Epigenetic alterations in creatine transporter deficiency: a new marker for dodecyl creatine ester therapeutic efficacy monitoring.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study, we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients' cells. We also investigated the effect of dodecyl creatine ester (DCE), a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation, unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy.

Neuroprotective efficacy of hypothermia and Inter-alpha Inhibitor Proteins after hypoxic ischemic brain injury in neonatal rats.

Therapeutic hypothermia is the standard of care for hypoxic-ischemic (HI) encephalopathy. Inter-alpha Inhibitor Proteins (IAIPs) attenuate brain injury after HI in neonatal rats. Human (h) IAIPs (60 â€‹mg/kg) or placebo (PL) were given 15 â€‹min, 24 and 48 â€‹h to postnatal (P) day-7 rats after carotid ligation and 8% oxygen for 90 â€‹min with (30 â€‹°C) and without (36 â€‹°C) exposure to hypothermia 1.5 â€‹h after HI for 3 â€‹h. Hemispheric volume atrophy (P14) and neurobehavioral tests including righting reflex (P8-P10), small open field (P13-P14), and negative geotaxis (P14) were determined. Hemispheric volume atrophy in males was reduced (P â€‹< â€‹0.05) by 41.9% in the normothermic-IAIP and 28.1% in the hypothermic-IAIP compared with the normothermic-PL group, and in females reduced (P â€‹< â€‹0.05) by 30.3% in the normothermic-IAIP, 45.7% in hypothermic-PL, and 55.2% in hypothermic-IAIP compared with the normothermic-PL group after HI. Hypothermia improved (P â€‹< â€‹0.05) the neuroprotective effects of hIAIPs in females. The neuroprotective efficacy of hIAIPs was comparable to hypothermia in female rats (P â€‹= â€‹0.183). Treatment with hIAIPs, hypothermia, and hIAIPs with hypothermia decreased (P â€‹< â€‹0.05) the latency to enter the peripheral zone in the small open field test in males. We conclude that hIAIPs provide neuroprotection from HI brain injury that is comparable to the protection by hypothermia, hypothermia increases the effects of hIAIPs in females, and hIAIPs and hypothermia exhibit some sex-related differential effects.

Deciphering neuronal deficit and protein profile changes in human brain organoids from patients with creatine transporter deficiency.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3ß, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3ß, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.

High-mobility group box-1 and inter-alpha inhibitor proteins: In vitro binding and co-localization in cerebral cortex after hypoxic-ischemic injury.

The high-mobility group box-1 (HMGB1) protein is a transcription-regulating protein located in the nucleus. However, it serves as a damage-associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter-alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro-inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic-ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti-inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co-localization in vivo in cerebral cortices after exposure to HI injury. Solid-phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs-HMGB1 in neonatal rat cortex were visualized by double labeling with anti-IAIPs and anti-HMGB1 antibodies. Solid-phase binding and SPR demonstrated specific binding between IAIPs and HMGB1 in vitro. Cortical cytoplasmic and nuclear co-localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3 hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co-localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs.

Author Correction: A Small Compound Targeting Prohibitin with Potential Interest for Cognitive Deficit Rescue in Aging mice and Tau Pathology Treatment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lipopolysaccharide-induced changes in the neurovascular unit in the preterm fetal sheep brain.

BACKGROUND: Exposure to inflammation during pregnancy can predispose to brain injury in premature infants. In the present study, we investigated the effects of prolonged exposure to inflammation on the cerebrovasculature of preterm fetal sheep. METHODS: Chronically instrumented fetal sheep at 103-104 days of gestation (full term is ~ 147 days) received continuous low-dose lipopolysaccharide (LPS) infusions (100 ng/kg over 24 h, followed by 250 ng/kg/24 h for 96 h plus boluses of 1 µg LPS at 48, 72, and 96 h) or the same volume of normal saline (0.9%, w/v). Ten days after the start of LPS exposure at 113-114 days of gestation, the sheep were killed, and the fetal brain perfused with formalin in situ. Vessel density, pericyte and astrocyte coverage of the blood vessels, and astrogliosis in the cerebral cortex and white matter were determined using immunohistochemistry. RESULTS: LPS exposure reduced (P < 0.05) microvascular vessel density and pericyte vascular coverage in the cerebral cortex and white matter of preterm fetal sheep, and increased the activation of perivascular astrocytes, but decreased astrocytic vessel coverage in the white matter. CONCLUSIONS: Prolonged exposure to LPS in preterm fetal sheep resulted in decreased vessel density and neurovascular remodeling, suggesting that chronic inflammation adversely affects the neurovascular unit and, therefore, could contribute to long-term impairment of brain development.

Hypoxic-ischemic-related cerebrovascular changes and potential therapeutic strategies in the neonatal brain.

