Raisa Serpi

Transmembrane prolyl 4-hydroxylase (P4H-TM) is an enigmatic enzyme whose cellular function and primary substrate remain to be identified. Its loss-of-function mutations cause a severe neurological HIDEA syndrome with hypotonia, intellectual disability, dysautonomia and hypoventilation. Previously, P4H-TM deficiency in mice was associated with reduced atherogenesis and lower serum triglyceride levels. Here, we characterized the glucose and lipid metabolism of P4h-tm^−/− mice in physiological and tissue analyses. P4h-tm^−/− mice showed variations in 24-h oscillations of energy expenditure, VO₂ and VCO₂ and locomotor activity compared to wild-type (WT) mice. Their rearing activity was reduced, and they showed significant muscle weakness and compromised coordination. Sedated P4h-tm^−/− mice had better glucose tolerance, lower fasting insulin levels, higher fasting lactate levels and lower fasting free fatty acid levels compared to WT. These alterations were not present in conscious P4h-tm^−/− mice. Fasted P4h-tm^−/− mice presented with faster hepatic glycogenolysis. The respiratory rate of conscious P4h-tm^−/− mice was significantly lower compared to the WT, the decrease being further exacerbated by sedation and associated with acidosis and a reduced ventilatory response to both hypoxia and hypercapnia. P4H-TM deficiency in mice is associated with alterations in whole-body energy metabolism, day-night rhythm of activity, glucose homeostasis and neuromuscular and respiratory functions. Although the underlying mechanism(s) are not yet fully understood, the phenotype appears to have neurological origins, controlled by brain and central nervous system circuits. The phenotype of P4h-tm^−/− mice recapitulates some of the symptoms of HIDEA patients, making this mouse model a valuable tool to study and develop tailored therapies.

Introduction: A better understanding of the earliest stages of Alzheimer’s disease (AD) could expedite the development or administration of treatments. Large population biobanks hold the promise to identify individuals at an elevated risk of AD and related dementias based on health registry information. Here, we establish the protocol for an observational clinical recall and biomarker study called TWINGEN with the aim to identify individuals at high risk of AD by assessing cognition, health and AD-related biomarkers. Suitable candidates were identified and invited to participate in the new study among Finnish biobank donors according to TWINGEN study criteria. Methods and analysis: A multi-center study (n=800) to obtain blood-based biomarkers, telephone-administered and web-based memory and cognitive parameters, questionnaire information on lifestyle, health and psychological factors, and accelerometer data for measures of physical activity, sedentary behavior and sleep. A sub-cohort are being asked to participate in an in-person neuropsychological assessment (n=200) and wear an Oura ring (n=50). All participants in the TWINGEN study have genome-wide genotyping data and up to 48 years of follow-up data from the population-based older Finnish Twin Cohort (FTC) study of the University of Helsinki. TWINGEN data will be transferred to Finnish Institute of Health and Welfare (THL) biobank and we aim to further to transfer it to the FinnGen study where it will be combined with health registry data for prediction of AD. Ethics and dissemination: This recall study consists of FTC/THL/FinnGen participants whose data were acquired in accordance with the Finnish Biobank Act. The recruitment protocols followed the biobank protocols approved by Finnish Medicines Agency. The TWINGEN study plan was approved by the Ethics Committee of Hospital District of Helsinki and Uusimaa (number 16831/2022). THL Biobank approved the research plan with the permission no: THLBB2022_83. Article summary: Strengths and limitations of this study: A large sample of individuals is recruited from a representative biobank database Using health registry information, we exclude those with documented AD or other neurological or psychiatric diseases that can affect cognition. Pre-screening limits the sending of unnecessary invitations and saves costs Participants have up to 48 years of follow-up questionnaire and clinical data from the Finnish Twin Cohort study and these data can be combined with multifaceted Finnish health registry information. Previous genotype data is available in the biobank from all TWINGEN study participants. We assess the feasibility of remote cognitive testing and blood samples in large-scale screening of AD risk, translating to the requirements of intervention trials and clinical practice Limitations of the study are a lack of gold standard biomarkers (cerebrospinal fluid, positron emission tomography imaging) and neurological examinations

Activation of the hypoxia-inducible factor (HIF) pathway reprograms energy metabolism. Hemoglobin (Hb) is the main carrier of oxygen. Using its normal variation as a surrogate measure for hypoxia, we explored whether lower Hb levels could lead to healthier metabolic profiles in mice and humans (n = 7175) and used Mendelian randomization (MR) to evaluate potential causality (n = 173,480). The results showed evidence for lower Hb levels being associated with lower body mass index, better glucose tolerance and other metabolic profiles, lower inflammatory load, and blood pressure. Expression of the key HIF target genes SLC2A4 and Slc2a1 in skeletal muscle and adipose tissue, respectively, associated with systolic blood pressure in MR analyses and body weight, liver weight, and adiposity in mice. Last, manipulation of murine Hb levels mediated changes to key metabolic parameters. In conclusion, low-end normal Hb levels may be favorable for metabolic health involving mild chronic activation of the HIF response.

