Neonatal Mouse Brain Research Articles

Sex differences in neonatal mouse brain injury after hypoxia‐ischemia and adaptaquin treatment

Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-PHDs) are important targets against oxidative stress. We hypothesized that inhibition HIF-PHD by adaptaquin reduces hypoxic-ischemic brain injury in a neonatal mouse model. The pups were treated intraperitoneally immediately with adaptaquin after hypoxia-ischemia (HI) and then every 24h for 3days. Adaptaquin treatment reduced infarction volume by an average of 26.3% at 72h after HI compared to vehicle alone, and this reduction was more pronounced in males (34.8%) than in females (11.7%). The protection was also more pronounced in the cortex. The subcortical white matter injury as measured by tissue loss volume was reduced by 24.4% in the adaptaquin treatment group, and this reduction was also more pronounced in males (28.4%) than in females (18.9%). Cell death was decreased in the cortex as indicated by Fluoro-Jade labeling, but not in other brain regions with adaptaquin treatment. Furthermore, in the brain injury area, adaptaquin did not alter the number of cells positive for caspase-3 activation or translocation of apoptosis-inducing factor to the nuclei. Adaptaquin treatment increased glutathione peroxidase 4 mRNA expression in the cortex but had no impact on 3-nitrotyrosine, 8-hydroxy-2 deoxyguanosine, or malondialdehyde production. Hif1α mRNA expression increased after HI, and adaptaquin treatment also stimulated Hif1α mRNA expression, which was also more pronounced in males than in females. However, nuclear translocation of HIF1α protein was decreased after HI, and adaptaquin treatment had no influence on HIF1α expression in the nucleus. These findings demonstrate that adaptaquin treatment is neuroprotective, but the potential mechanisms need further investigation. Read the Editorial Highlight for this article on page 645.

P1‐205: INTRANASAL ADMINISTRATION OF INSULIN PREVENTS CHRONIC COGNITIVE IMPAIRMENT AND NEURONAL APOPTOSIS INDUCED BY GENERAL ANESTHESIA IN NEONATAL MICE

General anesthesia increases the risk for cognitive impairment post operation, especially in children and elderly individuals. To date, there is no treatment to prevent the anesthesia-induced neurotoxicity and behavioral changes. We administered insulin through intranasal delivery to 7-day-old neonatal mice, followed by anesthesia with sevoflurane for 3 hrs per day for three consecutive days or once for 6 hrs. The mice were then assessed for cognitive function by using open field test, one-trial novel object recognition test, Morris water maze, and fear conditioning test when they reached to adult age. The neonatal mouse brains were also studied biochemically and immunohistochemically for changes in synaptic proteins, neuroinflammation, and neuronal apoptosis in the brain. We found that the anesthesia to neonatal mice led to mild behavioral abnormalities later in life that were detectable by using novel object recognition test, Morris water maze, and fear conditioning test. Biochemical and immunohistochemical studies indicate that anesthesia induced a decrease in brain level of postsynaptic density 95 (PSD95), a postsynaptic marker, and marked activation of neuronal apoptosis in neonatal mice. Importantly, insulin administered through intranasal delivery prior to anesthesia was found to prevent the anesthesia-induced long-term behavioral abnormalities, reduction of PSD95, and activation of neuronal apoptosis. Our results indicate that anesthesia exposure during development may be a risk factor for cognitive impairment later in life and that intranasal treatment with insulin can prevent this risk. These findings suggest that intranasal insulin administration could be an effective approach to prevent the increased risk of neurotoxicity and chronic damage caused by anesthesia to the developing brain.

Multiomics Investigation Reveals Benzalkonium Chloride Disinfectants Alter Sterol and Lipid Homeostasis in the Mouse Neonatal Brain.

Lipids are critical for neurodevelopment; therefore, disruption of lipid homeostasis by environmental chemicals is expected to have detrimental effects on this process. Previously, we demonstrated that the benzalkonium chlorides (BACs), a class of commonly used disinfectants, alter cholesterol biosynthesis and lipid homeostasis in neuronal cell cultures in a manner dependent on their alkyl chain length. However, the ability of BACs to reach the neonatal brain and alter sterol and lipid homeostasis during neurodevelopment in vivo has not been characterized. Therefore, the goal of this study was to use targeted and untargeted mass spectrometry and transcriptomics to investigate the effect of BACs on sterol and lipid homeostasis and to predict the mechanism of toxicity of BACs on neurodevelopmental processes. After maternal dietary exposure to 120 mg BAC/kg body weight/day, we quantified BAC levels in the mouse neonatal brain, demonstrating for the first time that BACs can cross the blood-placental barrier and enter the developing brain. Transcriptomic analysis of neonatal brains using RNA sequencing revealed alterations in canonical pathways related to cholesterol biosynthesis, liver X receptor-retinoid X receptor (LXR/RXR) signaling, and glutamate receptor signaling. Mass spectrometry analysis revealed decreases in total sterol levels and downregulation of triglycerides and diglycerides, which were consistent with the upregulation of genes involved in sterol biosynthesis and uptake as well as inhibition of LXR signaling. In conclusion, these findings demonstrate that BACs target sterol and lipid homeostasis and provide new insights for the possible mechanisms of action of BACs as developmental neurotoxicants.

Effects of hypoxia ischemia on caspase-3 expression and neuronal apoptosis in the brain of neonatal mice.

Effects of hypoxia ischemia on caspase-3 expression and neuronal apoptosis in the brain of neonatal mice were investigated. Twenty-five neonatal CD1 mice aged 1 week were selected and randomly divided into sham-operation group (n=8) and newborn hypoxia ischemia encephalopathy (NHIE) model group (n=17). The messenger ribonucleic acid (mRNA) expression levels of caspase-3 and Fas ligand (FasL) in brain tissues of mice in both groups were detected via reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of caspase-3 and FasL in mice in both groups were detected via western blotting. Moreover, apoptosis of brain tissues was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 protein expression level in brain tissues was detected using immunohistochemical methods. Results of RT-PCR and western blotting revealed that compared with those in sham-operation group, caspase-3 and FasL expression levels in model group were significantly increased. Results of TUNEL showed that the number of apoptotic neurons in model group was significantly increased. Besides, results of immunohistochemical detection manifested that the caspase-3 protein expression level in model group was obviously increased. Hypoxia ischemia can lead to significant increase of caspase-3 expression and increase of neuronal apoptosis in the brain of neonatal mice.

CD4 T cells are required for maintenance of CD8 TRM cells and virus control in the brain of MCMV-infected newborn mice.

Cytomegalovirus (CMV) infection is a significant public health problem. Congenital CMV infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and sensorineural hearing loss. Immune protection against mouse cytomegalovirus (MCMV) is primarily mediated by NK cells and CD8+ T cells, while CD4+ T cells are not needed for control of MCMV in majority of organs in immunocompetent adult mice. Here, we set out to determine the role of CD4+ T cells upon MCMV infection of newborn mice. We provide evidence that CD4+ T cells are essential for clearance of MCMV infection in brain of neonatal mice and for prevention of recurrence of latent MCMV. In addition, we provide evidence that CD4+ T cells are required for induction and maintenance of tissue-resident memory CD8+ T cells in the brain of mice perinatally infected with MCMV.

Evidence for Sexual Dimorphism in the Response to TLR3 Activation in the Developing Neonatal Mouse Brain: A Pilot Study.

Toll-like receptor (TLR)3 activation during the neonatal period produces responses linked to the origins of neuropsychiatric disorders. Although there is sexual dimorphism in neuropsychiatric disorders, it is unknown if brain responses to TLR3 activation are sex-specific. We hypothesized that poly I:C in a post-natal day (P)8 model induces a sexually dimorphic inflammatory responses. C57BL6 mice received intraperitoneal injection of poly I:C (10 mg/kg) or vehicle [normal saline (NS)] at P8. Pups were killed at 6 or 14 h for caspase 3 and 8 activity assays, NFkB ELISA, IRF3, AP1, and GFAP western blotting and cytokines/chemokines gene expression real time qRT-PCR (4–6/group). A second group of pups were killed at 24 h (P9) or 7 days (P15) after poly I:C to assess astrocytic (GFAP) and microglia (Iba1) activation in the hippocampus, thalamus and cortex using immunohistochemistry, and gene and protein expression of cytokines/chemokines using real time RT-PCR and MSD, respectively (4–6/group). Non-parametric analysis was applied. Six hours after poly I:C, caspase-3 and -8 activities in cytosolic fractions were 1.6 and 2.8-fold higher in poly I:C-treated than in NS-treated female mice, respectively, while gene expressions of pro-inflammatory cytokines were upregulated in both sexes. After poly I:C, IRF3 nuclear translocation occurred earlier (6 h) in female mice and later (14 h) in male mice. At 14 h after poly I:C, only male mice also had increased nuclear NFκB levels (88%, p < 0.001) and GFAP expression coinciding with persistent IL-6 and FAS gene upregulation (110 and 77%, respectively; p < 0.001) and IL-10 gene downregulation (-42%, p < 0.05). At 24 h after poly I:C, IL-1β, CXCL-10, TNF-α, and MCP-1 were similarly increased in both sexes but at 7 days after exposure, CXCL-10 and INFγ were increased and IL-10 was decreased only in female mice. Accordingly, microglial activation persisted at 7 days after poly I:C in the hippocampus, thalamus and cortex of female mice. This preliminary study suggests that TLR3 activation may produce in the developing neonatal mouse brain a sexually dimorphic response with early activation of caspase-dependent pathways in female mice, activation of inflammatory cascades in both sexes, which then persists in female mice. Further well-powered studies are essential to confirm these sex-specific findings.

Choroid plexus transcriptome and ultrastructure analysis reveals a TLR2-specific chemotaxis signature and cytoskeleton remodeling in leukocyte trafficking

Perinatal infection and inflammation are major risk factors for injury in the developing brain, however, underlying mechanisms are not fully understood. Leukocyte migration to the cerebrospinal fluid (CSF) and brain is a hallmark of many pathologies of the central nervous system including those in neonates. We previously reported that systemic activation of Toll-like receptor (TLR) 2, a major receptor for gram-positive bacteria, by agonist Pam3CSK4 (P3C) resulted in dramatic neutrophil and monocyte infiltration to the CSF and periventricular brain of neonatal mice, an effect that was absent by the TLR4 agonist, LPS. Here we first report that choroid plexus is a route of TLR2-mediated leukocyte infiltration to the CSF by performing flow cytometry and transmission electron microscopy (TEM) of the choroid plexus. Next, we exploited the striking discrepancy between P3C and LPS effects on cell migration to determine the pathways regulating leukocyte trafficking through the choroid plexus. We performed RNA sequencing on the choroid plexus after administration of P3C and LPS to postnatal day 8 mice. A cluster gene analysis revealed a TLR2-specific signature of chemotaxis represented by 80-fold increased expression of the gene Ccl3 and 1000-fold increased expression of the gene Cxcl2. Ingenuity pathway analysis (IPA) revealed TLR2-specific molecular signaling related to cytoskeleton organization (e.g. actin signaling) as well as inositol phospholipids biosynthesis and degradation. This included upregulation of genes such as Rac2 and Micall2. In support of IPA results, ultrastructural analysis by TEM revealed clefting and perforations in the basement membrane of the choroid plexus epithelial cells in P3C-treated mice. In summary, we show that the choroid plexus is a route of TLR2-mediated transmigration of neutrophils and monocytes to the developing brain, and reveal previously unrecognized mechanisms that includes a specific chemotaxis profile as well as pathways regulating cytoskeleton and basement membrane remodeling.

Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury

Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer’s sector of the Ammon’s horn (CA1). Analysis of bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.

Distinct response of adult neural stem cells to low versus high dose ionising radiation

Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible.Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50–500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation.

Grape Seed Proanthocyanidin Extract Down-Regulates Autophagic Proteins (LC3II and Beclin-1) in a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe brain disease that often leads to death or irreversible sequelae. The aim of this study was to determine the effect of a naturally active drug (grape seed proanthocyanidin extract [GSPE]) on the expression of autophagic proteins in a mouse model of neonatal hypoxic-ischemic brain injury. In this study immunofluorescence staining and Western blotting were used to detect the expression of autophagy markers (LC3II and beclin1) in the brains of neonatal mice with hypoxic-ischemic brain injuries after GSPE administration. Our study showed that GSPE pre-treatment down-regulates LC3II and beclin-1 expression, thus GSPE may be a potential drug for the treatment of HIE.

A novel image segmentation method for the evaluation of inflammation-induced cortical and hippocampal white matter injury in neonatal mice

The developing brain is very susceptible to environmental insults, and very immature infants often suffer from long-term neurological syndromes associated with white matter injuries such as periventricular leukomalacia. Infection and inflammation are important risk factors for neonatal brain white matter injuries, but the evaluation of white matter injury in animal models, especially the quantification of myelinated axons, has long been problematic due to the lack of ideal measurement methods. Here, we present an automated segmentation method, which we call MyelinQ, for the quantification of myelinated white matter in immunohistochemical DAB-stained sections of the neonatal mouse brain. Using MyelinQ, we show that a viral infection mimic agent, the Toll-like receptor 3 ligand Poly I:C, causes significant hypomyelination of white matter in the cortical and hippocampal fimbria regions, but not in the striatal caudoputamen region. We showed that MyelinQ can reliably produce results that are comparable to a method used in our previous publications. However, in comparison to the conventional method, MyelinQ has the advantages of being automated, objective and accurate. MyelinQ can analyze white matter in various specific brain regions and therefore provides a useful platform for the quantification of myelin and the evaluation of white matter injuries in animal models. MyelinQ and its code together with instructions for use can be found at: https://github.com/parham-ap/myelinq.

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions.

Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein 'bait'. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs was analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

A Chemical Recipe for Generation of Clinical-Grade Striatal Neurons from hESCs.

SummaryDifferentiation of human pluripotent stem cells (hPSCs) into striatal medium spiny neurons (MSNs) promises a cell-based therapy for Huntington's disease. However, clinical-grade MSNs remain unavailable. Here, we developed a chemical recipe named XLSBA to generate clinical-grade MSNs from embryonic stem cells (ESCs). We introduced the γ-secretase inhibitor DAPT into the recipe to accelerate neural differentiation, and replaced protein components with small molecules. Using this optimized protocol we could efficiently direct regular human ESCs (hESCs) as well as clinical-grade hESCs to lateral ganglionic eminence (LGE)-like progenitors and striatal MSNs within less than half of the time than previous protocols (within 14 days and 21 days, respectively). These striatal cells expressed appropriate MSN markers and electrophysiologically acted like authentic MSNs. Upon transplantation into brains of neonatal mice or mouse model of Huntington's disease, they exhibited sufficient safety and reasonable efficacy. Therefore, this quick and highly efficient derivation of MSNs offers unprecedented access to clinical application.

Control of occlusion of middle cerebral artery in perinatal and neonatal mice with magnetic force

Ischemic perinatal stroke (IPS) is common, resulting in significant mortality and morbidity. In such cases, the incidence of unilateral arterial cerebral infarction is often occluded in the middle cerebral artery (MCA), leading to focal ischemia. In adult rodents, blockage of MCA is the most frequently used strategy for ischemic stroke study. However, modeling MCA occlusion (MCAo) in postnatal day 0–7 (P0–7) mouse pups for IPS study has not been accomplished. Here we occluded the dMCA by inducing the accumulation of magnetic particles (MPs) administered through the superficial temporal vein of mice between P0 and P7, which we called neonatal or perinatal SIMPLE (Stroke Induced with Magnetic Particles). SIMPLE produced either permanent or transient occlusion in the dMCA of perinatal and neonatal mice. Permanent MCA occlusion with SIMPLE resulted in cerebral infarction and neuronal death in the brain. SIMPLE can also be used to reliably produce focal ischemic stroke in neonatal or perinatal mouse brains. As a result, SIMPLE allows the modeling of IPS or focal ischemic stroke for further mechanistic studies in mice, with particular utility for mimicking transient focal ischemia in human pre-term babies, which for the first time here has been accomplished in mice.

Foamy Virus Vectors Transduce Visceral Organs and Hippocampal Structures following In Vivo Delivery to Neonatal Mice

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

A Versatile Method for Viral Transfection of Calcium Indicators in the Neonatal Mouse Brain.

The first three postnatal weeks in rodents are a time when sensory experience drives the maturation of brain circuits, an important process that is not yet well understood. Alterations in this critical period of experience-dependent circuit assembly and plasticity contribute to several neurodevelopmental disorders, such as autism, epilepsy, and schizophrenia. Therefore, techniques for recording network activity and tracing neuronal connectivity over this time period are necessary for delineating circuit refinement in typical development and how it deviates in disease. Calcium imaging with GCaMP6 and other genetically encoded indicators is rapidly becoming the preferred method for recording network activity at the single-synapse and single-cell level in vivo, especially in genetically identified neuronal populations. We describe a protocol for intracortical injection of recombinant adeno-associated viruses in P1 neonatal mice and demonstrate its use for longitudinal imaging of GCaMP6s in the same neurons over several weeks to characterize the developmental desynchronization of cortical network activity. Our approach is ideally suited for chronic in vivo two-photon calcium imaging of neuronal activity from synapses to entire networks during the early postnatal period.

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Morpholino Antisense Oligomers as a Potential Therapeutic Option for the Correction of Alternative Splicing in PMD, SPG2, and HEMS

DNA variants of the proteolipid protein 1 gene (PLP1) that shift PLP1/DM20 alternative splicing away from the PLP1 form toward DM20 cause the allelic X-linked leukodystrophies Pelizaeus-Merzbacher disease (PMD), spastic paraplegia 2 (SPG2), and hypomyelination of early myelinating structures (HEMS). We designed a morpholino oligomer (MO-PLP) to block use of the DM20 5′ splice donor site, thereby shifting alternative splicing toward the PLP1 5′ splice site. Treatment of an immature oligodendrocyte cell line with MO-PLP significantly shifted alternative splicing toward PLP1 expression from the endogenous gene and from transfected human minigene splicing constructs harboring patient variants known to reduce the amount of the PLP1 spliced product. Additionally, a single intracerebroventricular injection of MO-PLP into the brains of neonatal mice, carrying a deletion of an intronic splicing enhancer identified in a PMD patient that reduces the Plp1 spliced form, corrected alternative splicing at both RNA and protein levels in the CNS. The effect lasted to post-natal day 90, well beyond the early post-natal spike in myelination and PLP production. Further, the single injection produced a sustained reduction of inflammatory markers in the brains of the mice. Our results suggest that morpholino oligomers have therapeutic potential for the treatment of PMD, SPG2, and HEMS.

Upregulation of cholesterol 24-hydroxylase following hypoxia-ischemia in neonatal mouse brain.

BackgroundMaintenance of cholesterol homeostasis is crucial for brain development. Brain cholesterol relies on de novo synthesis and is cleared primarily by conversion to 24S-hydroxycholesterol (24S-HC) with brain-specific cholesterol 24-hydroxylase (CYP46A1). We aimed to investigate the impact of hypoxia-ischemia (HI) on brain cholesterol metabolism in the neonatal mice.MethodsPostnatal day 9 C57BL/6 pups were subjected to HI using the Vannucci model. CYP46A1 expression was assessed by western blotting and its cellular localization was determined by immunofluorescence staining. The amount of brain cholesterol, 24S-HC in the cortex and in the serum was measured with ELISA.ResultsThere was a transient cholesterol loss at 6hr after HI. CYP46A1 was significantly up-regulated at 6hr and 24hr following HI with a concomitant increase of 24S-HC in the ipsilateral cortex and in the serum. The serum levels of 24S-HC correlated with those in the brain, as well as with necrotic and apoptotic cell death evaluated by the expression of spectrin breakdown products and cleaved caspase-3 at 6hr and 24hr after HI.ConclusionsEnhanced cholesterol turnover by activation of CYP46A1 represents disrupted brain cholesterol homeostasis early after neonatal HI. 24S-HC might be a novel blood biomarker for severity of hypoxic-ischemic encephalopathy with potential clinical application.