Perinatal hypoxic-ischemic (HI)-related brain injury is an important cause of morbidity and long-standing disability in newborns. The only currently approved therapeutic strategy available to reduce brain injury in the newborn is hypothermia. Therapeutic hypothermia can only be used to treat HI encephalopathy in full-term infants and survivors remain at high risk for a wide spectrum of neurodevelopmental abnormalities as a result of residual brain injury. Therefore, there is an urgent need for adjunctive therapeutic strategies. Inflammation and neurovascular damage are important factors that contribute to the pathophysiology of HI-related brain injury and represent exciting potential targets for therapeutic intervention. In this review, we address the role of each component of the neurovascular unit (NVU) in the pathophysiology of HI-related injury in the neonatal brain. Disruption of the blood-brain barrier (BBB) observed in the early hours after an HI-related event is associated with a response at the basal lamina level, which comprises astrocytes, pericytes, and immune cells, all of which could affect BBB function to further exacerbate parenchymal injury. Future research is required to determine potential drugs that could prevent or attenuate neurovascular damage and/or augment repair. However, some studies have reported beneficial effects of hypothermia, erythropoietin, stem cell therapy, anti-cytokine therapy and metformin in ameliorating several different facets of damage to the NVU after HI-related brain injury in the perinatal period.

A Small Compound Targeting Prohibitin with Potential Interest for Cognitive Deficit Rescue in Aging mice and Tau Pathology Treatment.

Neurodegenerative diseases, including Alzheimer's and Parkinson's disease, are characterized by increased protein aggregation in the brain, progressive neuronal loss, increased inflammation, and neurogenesis impairment. We analyzed the effects of a new purine derivative drug, PDD005, in attenuating mechanisms involved in the pathogenesis of neurodegenerative diseases, using both in vivo and in vitro models. We show that PDD005 is distributed to the brain and can rescue cognitive deficits associated with aging in mice. Treatment with PDD005 prevents impairment of neurogenesis by increasing sex-determining region Y-box 2, nestin, and also enhances synaptic function through upregulation of synaptophysin and postsynaptic density protein 95. PDD005 treatment also reduced neuro-inflammation by decreasing interleukin-1ß expression, activation of astrocytes, and microglia. We identified prohibitin as a potential target in mediating the therapeutic effects of PDD005 for the treatment of cognitive deficit in aging mice. Additionally, in the current study, glycogen synthase kinase appears to attenuate tau pathology.

Anti-Cytokine Therapy to Attenuate Ischemic-Reperfusion Associated Brain Injury in the Perinatal Period.

Perinatal brain injury is a major cause of morbidity and long-standing disability in newborns. Hypothermia is the only therapy approved to attenuate brain injury in the newborn. However, this treatment is unfortunately only partially neuroprotective and can only be used to treat hypoxic-ischemic encephalopathy in full term infants. Therefore, there is an urgent need for adjunctive therapeutic strategies. Post-ischemic neuro-inflammation is a crucial contributor to the evolution of brain injury in neonates and constitutes a promising therapeutic target. Recently, we demonstrated encouraging neuroprotective capacities of anti-cytokine monoclonal antibodies (mAbs) in an ischemic-reperfusion (I/R) model of brain injury in the ovine fetus. The purpose of this review is to summarize the current knowledge regarding the inflammatory response in the perinatal sheep brain after I/R injury and to review our recent findings regarding the beneficial effects of treatment with anti-cytokine mAbs.

Alterations in inter-alpha inhibitor protein expression after hypoxic-ischemic brain injury in neonatal rats.

Hypoxic-ischemic (HI) brain injury is frequently associated with premature and/or full-term birth-related complications that reflect widespread damage to cerebral cortical structures. Inflammation has been implicated in the long-term evolution and severity of HI brain injury. Inter-Alpha Inhibitor Proteins (IAIPs) are immune modulator proteins that are reduced in systemic neonatal inflammatory states. We have shown that endogenous IAIPs are present in neurons, astrocytes and microglia and that exogenous treatment with human plasma purified IAIPs decreases neuronal injury and improves behavioral outcomes in neonatal rats with HI brain injury. In addition, we have shown that endogenous IAIPs are reduced in the brain of the ovine fetus shortly after ischemic injury. However, the effect of HI on changes in circulating and endogenous brain IAIPs has not been examined in neonatal rats. In the current study, we examined changes in endogenous IAIPs in the systemic circulation and brain of neonatal rats after exposure to HI brain injury. Postnatal day 7 rats were exposed to right carotid artery ligation and 8% oxygen for 2h. Sera were obtained immediately, 3, 12, 24, and 48h and brains 3 and 24h after HI. IAIPs levels were determined by a competitive enzyme-linked immunosorbent assay (ELISA) in sera and by Western immunoblots in cerebral cortices. Serum IAIPs were decreased 3h after HI and remained lower than in non-ischemic rats up to 7days after HI. IAIP expression increased in the ipsilateral cerebral cortices 24h after HI brain injury and in the hypoxic contralateral cortices. However, 3h after hypoxia alone the 250kDa IAIP moiety was reduced in the contralateral cortices. We speculate that changes in endogenous IAIPs levels in blood and brain represent constituents of endogenous anti-inflammatory neuroprotective mechanism(s) after HI in neonatal rats.

