Neonatal Mice Research Articles

Effects of FGFR2b-ligand signaling on pancreatic branching morphogenesis and postnatal islet function.

Pancreatic development is a complex process vital for maintaining metabolic balance, requiring intricate interactions among different cell types and signaling pathways. Fibroblast growth factor receptors 2b (FGFR2b)-ligands signaling from adjacent mesenchymal cells is crucial in initiating pancreatic development and differentiating exocrine and endocrine cells through a paracrine mechanism. However, the precise critical time window that affects pancreatic development remains unclear. To explore the roles of FGFR2b-ligands and identify the narrow window of time during which FGFR2b-ligand signaling affects pancreatic development, we used an inducible mouse model to control the expression of soluble dominant-negative FGFR2b (sFGFR2b) at various stages of pancreatic development. Our findings revealed a significant effect of FGFR2b-ligand signaling on epithelial morphology, lumen formation, and pancreatic branching during primary and secondary transition stages. Additionally, sFGFR2b expression reduced the number of Pdx1+ progenitor cells and altered the pancreatic islet structure. Furthermore, we examined the effect of mutation in FGF10, an FGFR2b ligand, on embryonic pancreatic β-cell function. FGF10 null mutant embryos exhibited reduced pancreatic size and a decrease number of islet-like structure. Although neonatal mice with haploinsufficiency for FGF10 exhibited abnormal glucose tolerance test results, indicating a potential diabetes predisposition, these abnormalities normalized with age, aligning with observations in wild type mice. Our study underscores the critical role of FGFR2b-ligand signaling in pancreatic development and postnatal islet function, offering insights into potential therapeutic targets for pancreatic disorders.

Lipopolysaccharide differentially alters systemic and brain glucocorticoid levels in neonatal and adult mice.

Glucocorticoids (GCs) are secreted by the adrenal glands and increase in response to stressors (e.g., infection). The brain regulates local GC levels via GC synthesis, regeneration and/or metabolism. Little is known about local GC regulation within discrete brain regions at baseline or in response to stress. We treated male and female C57BL/6J mice at postnatal day 5 (PND5) or PND90 with lipopolysaccharide (LPS; 50 μg/kg bw i.p.) or vehicle and collected blood and brain after 4 h. We microdissected the prefrontal cortex, hippocampus, hypothalamus and amygdala. We measured seven steroids, including corticosterone, via liquid chromatography-tandem mass spectrometry and measured transcripts for key steroidogenic enzymes (Cyp11b1, Hsd11b1, Hsd11b2) via qPCR. At both ages, LPS increased GC levels in blood and all brain regions; however, the increases were much greater at PND90 than at PND5. Interestingly, PND5 corticosterone levels were lower in prefrontal cortex than in blood, but higher in amygdala than in blood. These changes in corticosterone levels align with local changes in steroidogenic enzyme expression, demonstrating robust regional heterogeneity and a possible mechanism for the region-specific effects of early-life stress. In contrast, PND90 corticosterone levels were lower in all brain regions than in blood and similar among regions, and steroidogenic enzyme mRNA levels were generally not affected by LPS. Together, these data indicate that local GC levels within discrete brain regions are more heterogeneous at baseline and in response to LPS at PND5 than at PND90, as a result of increased local GC production and metabolism in the neonatal brain.

Unveiling the Emerging Role of Extracellular Vesicle–Inflammasomes in Hyperoxia-Induced Neonatal Lung and Brain Injury

Extremely premature infants are at significant risk for developing bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment (NDI). Although BPD is a predictor of poor neurodevelopmental outcomes, it is currently unknown how BPD contributes to brain injury and long-term NDI in pre-term infants. Extracellular vesicles (EVs) are small, membrane-bound structures released from cells into the surrounding environment. EVs are involved in inter-organ communication in diverse pathological processes. Inflammasomes are large, multiprotein complexes that are part of the innate immune system and are responsible for triggering inflammatory responses and cell death. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly and activating inflammatory caspase-1. Activated caspase-1 cleaves gasdermin D (GSDMD) to release a 30 kD N-terminal domain that can form membrane pores, leading to lytic cell death, also known as pyroptosis. Activated caspase-1 can also cleave pro-IL-1β and pro-IL-18 to their active forms, which can be rapidly released through the GSDMD pores to induce inflammation. Recent evidence has emerged that activation of inflammasomes is associated with neonatal lung and brain injury, and inhibition of inflammasomes reduces hyperoxia-induced neonatal lung and brain injury. Additionally, multiple studies have demonstrated that hyperoxia stimulates the release of lung-derived EVs that contain inflammasome cargos. Adoptive transfer of these EVs into the circulation of normal neonatal mice and rats induces brain inflammatory injury. This review focuses on EV–inflammasomes’ roles in mediating lung-to-brain crosstalk via EV-dependent and EV-independent mechanisms critical in BPD, brain injury, and NDI pathogenesis. EV–inflammasomes will be discussed as potential therapeutic targets for neonatal lung and brain injury.