Objective: Atherosclerosis is a key component of cardiovascular diseases. We set out to study here whether genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) could protect against atherosclerosis as does inhibition of the other 3 classical HIF-P4Hs (hypoxia-inducible factor prolyl 4-hydroxylases). Approach and Results: We generated a double knockout mouse line deficient in P4H-TM and LDL (low-density lipoprotein) receptor ( P4h-tm ^−/− /Ldlr ^−/− ) and subjected these mice to a high-fat diet for 13 weeks. The double knockout mice had less atherosclerotic plaques in their full-length aorta than their P4h-tm ^+/+ /Ldlr ^−/− counterparts and also had lower serum triglyceride levels on standard laboratory diet and high-fat diet, higher levels of IgM autoantibodies against Ox-LDL (oxidized LDL), and significantly higher LPL (lipoprotein lipase) protein levels in white adipose tissue and sera. RNA-sequencing analysis revealed changes in expression of mRNAs in multiple pathways including lipid metabolism and immunologic response in the P4h-tm ^−/− / Ldlr ^−/− livers as compared with P4h-tm ^+/+ / Ldlr ^−/− . Conclusions: Our data identify P4H-TM inhibition as a potential novel immuno-metabolic mechanism for intervening in the pathology of atherosclerosis, as hypertriglyceridemia is an individual risk factor for atherosclerosis, and IgM antibodies to Ox-LDL and increased lipoprotein lipase have been associated with protection against it.

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4–NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4–NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II -mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro- and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4–NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries.

Identification of the adult cardiac stem cells (CSCs) has offered new therapeutic possibilities for treating ischemic myocardium. CSCs positive for the cell surface antigen c-Kit are known as the primary source for cardiac regeneration. Accumulating evidence shows that chemokines play important roles in stem cell homing. Here we investigated molecular targets to be utilized in modulating the mobility of endogenous CSCs. In a four week follow-up after experimental acute myocardial infarction (AMI) with ligation of the left anterior descending (LAD) coronary artery of Sprague-Dawley rats c-Kit+ CSCs redistributed in the heart. The number of c-Kit+ CSCs in the atrial c-Kit niche was diminished, whereas increased amount was observed in the left ventricle and apex. This was associated with increased expression of stromal cell-derived factor 1 alpha (SDF1α), and a significant positive correlation was found between c-Kit+ CSCs and SDF1α expression in the heart. Moreover, the migratory capacity of isolated c-Kit+ CSCs was induced by SDF1 treatment in vitro. We conclude that upregulation of SDF1α after AMI associates with increased expression of endogenous c-Kit+ CSCs in the injury area, and show induced migration of c-Kit+ cells by SDF1.

Transcription factors are pivotal regulators of gene transcription, and many diseases are associated with the deregulation of transcriptional networks. In the heart, the transcription factors GATA4 and NKX2-5 are required for cardiogenesis. GATA4 and NKX2-5 interact physically, and the activation of GATA4, in cooperation with NKX2-5, is essential for stretch-induced cardiomyocyte hypertrophy. Here, we report the identification of four small molecule families that either inhibit or enhance the GATA4–NKX2-5 transcriptional synergy. A fragment-based screening, reporter gene assay, and pharmacophore search were utilized for the small molecule screening, identification, and optimization. The compounds modulated the hypertrophic agonist-induced cardiac gene expression. The most potent hit compound, N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide (3, IC₅₀ = 3 μM), exhibited no activity on the protein kinases involved in the regulation of GATA4 phosphorylation. The identified and chemically and biologically characterized active compound, and its derivatives may provide a novel class of small molecules for modulating heart regeneration.

Erythrocytosis is mainly driven by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2/PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable for extramedullary erythropoiesis. We studied HIF-P4H-2 deficient mice (Hif-p4h-2^gt/gt) which show slightly induced erythropoiesis upon aging despite non-increased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased BFU-E growth and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligands and target Jag1, Jag2, Dll1 and Hes1 became downregulated upon aging HIF-2α-dependently. Inhibition of notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling.