Newly developed targeted anticancer drugs inhibit signaling pathways commonly altered in adult and pediatric cancers. However, as these pathways are also essential for normal brain development, ...concerns have emerged of neurologic sequelae resulting specifically from their application in pediatric cancers. The neural substrates and age dependency of these drug-induced effects
are unknown, and their long-term behavioral consequences have not been characterized. This study defines the age-dependent cellular and behavioral effects of these drugs on normally developing brains and determines their reversibility with post-drug intervention. Mice at different postnatal ages received short courses of molecularly targeted drugs in regimens analagous to clinical treatment. Analysis of rapidly developing brain structures important for sensorimotor and cognitive function showed that, while adult administration was without effect, earlier neonatal administration of targeted therapies attenuated white matter oligodendroglia and hippocampal neuronal development more profoundly than later administration, leading to long-lasting behavioral deficits. This functional impairment was reversed by rehabilitation with physical and cognitive enrichment. Our findings demonstrate age-dependent, reversible effects of these drugs on brain development, which are important considerations as treatment options expand for pediatric cancers.
Targeted therapeutics elicit age-dependent long-term consequences on the developing brain that can be ameliorated with environmental enrichment.
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To characterize the mechanism of Zika virus (ZIKV)‐associated microcephaly, we performed immunolabeling on brain tissue from a 20‐week fetus with intrauterine ZIKV infection. Although ZIKV ...demonstrated a wide range of neuronal and non‐neuronal tropism, the infection rate was highest in intermediate progenitor cells and immature neurons. Apoptosis was observed in both infected and uninfected bystander cortical neurons, suggesting a role for paracrine factors in induction of neuronal apoptosis. Our results highlight differential neuronal susceptibility and neuronal apoptosis as potential mechanisms in the development of ZIKV‐associated microcephaly, and may provide insights into the design and best timing of future therapy. Ann Neurol 2017;82:121–127
Down syndrome (DS), the most frequent genetic cause of intellectual disability and developmental delay, results from impaired neural stem cell proliferation and differentiation. Impaired neurogenesis ...in the neocortex, hippocampus and cerebellum is believed to be the underlying cause of learning and behavioral deficits in the Ts65Dn mouse model of DS. Aggressive sensorimotor and cognitive therapies have shown promise in mitigating the cognitive disabilities in DS but these behavioral therapies have not yet been investigated at the cellular level. Here, using the Ts65Dn mouse model of DS, we demonstrate that a combination of environmental enrichment and physical exercise starting in juvenile mice (postnatal day 18) markedly increases cell proliferation, neurogenesis and gliogenesis in the hippocampal dentate gyrus (DG) and the forebrain subventricular zone (SVZ) of both male and female mice. Enrichment and exercise increased the rate of Ts65Dn DG neurogenesis to be comparable to that of the nonenriched euploid group, while the effect on SVZ neurogenesis was reduced and seen only after prolonged exposure. These results clearly indicate that in a comprehensive stimulatory environment, the postnatal DS brain has the intrinsic capability of improving neurogenesis and gliogenesis to the levels of normal matched controls and that this cellular response underlies the cognitive improvement seen following behavioral therapies.
The study of cerebral metabolites relies heavily on detection methods and sample preparation. Animal experiments in vivo require anesthetic agents that can alter brain metabolism, whereas ex vivo ...experiments demand appropriate fixation methods to preserve the tissue from rapid postmortem degradation. In this study, the metabolic profiles of mouse hippocampi using proton magnetic resonance spectroscopy (1H‐MRS) were compared in vivo and in situ with or without focused beam microwave irradiation (FBMI) fixation. Ten major brain metabolites, including lactate (Lac), N‐acetylaspartate (NAA), total choline (tCho), myo‐inositol (mIns), glutamine (Gln), glutamate (Glu), aminobutyric acid (GABA), glutathione (GSH), total creatine (tCr) and taurine (Tau), were analyzed using LCModel. After FBMI fixation, the concentrations of Lac, tCho and mIns were comparable with those obtained in vivo under isoflurane, whereas other metabolites were significantly lower. Except for a decrease in NAA and an increase in Tau, all the other metabolites remained stable over 41 hours in FBMI‐fixed brains. Without FBMI, the concentrations of mIns (before 2 hours), tCho and GABA were close to those measured in vivo. However, higher Lac (P < .01) and lower NAA, Gln, Glu, GSH, tCr and Tau were observed (P < .01). NAA, Gln, Glu, GSH, tCr and Tau exhibited good temporal stability for at least 20 hours in the unfixed brain, whereas a linear increase of tCho, mIns and GABA was observed. Possible mechanisms of postmortem degradation are discussed. Our results indicate that a proper fixation method is required for in situ detection depending on the targeted metabolites of specific interests in the brain.
