limited. Therefore, more effective medical treatment options are urgently needed. Numerous clinical trials investigated the therapeutic potential of angiotensin receptor blockers (ARBs) and ß-blockers in patients suffering from syndromic TAAs. This review highlights the contribution of TGF-ß signaling, RAS, and impaired mechanosensing abilities of aortic VSMCs in TAA formation. Furthermore, it critically discusses the most recent clinical evidence regarding the possible therapeutic benefit of ARBs and ß-blockers in syndromic TAA patients and provides future research perspectives and therapeutic implications.

In Vivo Renin Activity Imaging in the Kidney of Progeroid Ercc1 Mutant Mice.

Changes in the renin-angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin-angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/- mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/- mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/- mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/- compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging.

Multi-Omics Profiling in Marfan Syndrome: Further Insights into the Molecular Mechanisms Involved in Aortic Disease.

Thoracic aortic aneurysm is a potentially life-threatening disease with a strong genetic contribution. Despite identification of multiple genes involved in aneurysm formation, little is known about the specific underlying mechanisms that drive the pathological changes in the aortic wall. The aim of our study was to unravel the molecular mechanisms underlying aneurysm formation in Marfan syndrome (MFS). We collected aortic wall samples from FBN1 variant-positive MFS patients (n = 6) and healthy donor hearts (n = 5). Messenger RNA (mRNA) expression levels were measured by RNA sequencing and compared between MFS patients and controls, and between haploinsufficient (HI) and dominant negative (DN) FBN1 variants. Immunohistochemical staining, proteomics and cellular respiration experiments were used to confirm our findings. FBN1 mRNA expression levels were highly variable in MFS patients and did not significantly differ from controls. Moreover, we did not identify a distinctive TGF-ß gene expression signature in MFS patients. On the contrary, differential gene and protein expression analysis, as well as vascular smooth muscle cell respiration measurements, pointed toward inflammation and mitochondrial dysfunction. Our findings confirm that inflammatory and mitochondrial pathways play important roles in the pathophysiological processes underlying MFS-related aortic disease, providing new therapeutic options.

Local endothelial DNA repair deficiency causes aging-resembling endothelial-specific dysfunction.

We previously identified genomic instability as a causative factor for vascular aging. In the present study, we determined which vascular aging outcomes are due to local endothelial DNA damage, which was accomplished by genetic removal of ERCC1 (excision repair cross-complementation group 1) DNA repair in mice (EC-knockout (EC-KO) mice). EC-KO showed a progressive decrease in microvascular dilation of the skin, increased microvascular leakage in the kidney, decreased lung perfusion, and increased aortic stiffness compared with wild-type (WT). EC-KO showed expression of DNA damage and potential senescence marker p21 exclusively in the endothelium, as demonstrated in aorta. Also the kidney showed p21-positive cells. Vasodilator responses measured in organ baths were decreased in aorta, iliac and coronary artery EC-KO compared with WT, of which coronary artery was the earliest to be affected. Nitric oxide-mediated endothelium-dependent vasodilation was abolished in aorta and coronary artery, whereas endothelium-derived hyperpolarization and responses to exogenous nitric oxide (NO) were intact. EC-KO showed increased superoxide production compared with WT, as measured in lung tissue, rich in endothelial cells (ECs). Arterial systolic blood pressure (BP) was increased at 3 months, but normal at 5 months, at which age cardiac output (CO) was decreased. Since no further signs of cardiac dysfunction were detected, this decrease might be an adaptation to prevent an increase in BP. In summary, a selective DNA repair defect in the endothelium produces features of age-related endothelial dysfunction, largely attributed to loss of endothelium-derived NO. Increased superoxide generation might contribute to the observed changes affecting end organ perfusion, as demonstrated in kidney and lung.

Pre-Operative Fasting Provides Long Term Protection Against Chronic Renal Damage Induced by Ischaemia Reperfusion Injury in Wild Type and Aneurysm Prone Fibulin-4 Mice.

