Developmental iron deficiency dysregulates TET activity and DNA hydroxymethylation in the rat hippocampus and cerebellum

Iron deficiency (ID) during neurodevelopment is associated with lasting cognitive and socioemotional deficits, and increased risk for neuropsychiatric disease throughout the lifespan. These neurophenotypical changes are underlain by gene dysregulation in the brain that outlasts the period of ID; however, the mechanisms by which ID establishes and maintains gene expression changes are incompletely understood. The epigenetic modification 5-hydroxymethylcytosine (5hmC), or DNA hydroxymethylation, is one candidate mechanism because of its dependence on iron-containing TET enzymes. The aim of the present study was to determine the effect of fetal-neonatal ID on regional brain TET activity, Tet expression, and 5hmC in the developing rat hippocampus and cerebellum, and to determine whether changes are reversible with dietary iron treatment. Timed pregnant Sprague-Dawley rats were fed iron deficient diet (ID; 4 mg/kg Fe) from gestational day (G)2 to generate iron deficient anemic (IDA) offspring. Control dams were fed iron sufficient diet (IS; 200 mg/kg Fe). At postnatal day (P)7, a subset of ID-fed litters was randomized to IS diet, generating treated IDA (TIDA) offspring. At P15, hippocampus and cerebellum were isolated for subsequent analysis. TET activity was quantified by ELISA from nuclear proteins. Expression of Tet1, Tet2, and Tet3 was quantified by qPCR from total RNA. Global %5hmC was quantified by ELISA from genomic DNA. ID increased DNA hydroxymethylation (p=0.0105), with a corresponding increase in TET activity (p<0.0001) and Tet3 expression (p<0.0001) in the P15 hippocampus. In contrast, ID reduced TET activity (p=0.0016) in the P15 cerebellum, with minimal effect on DNA hydroxymethylation. Neonatal dietary iron treatment resulted in partial normalization of these changes in both brain regions. These results demonstrate that the TET/DNA hydroxymethylation system is disrupted by developmental ID in a brain region-specific manner. Differential regional disruption of this epigenetic system may contribute to the lasting neural circuit dysfunction and neurobehavioral dysfunction associated with developmental ID.

Promotion of the Differentiation of Dental Pulp Stem Cells into Oligodendrocytes by Knockdown of Heat-Shock Protein 27

Stem cell-based therapy has been evaluated in many different clinical trials for various diseases. This capability was applied in various neurodegenerative diseases, such as Alzheimer’s disease, which is characterized by synaptic damage accompanied by neuronal loss. Dental pulp stem cells (DPSCs) are mesenchymal stem cells from the oral cavity and have been studied with potential application for regeneration of different tissues. Heat shock protein 27 (HSP27) is known to regulate neurogenesis in the process of neural differentiation of placenta-multipotent stem cells. Here, we hypothesize that HSP27 expression is also critical in neural differentiation of DPSCs. An evaluation of the possible role of HSP27 in differentiation of DPSCs was per-formed by gene knockdown and neural immunofluorescent staining. We found that HSP27 has a role in the differentiation of DPSCs and that knockdown of HSP27 in DPSCs renders cells to oligodendrocyte progenitors. In other words, shHSP27-DPSCs showed NG2-positive immunoreactivity and gave rise to oligodendrocytes or type-2 astrocytes. This neural differentiation of DPSCs may have clinical significance for treatment of patients with neurodegenerative diseases. In conclusion, our data provide an example of oligodendrocyte differentiation of a DPSCs model that may have potential application in human regenerative medicine.

Bioinformatic Analysis Reveals the Distinct Role of 5’UTR-specific m6A RNA Modification in Mice Developing Cerebral Cortices