Brain Inflammation, Blood Brain Barrier dysfunction and Neuronal Synaptophysin Decrease after Inhalation Exposure to Titanium Dioxide Nano-aerosol in Aging Rats.

Notwithstanding potential neurotoxicity of inhaled titanium dioxide nanoparticles (TiO2 NPs), the toxicokinetics and consequences on blood-brain barrier (BBB) function remain poorly characterized. To improve risk assessment, we need to evaluate the impact on BBB under realistic environmental conditions and take into account vulnerability status such as age. 12-13 week and 19-month-old male rats were exposed by inhalation to 10 mg/m3 of TiO2 nano-aerosol (6 hrs/day, 5 day/week, for 4 weeks). We showed an age-dependent modulation of BBB integrity parameters suggesting increased BBB permeability in aging rats. This alteration was associated with a significant increase of cytokines/chemokines in the brain, including interleukin-1ß, interferon-γ, and fractalkine as well as a decreased expression of synaptophysin, a neuronal activity marker. These observations, in absence of detectable titanium in the brain suggest that CNS-related effects are mediated by systemic-pathway. Moreover, observations in terms of BBB permeability and brain inflammation underline age susceptibility. Even if TiO2 NPs were not evidenced in the brain, we observed an association between the exposure to TiO2 NPs and the dysregulation of BBB physiology associated with neuroinflammation and decreased expression of neuronal activity marker, which was further exacerbated in the brain of aged animal's.

Biopersistence and translocation to extrapulmonary organs of titanium dioxide nanoparticles after subacute inhalation exposure to aerosol in adult and elderly rats.

The increasing industrial use of nanoparticles (NPs) has raised concerns about their impact on human health. Since aging and exposure to environmental factors are linked to the risk for developing pathologies, we address the question of TiO2 NPs toxicokinetics in the context of a realistic occupational exposure. We report the biodistribution of titanium in healthy young adults (12-13-week-old) and in elderly rats (19-month-old) exposed to 10mg/m3 of a TiO2 nanostructured aerosol 6h/day, 5days/week for 4 weeks. We measured Ti content in major organs using inductively coupled plasma mass spectrometry immediately and up to 180days after the end of exposure. Large amounts of titanium were initially found in lung which were slowly cleared during the post-exposure period. From day 28, a small increase of Ti was found in the spleen and liver of exposed young adult rats. Such an increase was however never found in their blood, kidneys or brain. In the elderly group, translocation to extra-pulmonary organs was significant at day 90. Ti recovered from the spleen and liver of exposed elderly rats was higher than in exposed young adults. These data suggest that TiO2 NPs may translocate from the lung to extra-pulmonary organs where they could possibly promote systemic health effects.

Tissue biodistribution of intravenously administrated titanium dioxide nanoparticles revealed blood-brain barrier clearance and brain inflammation in rat.

BACKGROUND: Notwithstanding increasing knowledge of titanium dioxide nanoparticles (TiO2 NPs) passing through biological barriers, their biodistribution to the central nervous system (CNS) and potential effects on blood-brain barrier (BBB) physiology remain poorly characterized. METHODS: Here, we report time-related responses from single-dose intravenous (IV) administration of 1 mg/kg TiO2 NPs to rats, with particular emphasis on titanium (Ti) quantification in the brain. Ti content in tissues was analyzed using inductively coupled plasma mass spectrometry. Integrity and functionality of the BBB as well as brain inflammation were characterized using a panel of methods including RT-PCR, immuno-histo chemistry and transporter activity evaluation. RESULTS: Biokinetic analysis revealed Ti biopersistence in liver, lungs and spleen up to one year after TiO2 NPs administration. A significant increase of Ti in the brain was observed at early end points followed by a subsequent decrease. In-depth analysis of Ti in the total brain demonstrated quantitative Ti uptake and clearance by brain microvasculature endothelial cells (BECs) with minimal translocation in the brain parenchyma. The presence of Ti in the BECs did not affect BBB integrity, despite rapid reversible modulation of breast cancer resistance protein activity. Ti biopersistence in organs such as liver was associated with significant increases of tight junction proteins (claudin-5 and occludin), interleukin 1ß (IL-1ß), chemokine ligand 1 (CXCL1) and γ inducible protein-10 (IP-10/CXCL10) in BECs and also increased levels of IL-1ß in brain parenchyma despite lack of evidence of Ti in the brain. These findings mentioned suggest potential effect of Ti present at a distance from the brain possibly via mediators transported by blood. Exposure of an in vitro BBB model to sera from TiO2 NPs-treated animals confirmed the tightness of the BBB and inflammatory responses. CONCLUSION: Overall, these findings suggest the clearance of TiO2 NPs at the BBB with persistent brain inflammation and underscore the role of Ti biopersistence in organs that can exert indirect effects on the CNS dependent on circulating factors.