Reduced Neurite Arborization in Primary Dopaminergic Neurons in Autism-Like Shank3B-Deficient Mice.

Despite many studies on dopamine changes in autism, specific alterations in midbrain dopamine neurons projecting to the striatum and cortex remain unclear. Mouse models with diverse SH3 domain and ankyrin repeat containing protein 3 (Shank3) deficiencies are used for investigating autistic symptoms and underlying neurobiological mechanisms. SHANK3 belongs to postsynaptic proteins crucial for synapse formation during development, and disruptions in SHANK3 structure could lead to impaired neurite outgrowth and altered dendritic arborization and morphology. Therefore, we aimed to investigate whether Shank3 deficiency (Shank3B) leads to changes in the morphology of primary neuronal cell cultures from dopaminergic brain regions of neonatal mouse pups and whether it results in alterations in synaptic proteins in dopaminergic nerve pathway projection areas (striatum, frontal cortex). Significantly reduced neurite outgrowth was observed in primary dopaminergic neurons from the midbrain and striatum of Shank3-deficient compared to WT mice. A decrease in Synapsin I immunofluorescence signal in the cortical neurons isolated from Shank3-deficient mice was found, although neurite arborization changes were less severe. Importantly, the deficit in the length of the longest neurite was confirmed in primary cortical neurons isolated from Shank3-deficient mice. No changes in the gene expression of synaptic proteins were observed in the striatum and frontal cortex of Shank3-deficient mice, but an altered gene expression profile of dopaminergic receptors was found. These results show structural changes of dopaminergic neurons, which may explain autistic symptomatology in the used model and provide a basis for understanding the long-term development of autistic symptoms.

Nicotinamide mononucleotide protects septic hearts in mice via preventing cyclophilin F modification and lysosomal dysfunction.

Myocardial dysfunction is a decisive factor of death in septic patients. Cyclophilin F (PPIF) is a major component of the mitochondrial permeability transition pore (mPTP) and acts as a critical mPTP sensitizer triggering mPTP opening. In sepsis, decreased NAD+ impairs Sirtuin 3 function, which may prevent PPIF de-acetylation. Repletion of NAD+ with nicotinamide mononucleotide (NMN) reduces myocardial dysfunction in septic mice. In addition, administration of the mPTP inhibitor cyclosporine-A attenuated sepsis-induced myocardial dysfunction, and deletion of PPIF reduced lung and liver injuries in sepsis, leading to increased survival. It is plausible that NAD+ repletion with NMN may prevent mPTP opening in protecting septic hearts through PPIF de-acetylation and/or inhibition of mitochondrial ROS-mediated PPIF oxidation. In this study we investigated how NMN alleviated myocardial dysfunction in septic mice. Sepsis was induced in mice by injection of LPS (4 mg/kg, i.p.). Then mice received NMN (500 mg/kg, i.p.) or mito-TEMPO (0.7 mg/kg, i.p.) right after LPS injection, and subjected to echocardiography for assessing myocardial function. At the end of experiment, the heart tissues and sera were collected for analyses. In vitro experiments were conducted in neonatal mouse cardiomyocytes treated with LPS (1 µg/mL) in the presence of NMN (500 µmol/L) or mito-TEMPO (25 nmol/L). We showed that LPS treatment markedly increased mitochondrial ROS production and induced lysosomal dysfunction and aberrant autophagy in cardiomyocytes and mouse hearts, leading to inflammatory responses and myocardial injury and dysfunction in septic mice. NMN administration attenuated LPS-induced deteriorative effects. Selective inhibition of mitochondrial superoxide production with mito-TEMPO attenuated lysosomal dysfunction and aberrant autophagy in septic mouse hearts. Notably, LPS treatment significantly increased acetylation and oxidation of PPIF, which was prevented by NMN in mouse hearts. Knockdown of PPIF replicated the beneficial effects of NMN or mito-TEMPO on ROS production, lysosomal dysfunction, aberrant autophagy, and myocardial injury/dysfunction in sepsis. In addition, administration of NMN abrogated LPS-induced ATP5A1 acetylation and increased ATP5A1 protein levels and ATP production in septic mouse hearts. This study demonstrates that NMN modulates the interplay of mitochondrial ROS and PPIF in maintaining normal lysosomal function and autophagy and protecting ATP5A1 and ATP production during sepsis.

Inhibition of lysophosphatidic acid receptor 2 attenuates neonatal chronic lung disease in mice by preserving vascular and alveolar development

AimBronchopulmonary dysplasia (BPD) is a common morbidity in extremely premature infants. Previous studies demonstrated the important role of lysophosphatidic acid (LPA) in inflammation in BPD. However, the role of LPA and its receptors in hyperoxia-induced vascular malformations in BPD remains to be elucidated. Methods and ResultsElevated plasma LPA levels were observed in mice with BPD compared to controls (792 vs. 607 ng/mL, p < 0.05). Inhibition of LPA signaling protected against hyperoxia-induced lung injury in neonatal mice, demonstrated by a 2.8-fold increase in pulmonary vascular density and a 14% reduction in alveolar enlargement. In vitro studies showed that LPA suppressed tube formation in human umbilical vein endothelial cells (HUVECs) by approximately 50%. LPA receptor 2 (LPA2) was identified as a functional LPA receptor in primary endothelial cells from the lungs of hyperoxic mice and in HUVECs under hyperoxic conditions. The LPA2 antagonist H2L5186303 enhanced the tube formation ability of HUVECs exposed to LPA, both under normoxia (4-fold) and hyperoxia (5-fold). Moreover, H2L5186303 significantly protected against hyperoxia-induced vascular malformation (2-fold) and improved alveolarization in neonatal mice (12% decrease in mean linear intercept, MLI). Early growth response 1 (EGR1) was characterized as a downstream target of LPA2, silencing EGR1 restored tube formation in HUVECs exposed to LPA and hyperoxia. ConclusionsOur in vitro and in vivo findings demonstrate that the inhibition of LPA/LPA2 signaling mitigates hyperoxia-induced pulmonary vascular malformations, suggesting the LPA/LPA2-dependent signaling pathway has therapeutic potential for extremely premature infants with BPD.

Maternal Lactobacillus johnsonii supplementation attenuates hyperoxia-induced lung injury in neonatal mice through microbiota regulation

Maternal Lactobacillus johnsonii supplementation attenuates hyperoxia-induced lung injury in neonatal mice through microbiota regulation

Cardiomyocyte-derived small extracellular vesicle-transported let-7b-5p modulates cardiac remodeling viaTLR7 signaling pathway.

Cardiac remodeling is the major pathological change of heart failure. And let-7 family has been implicated in the development and pathogenesis of cardiovascular diseases. However, the mechanisms underlying let-7b-5p-mediated cellular pathogenesis of cardiac remodeling are not well understood. The present study aimed to explore the effects of let-7b-5p on cardiac remodeling and the corresponding regulatory mechanism. Invivo results indicated that cardiac let-7b-5p was upregulated in the mouse model of Angiotensin II (Ang II)-induced cardiac remodeling. Additionally, let-7b-5p knockdown ameliorated the effects of Ang II-induced cardiac remodeling, whereas let-7b-5p overexpression facilitated cardiac remodeling. Invitro, let-7b-5p mimics induced cardiomyocyte hypertrophy, fibroblast transdifferentiation, and the expression of inflammatory factors in neonatal mouse cardiomyocytes (NMCMs), neonatal mouse cardiac fibroblasts (NMCFs), and bone marrow-derived macrophages (BMDMs), respectively. Furthermore, let-7b-5p exerted its cardiac pro-remodeling effects at least partially through a small extracellular vesicle (SEV)-based delivery strategy. We found that let-7b-5p was enriched in SEVs derived from Ang II-treated NMCMs (NMCM-SEV) but not from Ang II-treated NMCFs (NMCF-SEV). Mechanistic analyses revealed that NMCM-SEV promoted TLR7 and MyD88 expression, which increased NF-κB phosphorylation levels. Knockdown of let-7b-5p, TLR7 or MyD88 in NMCMs, NMCFs, and BMDMs abolished the cardiac pro-remodeling effects of NMCM-SEV. These results uncover that let-7b-5p-containing NMCM-SEVs promote cardiac remodeling via the TLR7/MyD88/NF-κB pathway, implicating let-7b-5p as a potential therapeutic target for heart failure.