OBJECTIVE: Renal ischaemia reperfusion injury (IRI) is inevitable during open repair of pararenal aortic aneurysms. Pre-operative fasting potently increases resistance against IRI. The effect of fasting on IRI was examined in a hypomorphic Fibulin-4 mouse model (Fibulin-4+/R), which is predisposed to develop aortic aneurysms. METHODS: Wild type (WT) and Fibulin-4+/R mice were either fed ad libitum (AL) or fasted for two days before renal IRI induction by temporary clamping of the renal artery and vein of both kidneys. Six hours, 48 h, and seven days post-operatively, serum urea levels, renal histology, and mRNA expression levels of inflammatory and injury genes were determined to assess kidney function and damage. Additionally, matrix metalloproteinase activity in the kidney was assessed six months after IRI. RESULTS: Two days of fasting improved survival the first week after renal IRI in WT mice compared with AL fed mice. Short term AL fed Fibulin-4+/R mice showed improved survival and kidney function compared with AL fed WT mice, which could not be further enhanced by fasting. Both fasted WT and Fibulin-4+/R mice showed improved survival, kidney function and morphology compared with AL fed mice six months after renal IRI. Fibulin-4+/R kidneys of fasted mice showed reduced apoptosis together with increased matrix metalloprotease activity levels compared with AL fed Fibulin-4+/R mice, indicative of increased matrix remodelling. CONCLUSION: Fibulin-4+/R mice are naturally protected against the short-term, but not long-term, consequences of renal IRI. Pre-operative fasting protects against renal IRI and prevents (long-term) deterioration of kidney function and morphology in both WT and Fibulin-4+/R mice. These data suggest that pre-operative fasting may decrease renal damage in patients undergoing open abdominal aneurysm repair.

Chronic Sildenafil Treatment Improves Vasomotor Function in a Mouse Model of Accelerated Aging.

Aging leads to a loss of vasomotor control. Both vasodilation and vasoconstriction are affected. Decreased nitric oxide-cGMP-mediated relaxation is a hallmark of aging. It contributes to vascular disease, notably hypertension, infarction, and dementia. Decreased vasodilation can be caused by aging independently from cardiovascular risk factors. This process that can be mimicked in mice in an accelerated way by activation of the DNA damage response. Genetic deletion of the DNA repair enzyme ERCC1 endonuclease in mice, as in the case of Ercc1Δ/- mice, can be used as a tool to accelerate aging. Ercc1Δ/- mice develop age-dependent vasomotor dysfunction from two months after birth. In the present study we tested if chronic treatment with sildenafil, a phosphodiesterase 5 inhibitor that augments NO-cGMP signaling, can reduce the development of vasomotor dysfunction in Ercc1Δ/- mice. Ercc1Δ/- mice and wild-type littermates were treated with 10 mg/kg/d of sildenafil from the age of 6 to the age of 14 weeks. Blood pressure and in vivo and ex vivo vasomotor responses were measured at the end of the treatment period. Ercc1Δ/- mice developed decreased reactive hyperemia, and diminished NO-cGMP-dependent acetylcholine responses. The diminished acetylcholine response involved both endothelial and vascular smooth muscle cell signaling. Chronic sildenafil exclusively improved NO-cGMP signaling in VSMC, and had no effect on endothelium-derived hyperpolarization. Sildenafil also improved KCl hypocontractility in Ercc1Δ/- mice. All effects were blood pressure-independent. The findings might be of clinical importance for prevention of morbidities related to vascular aging as well as for progeria patients with a high risk of cardiovascular disease.

Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model (Ercc1Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating endothelial dysfunction, by preserving genomic integrity. In this mouse mutant displaying prominent accelerated, age-dependent endothelial dysfunction we investigated the signaling pathways involved in improved endothelium-mediated vasodilation by DR, and explore the potential role of the renin-angiotensin system (RAS). Ercc1Δ/- mice showed increased blood pressure and decreased aortic relaxations to acetylcholine (ACh) in organ bath experiments. Nitric oxide (NO) signaling and phospho-Ser1177-eNOS were compromised in Ercc1Δ/- DR improved relaxations by increasing prostaglandin-mediated responses. Increase of cyclo-oxygenase 2 and decrease of phosphodiesterase 4B were identified as potential mechanisms. DR also prevented loss of NO signaling in vascular smooth muscle cells and normalized angiotensin II (Ang II) vasoconstrictions, which were increased in Ercc1Δ/- mice. Ercc1Δ/- mutants showed a loss of Ang II type 2 receptor-mediated counter-regulation of Ang II type 1 receptor-induced vasoconstrictions. Chronic losartan treatment effectively decreased blood pressure, but did not improve endothelium-dependent relaxations. This result might relate to the aging-associated loss of treatment efficacy of RAS blockade with respect to endothelial function improvement. In summary, DR effectively prevents endothelium-dependent vasodilator dysfunction by augmenting prostaglandin-mediated responses, whereas chronic Ang II type 1 receptor blockade is ineffective.

Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency.

As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

Cockayne syndrome group B (Csb) and group a (Csa) deficiencies predispose to hearing loss and cochlear hair cell degeneration in mice.

Sensory hair cells in the cochlea, like most neuronal populations that are postmitotic, terminally differentiated, and non-regenerating, depend on robust mechanisms of self-renewal for lifelong survival. We report that hair cell homeostasis requires a specific sub-branch of the DNA damage nucleotide excision repair pathway, termed transcription-coupled repair (TCR). Cockayne syndrome (CS), caused by defects in TCR, is a rare DNA repair disorder with a broad clinical spectrum that includes sensorineural hearing loss. We tested hearing and analyzed the cellular integrity of the organ of Corti in two mouse models of this disease with mutations in the Csb gene (CSB(m/m) mice) and Csa gene (Csa(-/-) mice), respectively. Csb(m/m) and Csa(-/-) mice manifested progressive hearing loss, as measured by an increase in auditory brainstem response thresholds. In contrast to wild-type mice, mutant mice showed reduced or absent otoacoustic emissions, suggesting cochlear outer hair cell impairment. Hearing loss in Csb(m/m) and Csa(-/-) mice correlated with progressive hair cell loss in the base of the organ of Corti, starting between 6 and 13 weeks of age, which increased by 16 weeks of age in a basal-to-apical gradient, with outer hair cells more severely affected than inner hair cells. Our data indicate that the hearing loss observed in CS patients is reproduced in mouse models of this disease. We hypothesize that accumulating DNA damage, secondary to the loss of TCR, contributes to susceptibility to hearing loss.

Spatio-temporal analysis of molecular determinants of neuronal degeneration in the aging mouse cerebellum.

The accumulation of cellular damage, including DNA damage, is hypothesized to contribute to aging-related neurodegenerative changes. DNA excision repair cross-complementing group 1 (Ercc1) knock-out mice represent an accepted model of neuronal aging, showing gradual neurodegenerative changes, including loss of synaptic contacts and cell body shrinkage. Here, we used the Purkinje cell-specific Ercc1 DNA-repair knock-out mouse model to study aging in the mouse cerebellum. We performed an in-depth quantitative proteomics analysis, using stable isotope dimethyl labeling, to decipher changes in protein expression between the early (8 weeks), intermediate (16 weeks), and late (26 weeks) stages of the phenotypically aging Ercc1 knock-out and healthy littermate control mice. The expression of over 5,200 proteins from the cerebellum was compared quantitatively, whereby 79 proteins (i.e. 1.5%) were found to be substantially regulated during aging. Nearly all of these molecular markers of the early aging onset belonged to a strongly interconnected network involved in excitatory synaptic signaling. Using immunohistological staining, we obtained temporal and spatial profiles of these markers confirming not only the proteomics data but in addition revealed how the change in protein expression correlates to synaptic changes in the cerebellum. In summary, this study provides a highly comprehensive spatial and temporal view of the dynamic changes in the cerebellum and Purkinje cell signaling in particular, indicating that synapse signaling is one of the first processes to be affected in this premature aging model, leading to neuron morphological changes, neuron degeneration, inflammation, and ultimately behavior disorders.

Age-related neuronal degeneration: complementary roles of nucleotide excision repair and transcription-coupled repair in preventing neuropathology.

Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR-deficient Csa(-/-) and Csb(-/-) CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER-deficient Xpa(-/-) and Xpc(-/-) XP mice, but also occurred in Xpd(XPCS) mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR-deficient mice are compatible with focal dysmyelination in CS patients. Both TCR-deficient and NER-deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa(-/-), Csb(-/-)) or highly sporadic (Xpa(-/-), Xpc(-/-)) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR-deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa(-/-) and Csb(-/-) TCR-deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR-deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival.

The Extracellular Matrix and Cardiac Pressure Overload: Focus on Novel Treatment Targets.

Heart failure is a significant health issue in developed countries, often stemming from conditions like hypertension, which imposes a pressure overload on the heart. Despite various treatment strategies for heart failure, many lack long-term effectiveness. A critical aspect of cardiac disease is the remodeling of the heart, where compensatory changes in the extracellular matrix exacerbate disease progression. This review explores the processes and changes occurring in the pressure-overloaded heart with respect to the extracellular matrix. It further summarizes current treatment strategies, and then focuses on novel treatment targets for maladaptive cardiac remodeling, derived from transverse aortic constriction-induced pressure overload animal models.

Self-adaptive deep learning-based segmentation for universal and functional clinical and preclinical CT image analysis.

BACKGROUND: Methods to monitor cardiac functioning non-invasively can accelerate preclinical and clinical research into novel treatment options for heart failure. However, manual image analysis of cardiac substructures is resource-intensive and error-prone. While automated methods exist for clinical CT images, translating these to preclinical µCT data is challenging. We employed deep learning to automate the extraction of quantitative data from both CT and µCT images. METHODS: We collected a public dataset of cardiac CT images of human patients, as well as acquired µCT images of wild-type and accelerated aging mice. The left ventricle, myocardium, and right ventricle were manually segmented in the µCT training set. After template-based heart detection, two separate segmentation neural networks were trained using the nnU-Net framework. RESULTS: The mean Dice score of the CT segmentation results (0.925 ± 0.019, n = 40) was superior to those achieved by state-of-the-art algorithms. Automated and manual segmentations of the µCT training set were nearly identical. The estimated median Dice score (0.940) of the test set results was comparable to existing methods. The automated volume metrics were similar to manual expert observations. In aging mice, ejection fractions had significantly decreased, and myocardial volume increased by age 24 weeks. CONCLUSIONS: With further optimization, automated data extraction expands the application of (µ)CT imaging, while reducing subjectivity and workload. The proposed method efficiently measures the left and right ventricular ejection fraction and myocardial mass. With uniform translation between image types, cardiac functioning in diastolic and systolic phases can be monitored in both animals and humans.

The mouse Social Frailty Index (mSFI): a novel behavioral assessment for impaired social functioning in aging mice.

Various approaches exist to quantify the aging process and estimate biological age on an individual level. Frailty indices based on an age-related accumulation of physical deficits have been developed for human use and translated into mouse models. However, declines observed in aging are not limited to physical functioning but also involve social capabilities. The concept of "social frailty" has been recently introduced into human literature, but no index of social frailty exists for laboratory mice yet. To fill this gap, we developed a mouse Social Frailty Index (mSFI) consisting of seven distinct assays designed to quantify social functioning which is relatively simple to execute and is minimally invasive. Application of the mSFI in group-housed male C57BL/6 mice demonstrated a progressively elevated levels of social frailty through the lifespan. Conversely, group-housed females C57BL/6 mice manifested social frailty only at a very old age. Female mice also showed significantly lower mSFI score from 10 months of age onward when compared to males. We also applied the mSFI in male C57BL/6 mice under chronic subordination stress and in chronic isolation, both of which induced larger increases in social frailty compared to age-matched group-housed males. Lastly, we show that the mSFI is enhanced in mouse models that show accelerated biological aging such as progeroid Ercc1-/Δ and Xpg-/- mice of both sexes compared to age matched littermate wild types. In summary, the mSFI represents a novel index to quantify trajectories of biological aging in mice and may help elucidate links between impaired social behavior and the aging process.