N6-methyladenosine (m6A) abundantly exists in the cerebral cortex, and is emerging as an essential factor in cortical development and function. As the m6A binding site appears to be dynamically methylated in different RNA regions at the temporal-specific developing stage, it is of value to distinguish the unique character of region- and temporal-specific m6A. Herein, we analyzed the status of temporal-specific m6A within RNA 5’ untranslated region (5’UTR) using m6A-methylated sequencing data and transcriptomic sequencing data from 12.5-13-day embryonic cerebral cortices and 14-day postnatal ones. We identified sorts of RNAs that are uniquely m6A-methylated in the 5’UTR region and sorted them into specific neurological processes. Compared with 3’UTR-m6A-methylated RNAs, 5’UTR-m6A-methylated RNAs showed unique functions and mechanisms in regulating cortical development, especially through the pathway of mRNA transport and surveillance. Moreover, the 5’UTR-specific m6A was associated with neurological disorders as well. The FoxO signaling pathway was then focused by these pathogenic 5’UTR-m6A-methylated RNAs, and explored to be involved in the determination of neurological disorders. Additionally, the 5’UTR-m6A-modification patterns and transcriptional patterns play independent but cohesive roles in the developing cortices. Our study emphasizes the importance of 5’UTR-specific m6A in the developing cortex and provides an informative reference for future studies of 5’UTR-specific m6A in normal cortical development and neurological disorders.

L-Ascorbic Acid and Curcumin prevents Brain Damage-induced via Lead Acetate in Rats: Possible Mechanisms

Lead acetate (lead ac.) is a widespread ecological toxicant that can cause marked neurotoxicity and decline in brain functions. This study aimed to evaluate the possible neuroprotective role of L-ascorbic (ASCR) and curcumin (CRCM) alone or together against lead ac.-induced neurotoxicity. Rats were injected with lead ac. then treated orally with ASCR and CRCM alone or in combination for seven days. Lead ac. caused elevation in brain tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), caspase-3, and malondialdehyde (MDA) levels, while superoxide dismutase (SOD), reduced glutathione (GSH) as well as the expression of brain-derived neurotrophic factor (BDNF), cAMP response element-binding (CREB) and Beclin1 were down-regulated. Expressions of C/EBP homologous protein (CHOP) and mammalian Target of rapamycin kinase (mTOR) were upregulated in brain tissues matched with the control group. Histopathological examination supported the previously mentioned parameters, the administration of the antioxidants in question modulated all the altered previous parameters. The combination regimen achieved the superlative results in the antagonizing lead ac.-induced neurotoxicity via its antioxidant and anti-apoptotic activities.

Exosome miR-23a-3p From Osteoblast Alleviates Spinal Cord Ischemia/Reperfusion Injury by Down-regulating KLF3-activated CCNL2 Transcription

Background: Spinal cord ischemia/reperfusion injury (SCIRI) is usually caused by spinal surgery or aortic aneurysm surgery and can eventually lead to paralysis or paraplegia and neurological dysfunction. Exosomes are considered as one of the most promising therapeutic strategies for SCIRI as they can pass the blood-spinal barrier. Previous studies have proved that exosomes secreted by osteocytes have a certain slowing effect on SCIRI. Aim: We aimed to explore the effect of osteoblast secreted exosomes on SCIRI. Methods: Firstly, neurons and osteoblasts were co-cultured under different conditions. GEO database was utilized to detect the expression of miR-23a-3p in osteoblast exosomes. SCIRI cells were treated with exosomes, and the detection was taken to prove whether miR-23a-3p could slow the progression of SCIRI. Downstream gene and the potential regulatory mechanism were explored through database and functional experiments. Results: MiR-23a-3p was highly expressed in exosomes and it slowed down the process of SCIRI. Downstream mRNA KLF3 could bind to miR-23a-3p and was highly expressed in IRI. Moreover, CCNL2 was regulated by KLF3 and was highly expressed in IRI. Rescue experiments verified that miR-23a-3p suppressed the transcription of CCNL2 by targeting KLF3. Conclusion: Exosome miR-23a-3p from osteoblast alleviates SCIRI by down-regulating KLF3-activated CCNL2 transcription.

Is late prevention of cerebral palsy in extremely preterm infants plausible?

Preterm birth continues to be associated with neurodevelopmental problems including cerebral palsy. Cystic white matter injury is still the major neuropathology underlying cerebral palsy, affecting 1-3% of preterm infants. Although rates have gradually fallen over time, the pathogenesis and evolution of cystic white matter injury are still poorly understood. Hypoxia-ischemia (HI) remains an important contributor yet there is no established treatment to prevent injury. Clinically, serial ultrasound and magnetic resonance imaging studies typically show delayed development of cystic lesions 2 to 4 weeks after birth. This raises the important and unresolved question as to whether this represents slow evolution of injury occurring around the time of birth, or repeated injury over many weeks after birth. There is increasing evidence that tertiary injury after HI can contribute to impairment of white and grey matter maturation. In the present review, we discuss preclinical evidence that severe, cystic white matter injury can evolve for many weeks after acute HI and is associated with microglia activity. This suggests the intriguing hypothesis that the tertiary phase of injury is not as subtle as often thought and that there may be a window of therapeutic opportunity for one to two weeks after hypoxic-ischemic injury to prevent delayed cystic lesions and so further reduce the risk of cerebral palsy after preterm birth.

Intrauterine growth restriction disrupts the postnatal critical period of synaptic plasticity in the mouse dorsal hippocampus in a model of hypertensive disease of pregnancy

Introduction: Intrauterine growth restriction (IUGR) from hypertensive disease of pregnancy complicates up to 10% of all pregnancies. Significant hippocampal-dependent cognitive and memory impairments as well as neuropsychiatric disorders have been linked to IUGR. Because disturbance of hippocampal critical period (CPd) of synaptic plasticity leads to impairments similar to those described in IUGR human offspring, we hypothesized that IUGR would perturb the CPd of synaptic plasticity in the mouse hippocampus in our model. Methods: IUGR was produced by a micro-osmotic pump infusion of the potent vasoconstrictor U-46619, a thromboxane A2-agonist (TXA2), at embryonic day (E) 12.5 in C57BL/6J mouse dams to precipitate hypertensive disease of pregnancy and IUGR. Sham-operated mice acted as controls. At P10, P18, and P40, we assessed astrogliosis using GFAP-IHC. In dorsal CA1 and CA3 subfields, we assessed the immunoreactivities (IR) (IF-IHC) to: i) parvalbumin (PV) and glutamate decarboxylase (GAD) 65/67, involved in CPd onset; ii) PSA-NCAM, that antagonizes CPd onset; iii) NPTX2, necessary for excitatory synapse formation and engagement of CPd; and iv) MBP and WFA, staining perineural nets (PNNs), marking CPd closure. ImageJ/Fiji and IMARIS were used for image processing and SPSS v24 for statistical analysis. Results: Although PV+ interneuron (IN) numbers and IR intensity were unchanged, development of GAD65/67+ synaptic boutons was accelerated at P18 IUGR mice, and inversely correlated with decreased expression of PSA-NCAM in the CA of P18 IUGR mice at P18. NPTX2 + puncta and total volume were persistently decreased in the CA3 pyramidal and radiatum layers of IUGR mice from P18 to P40. At P40, axonal myelination (MBP+) in CA3 of IUGR mice was decreased and correlated with NPTX2 deficits. Lastly, the volume and integrity of the PNNs in the dorsal CA was disrupted in IUGR mice at P40. Discussion/Conclusion: IUGR disrupts the molecular and structural initiation, consolidation and closure of the CPd of synaptic plasticity in the mouse hippocampus in our model, which may explain the learning and memory deficits observed in juvenile IUGR mice and the cognitive disorders seen in human IUGR offspring. The mechanistic links warrant further investigation, to identify therapeutic targets to prevent neurodevelopmental deficits in patients affected by IUGR.

Circ-Ctnnb1 regulates neuronal injury in spinal cord injury through Wnt/β-catenin signaling pathway

Study design: Spinal cord injury (SCI) rat model and cell model were established for in vivo and in vitro experiments. Functional assays were utilized to explore the role of the circRNAs derived from catenin beta 1 (mmu_circ_0001859, circ-Ctnnb1 herein) in regulating neuronal cell viability and apoptosis. Bioinformatics analysis and mechanism experiments were conducted to assess the underlying molecular mechanism of circ-Ctnnb1. Objective: We aimed to probe into the biological function of circ-Ctnnb1 in neuronal cells of SCI. Methods: The rat model of SCI and hypoxia-induced cell model were constructed to examine circ-Ctnnb1 expression in SCI through quantitative reverse transcription real-time polymerase chain reaction (RT-qPCR). Basso, Beattie and Bresnahan (BBB) score was utilized for evaluating the neurological function. Terminal-deoxynucleoitidyl Transferase Mediated Nick End labeling (TUNEL) assays were performed to assess the apoptosis of neuronal cells. RNase R and Actinomycin D (ActD) were used to treat cells to evaluate the stability of circ-Ctnnb1. Results: Circ-Ctnnb1 was highly expressed in SCI rat models and hypoxia-induced neuronal cells, and its deletion elevated the apoptosis rate of hypoxia-induced neuronal cells. Furthermore, circ-Ctnnb1 activated the Wnt/β-catenin signaling pathway via sponging mircoRNA-205-5p (miR-205-5p) to up-regulate Ctnnb1 and Wnt family member 2B (Wnt2b). Conclusion: Circ-Ctnnb1 promotes SCI through regulating Wnt/β-catenin signaling via modulating the miR-205-5p/Ctnnb1/Wnt2b axis.

The deleterious effect of crossfostering in rat pups on hypoxic-ischemic injury tolerance and hypothermic neuroprotection

We study the effect of hypothermia (HT) following hypoxic-ischemic (HI) brain injury in postnatal day 7 (P7) rats. In 2015, new European Union animal transport regulations prompted a change in practice at the breeding facility, which henceforth crossfostered P3 litters to P8 older lactating dam prior to transportation. It is generally assumed that crossfostering does not significantly affect the experimental results. The aim of this study was to examine whether crossfostering affects our model consistency by modifying injury susceptibility and hypothermic neuroprotection. We analysed 219 pups (56 litters) from 11 experiments conducted between 2013 and 2015: 73 non-crossfostered and 146 crossfostered pups. At P7, all pups underwent unilateral common carotid artery ligation followed by 50min of hypoxia (8% O2, 36°C). Immediately after this mild insult, the pups were randomised to post-insult normothermia (NT) or HT treatment. Pups were culled at P14. Injury was assessed by area loss of the ipsilateral hemisphere and histopathology scoring of hippocampus, cortex, thalamus, and basal ganglia. Crossfostered pups had double the injury compared to non-crossfostered pups irrespective of treatment group. Hypothermic neuroprotection was statistically significant, but with a smaller and less consistent effect in crossfostered pups (relative neuroprotection 16% vs. 31% in non-crossfostered). These results demonstrate hypothermic neuroprotection following a mild HI insult. A representative subset of 41 animals were also assessed for evidence of microglial reactivity, however no detectable difference in microglial reactivity was observed between any of the groups. In conclusion, crossfostering alters outcomes in our established model through reduced insult tolerance and variable neuroprotection. Crossfostering as a common breeding practice is a largely unexplored variable in animal research that may result in invalid research conclusions if inadequately adjusted for by larger group sizes. As a result, crossfostering is likely to be inconsistent with the principles of replacement, reduction, and refinement.

Early appearance of dendritic alterations in neocortical pyramidal neurons of the Ts65Dn model of Down syndrome

Down syndrome (DS), which is due to triplication of chromosome 21, is constantly associated with intellectual disability (ID). ID can be ascribed to both neurogenesis impairment and dendritic pathology. These defects are replicated in the Ts65Dn mouse, a widely used model of DS. While neurogenesis impairment in DS is a fetal event, dendritic pathology occurs after the first postnatal months. Neurogenesis alterations across the lifespan have been extensively studied in the Ts65Dn mouse. In contrast, there is scarce information regarding dendritic alterations at early life stages in this and other models, although there is evidence for dendritic alterations in adult mouse models. Thus, the goal of the current study was to establish whether dendritic alterations are already present in the neonatal period in Ts65Dn mice. In Golgi-stained brains we quantified the dendritic arbors of layer II/III pyramidal neurons in the frontal cortex of Ts65Dn mice aged 2 (P2) and 8 (P8) days and their euploid littermates. In P2 Ts65Dn mice we found a moderate hypotrophy of the apical and collateral dendrites but a patent hypotrophy of the basal dendrites. In P8 Ts65Dn mice the distalmost apical branches were missing or reduced in number but there were no alterations in the collateral and basal dendrites. No genotype effects were detected on either somatic or dendritic spine density. This study shows dendritic branching defects that mainly involve the basal domain in P2 Ts65Dn mice, and the apical but not the other domains in P8 Ts65Dn mice. This suggests that dendritic defects may be related to dendritic compartment and age. The lack of a severe dendritic pathology in Ts65Dn pups is reminiscent of the delayed appearance of patent dendritic alterations in newborns with DS. This similarly highlights the usefulness of the Ts65Dn model for the study of the mechanisms underlying dendritic alterations in DS and the design of possible therapeutic interventions.