Right ventricular volume overload reboots cardiomyocyte proliferation via immune responses

BackgroundRight ventricular volume overload (RVVO) is one of the most important hemodynamic characteristics in children with congenital heart disease (CHD) and heart failure, and cardiomyocyte (CM) proliferation is one of the most vital factors for improving cardiac performance. However, whether and how RVVO reboots CM proliferation remains elusive.Methods and resultsWe first created a neonatal RVVO mouse model via abdominal aorta and inferior vena cava-fistula microsurgery at postnatal day 7 (P7), the edge of CM proliferation window. We subsequently performed bulk RNA-seq, single cell RNA-seq/flow cytometry, and immunofluorescence staining on the right ventricles (RV) of RVVO mice at P14/P21, defined as prepubertal stage, revealing that RVVO temporarily reboots prepubertal CM proliferation via immune responses.ConclusionsIn considering the importance of RVVO and CM proliferation, this study may bring an opportunity to create a novel paradigm to treat pediatric CHDs or heart failure.Graphical

The Shh-p38-NFATc1 signaling pathway is essential for osteoclastogenesis during tooth eruption

Tooth eruption, a critical stage in tooth development, is related to osteoclastogenesis. Intraperitoneal injection of Shh agonists into neonatal mice promoted tooth eruption at postnatal day (PN) 15, whereas treatment with the Shh inhibitor (LDE225) suppressed this process. When RAW264.7 osteoclast precursor cells were treated with RANKL, NFATc1 translocated from the cytoplasm to the nucleus and induced cell differentiation into TRAP+ osteoclasts; this process was activated by Shh but inhibited by LDE225. Treating RAW264.7 cells with the p38 inhibitor, BIRB796, also inhibited NFATc1 nuclear localization. p-p38 expression in the alveolar bone of PN3 and PN5 mice was decreased by treatment with LDE225, and RAW264.7 cell differentiation was reduced by BIRB796, regardless of treatment with Shh. Furthermore, Shh activated p38 signaling pathway in RAW264.7 cells, while p38 phosphorylation was reduced by LDE225, which ultimately inhibited osteoclast precursor differentiation. Therefore, we concluded that Shh promotes osteoclast precursor differentiation via the p38-NFATc1 signaling pathway.

Ciliopathy interacts with neonatal anesthesia to cause non-apoptotic caspase-mediated motor deficits.

Increasing evidence suggests that anesthesia may induce developmental neurotoxicity, yet the influence of genetic predispositions associated with congenital anomalies on this toxicity remains largely unknown. Children with congenital heart disease often exhibit mutations in cilia-related genes and ciliary dysfunction, requiring sedation for their catheter or surgical interventions during the neonatal period. Here we demonstrate that briefly exposing ciliopathic neonatal mice to ketamine causes motor skill impairments, which are associated with a baseline deficit in neocortical layer V neuron apical spine density and their altered dynamics during motor learning.. These neuromorphological changes were linked to augmented non-apoptotic neuronal caspase activation. Neonatal caspase suppression rescued the spine density and motor deficits, confirming the requirement for sublethal caspase signaling in appropriate spine formation and motor learning. Our findings suggest that ciliopathy interacts with ketamine to induce motor impairments, which is reversible through caspase inhibition. Furthermore, they underscore the potential for ketamine- induced sublethal caspase responses in shaping neurodevelopmental outcomes.

Impact of vitamin D on hyperoxic acute lung injury in neonatal mice

BackgroundProlonged exposure to hyperoxia can lead to hyperoxic acute lung injury (HALI) in preterm neonates. Vitamin D (VitD) stimulates lung maturation and acts as an anti-inflammatory agent. Our objective was to determine if VitD provides a dose-dependent protective effect against HALI by reducing inflammatory cytokine expression and improving alveolarization and lung function in neonatal mice.MethodsC57BL/6 mouse neonates were randomized and placed in room air or hyperoxic (85% O2) conditions for 6 days. Control, low (5 ng/neonate) and high (25 ng/neonate) doses of VitD were administered daily beginning at day 2 via oral gavage. Lung tissue was analyzed for edema, changes in pulmonary structure and function, and inflammatory cytokine expression.ResultsNeonatal mice treated with VitD in hyperoxic conditions had improved weight gain, reduced pulmonary edema and increased alveolar surface area compared to untreated pups in hyperoxia. No significant changes in cytokine expression were observed between untreated and VitD neonates. While changes in surfactant protein mRNA expression were impacted by hyperoxia and VitD administration, no significant changes in alveolar type II cell percentages were observed. At 3 weeks, mice in hyperoxia treated with VitD had greater lung compliance, diminished airway reactivity and improved weight gain.ConclusionsHigh dose VitD significantly limited harmful effects of HALI. These results suggest that supplementation of VitD to neonatal mice during hyperoxia exposure provides both short-term and long-term protective benefits against HALI.

IL-17A is a key regulator of neuroinflammation and neurodevelopment in cognitive impairment induced by sevoflurane

Increasing numbers of animal studies have shown that repeat sevoflurane exposure during developmental stage may lead to long-term cognitive impairment. Nevertheless, the exact pathogenesis remains unclear. Interleukin 17A (IL-17A) has been associated with cognitive decline in various neurological disorders. Here we found that the expression of IL-17A was up-regulated in hippocampus of sevoflurane exposed neonatal mice. Genetic deletion of IL-17A or inhibition of IL-17A improved behavioral function and down-regulated neuroinflammation related genes, interleukin 1β (IL-1β), interleukin 6 (IL-6), Nicotinamide adenine dinucleotide phosphate(NADPH) oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4) in hippocampus of sevoflurane exposed neonatal mice. Moreover, negative regulation of IL-17A/Interleukin 17A receptor(IL-17RA) promoted the extracellular signal-regulated protein kinase (ERK) signaling pathway and nucleation of cyclic adenosine monophosphate (cAMP) response element-binding (CREB) in neurons of cognitive impaired mice. Knockdown of IL-17A in vivo identified neurons-localized IL-17A as a major factor in neuroinflammation and neurodevelopment. Collectively, our results suggested that IL-17A was required for the pathogenesis of neuroinflammatory response and identify IL-17A as a potential therapeutic target for cognitive impairment exposed by general anesthetics during infancy.

Cardiac effects of OPA1 protein promotion in a transgenic animal model.

Mitochondria form a dynamic network in cells, regulated by the balance between mitochondrial fusion and fission. The inhibition of mitochondrial fission can have positive effects in acute ischemic/reperfusion injury models by preventing the fall in mitochondrial membrane potential associated with fission processes. However, inhibition of fission in chronic models is disadvantageous because it obstructs the elimination of damaged mitochondrial fragments. OPA1, in view of previous results, is a possible therapeutic target as a fusion promoter and structure stabilizer protein. We used transgenic mice in which the OMA1 cleavage sites of OPA1 were deleted. This resulted in a higher representation of L-OPA1 compared to S-OPA1. After genotyping and model validation, all animals were examined by echocardiograph on two occasions, at weeks 11 and 36. Histological samples were taken from hearts to examine mitochondrial morphology and structure remodeling. The signaling pathways related to mitochondrial dynamic processes were evaluated. Cardiomyocytes were isolated from neonatal mice to determine the efficiency of mitochondrial respiration using the SeaHorse assay method. OPA1 protein promotion has a negative effect on systolic function during aging. We confirmed that volume overload and ventricular remodeling did not manifest. The reason behind the loss of pump function might be, at least partly, due to the energy deficit caused by mitochondrial respiratory failure and damage in mitochondrial quality control pathways.

Quercetin alleviates neonatal hypoxic-ischaemic brain injury by rebalancing microglial M1/M2 polarization through silent information regulator 1/ high mobility group box-1 signalling.

Neonatal hypoxic-ischaemic encephalopathy (HIE) remains one of the major causes of neonatal death and long-term neurological disability. Due to its complex pathogenesis, there are still many challenges in its treatment. In our previous studies, we found that quercetin can alleviate neurological dysfunction after hypoxic-ischaemic brain injury (HIBI) in neonatal mice. As demonstrated through in vitro experiments, quercetin can inhibit the activation of the TLR4/MyD88/NF-κB signalling pathway and the inflammatory response in the microglial cell line BV2 after oxygen-glucose deprivation. However, the in-depth mechanism still needs to be further elucidated. In the present study, 7day-old neonatal ICR mice or BV2 cells were treated with quercetin with or without the SIRT1 inhibitor EX527 via neurobehavioural, histopathological and molecular methods. In vivo experiments have shown that quercetin can significantly improve the performance of HI mice in behavioural tests, such as the Morris water maze, rotarod test and pole climbing test, and reduce HI insult-induced structural brain damage, cell apoptosis and hippocampal neuron loss. Quercetin also inhibited the immunofluorescence intensity of the microglial M1 marker CD16 + 32 and significantly downregulated the expression of the M1-related proteins iNOS, IL-1β and TNF-α. Moreover, quercetin increased the immunofluorescence intensity of the microglial M2 marker CD206 and significantly increased the expression of the M2-related proteins Arg-1 and IL-10. In addition, quercetin limits the nucleocytoplasmic translocation and release of microglial HMGB1 and further suppresses the activation of the downstream TLR4/MyD88/NF-κB signalling pathway. The above effects of quercetin are partially weakened by pretreatment with EX527. Similar results were found in in vitro experiments, and the mechanism further revealed that the rebalancing effect of quercetin on microglial polarization is achieved through the SIRT1-mediated reduction in HMGB1 acetylation levels. This study provides new and complementary insights into the neuroprotective effects of quercetin and a new direction for the treatment of neonatal HIE.

Inhibition of PAD4-mediated neutrophil extracellular traps formation attenuates hypoxic–ischemic brain injury in neonatal mice

Neonatal hypoxic–ischemic encephalopathy (HIE) is the primary cause of neonatal mortality and severe neurological sequelae. The interaction of neuroinflammation with the immune system represents a significant pathological mechanism underlying the development of HIE. Neutrophil extracellular traps (NETs) are a recently identified antimicrobial mechanism utilized by neutrophils. NETs can act as damage-associated molecular patterns, thereby amplifying the immune response and exerting proinflammatory effects. However, further research is needed to elucidate their role in the pathogenesis of HIE. In this study, we investigated the role of NETs in a hypoxic–ischemic brain injury (HIBI) model. We first reported that a pharmacological intervention to inhibit peptidylarginine deiminase type IV (PAD4) may constitute an effective strategy for reducing HI insult-induced neuroinflammation, neuronal apoptosis, and brain tissue destruction while also enhancing long-term neurobehavioral function in mice. These results support a pathological role for NETs in HIBI, and targeting PAD4 is a potential direction for the treatment of HIE.

POU3F4 up-regulates Gli1 expression and promotes neuronal differentiation and synaptic development of hippocampal neural stem cells

BackgroundNeural stem cells (NSCs) are considered to be the most promising cell type for cell replacement therapy in neurodegenerative diseases. However, their low neuronal differentiation ratio impedes their application in such conditions. Elucidating the molecular mechanism of NSC differentiation may provide the necessary experimental basis for expanding their application. Previous studies have indicated that POU3F4 can induce neuronal differentiation of NSCs, this study aims to underly the possible exact mechanism of POU3F4 on the NSC differentiation and development.MethodsNSCs were isolated and cultured from the hippocampus of neonatal mice. The frozen hippocampal sections were prepared for immunohistochemical staining. Synaptic development was assessed using electron microscopy. High-throughput sequencing was employed to analyze the gene expression profile following the overexpression of Brn4. Gene expression levels were determined through Western blotting and qRT-PCR. Cell cycle and differentiation were evaluated using flow cytometry and immunofluorescent staining.ResultsIt was found that POU3F4 promoted the neuronal differentiation of hippocampal NSCs and synapse development, and inhibited NSC proliferation. POU3F4-deficient mice exhibited impairments in learning and memory. RNA sequencing and ChIP assays confirmed that Gli1 was downstream of POU3F4. Loss and gain function experiments indicated that Gli1 mediated POU3F4 promoting neuronal differentiation and synapse development. Forced expression of Gli1 in hippocampus improved learning and memory function of animal models.ConclusionsThe results suggest that POU3F4 and Gli1 promote neuronal differentiation and synaptic development of NSCs, and that Gli1 partially mediates the effects of POU3F4.Graphical abstract

Postnatal maturation of serotonergic modulation of spinal RORβ interneurons in the medial deep dorsal horn.

Proprioceptive input is essential for coordinated locomotion and this input must be properly gated to ensure smooth and effective movement. Presynaptic inhibition mediated by GABAergic interneurons provides regulation of sensory afferent feedback. Serotonin not only promotes locomotion, but also modulates feedback from sensory afferents, both directly and indirectly, potentially by acting on the GABAergic interneurons that mediate presynaptic inhibition. Developmental disruptions in presynaptic inhibition can produce deficits in sensorimotor processing. Importantly, both presynaptic inhibition of proprioceptive afferents and serotonergic innervation of the spinal cord become mature and functional after the first postnatal week. However, little is known about the serotonergic receptors involved in the modulation of interneurons mediating presynaptic inhibition and when developmentally their actions mature. Here, we used whole-cell patch clamp recordings in lumbar spinal slices from neonatal and juvenile mice to assess the intrinsic properties and serotonergic modulation of deep dorsal horn GABAergic RORβ interneurons previously shown to mediate presynaptic inhibition of proprioceptive afferents. RORβ interneurons from juvenile cords displayed more mature membrane properties. Further, serotonin increased the excitability of RORβ interneurons via actions at 5-HT 2A , 5-HT 2B/2C , and 5-HT 7 receptors in juvenile but not early neonatal spinal cords. Our findings indicate that deep dorsal horn RORβ interneurons undergo postnatal maturation in both their intrinsic excitability and ability to respond to serotonin, concurrent with the maturation of serotonergic innervation of the dorsal horn. This information can prompt future targeted studies testing relationships between impairments of serotonergic development, proprioceptive processing disorders, and presynaptic inhibition mediated by RORβ interneurons.

N6-methyladenosine modification of SPOP relieves ferroptosis and diabetic cardiomyopathy by enhancing ubiquitination of VDAC3

Understanding the pathogenesis of diabetic cardiomyopathy (DCM), a common microvascular complication affecting the heart, is crucial for identifying new therapeutic targets and intervention strategies for DCM. Our study revealed a significant downregulation in Speckle-type POZ protein (SPOP) expression in DCM, while the overexpression of SPOP improved DCM-induced myocardial dysfunction, injury, fibrosis, hypertrophy, and ferroptosis. Mechanistically, SPOP facilitated the degradation of voltage-dependent anion channel 3 (VDAC3) by enhancing its ubiquitination. M6A demethylase AlkB homolog 5 (ALKBH5) reduced the mRNA stability of SPOP by decreasing m6A modification in its 3′UTR. The m6A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced the stability of SPOP mRNA through recognition of m6A-modified SPOP 3′UTR. Furthermore, ALKBH5 promoted ferroptosis by inhibiting SPOP-induced VDAC3 degradation, while IGF2BP2 inhibited ferroptosis via activation of SPOP-induced VDAC3 degradation in high glucose-treated neonatal mouse ventricular cardiomyocytes (NMVCs). Overall, our study has unveiled a novel role of SPOP in the pathogenesis of ferroptosis and DCM, thereby significantly advancing our understanding of the involvement of ferroptosis during the progression of DCM. Moreover, this discovery offers promising potential therapeutic interventions targeting DCM.

Pharmacogenomic screening identifies and repurposes leucovorin and dyclonine as pro-oligodendrogenic compounds in brain repair

Oligodendrocytes are critical for CNS myelin formation and are involved in preterm-birth brain injury (PBI) and multiple sclerosis (MS), both of which lack effective treatments. We present a pharmacogenomic approach that identifies compounds with potent pro-oligodendrogenic activity, selected through a scoring strategy (OligoScore) based on their modulation of oligodendrogenic and (re)myelination-related transcriptional programs. Through in vitro neural and oligodendrocyte progenitor cell (OPC) cultures, ex vivo cerebellar explants, and in vivo mouse models of PBI and MS, we identify FDA-approved leucovorin and dyclonine as promising candidates. In a neonatal chronic hypoxia mouse model mimicking PBI, both compounds promote neural progenitor cell proliferation and oligodendroglial fate acquisition, with leucovorin further enhancing differentiation. In an adult MS model of focal de/remyelination, they improve lesion repair by promoting OPC differentiation while preserving the OPC pool. Additionally, they shift microglia from a pro-inflammatory to a pro-regenerative profile and enhance myelin debris clearance. These findings support the repurposing of leucovorin and dyclonine for clinical trials targeting myelin disorders, offering potential therapeutic avenues for PBI and MS.