The mouse hippocampal metabolites after direct decapitation (no fixation), by focused beam microwave irradiation (FBMI) fixation over 41 hours, and in vivo condition under isoflurane anesthesia, were analyzed from 1H‐MRS. Metabolites showing various time curves under different conditions indicate that a proper fixation method is required depending on the targeted metabolites of specific interest.
Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to ...induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50). We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro. NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.
Abstract Background Neonatal Encephalopathy (NE) is a prominent cause of infant mortality and neurodevelopmental disability. Hypothermia is an effective neuroprotective therapy for newborns with ...encephalopathy. Post-hypothermia functional–anatomical correlation between neonatal neurobehavioral abnormalities and brain injury findings on MRI in encephalopathic newborns has not been previously described. Aim To evaluate the relationship between neonatal neurobehavioral abnormalities and brain injury on magnetic resonance imaging (MRI) in encephalopathic newborns treated with therapeutic hypothermia. Study design Neonates with hypoxic ischemic encephalopathy (HIE) referred for therapeutic hypothermia were prospectively enrolled in this observational study. Neurobehavioral functioning was assessed with the NICU network neurobehavioral scale (NNNS) performed at target age 14 days. Brain injury was assessed by MRI at target age 7–10 days. NNNS scores were compared between infants with and without severe MRI injury. Subjects & outcome measures Sixty-eight term newborns (62% males) with moderate to severe encephalopathy underwent MRI at median 8 days (range 5–16) and NNNS at median 12 days of life (range 5–20). Fifteen (22%) had severe injury on MRI. Results Overall Total Motor Abnormality Score and individual summary scores for Non-optimal Reflexes and Asymmetry were higher, while Total NNNS Z-score across cognitive/behavioral domains was lower (reflecting poorer performance) in infants with severe MRI injury compared to those without ( p < 0.05). Conclusions Neonatal neurobehavioral abnormalities identified by the NNNS are associated with MRI brain injury in encephalopathic newborns post-hypothermia. The NNNS can provide an early functional assessment of structural brain injury in newborns, which may guide rehabilitative therapies in infants after perinatal brain injury.
Diffuse white matter injury (DWMI) caused by hypoxia is associated with permanent neurodevelopmental disabilities in preterm infants. The cellular and molecular mechanisms producing DWMI are poorly ...defined. Using a mouse model of neonatal hypoxia, we demonstrate a biphasic effect on oligodendrocyte development, resulting in hypomyelination. Oligodendrocyte death and oligodendrocyte progenitor cell (OPC) proliferation during the week after hypoxia were followed by delayed oligodendrocyte differentiation and abnormal myelination, as demonstrated by electron microscopy. Cdk2 activation was essential for the regenerative OPC response after hypoxia and was accompanied by reduced FoxO1-dependent p27
Kip1
expression. p27
Kip1
was also reduced in OPCs in human infant white matter lesions after hypoxia. The negative effects of hypoxia on oligodendrogenesis and myelination were more pronounced in p27
Kip1
-null mice; conversely, overexpression of FoxO1 or p27
Kip1
in OPCs after hypoxia promoted oligodendrogenesis. Our studies demonstrate for the first time that neonatal hypoxia affects the Foxo1/p27
Kip1
pathway during white matter development. We also show that molecular manipulation of this pathway enhances oligodendrocyte regeneration during a critical developmental time window after DWMI. Thus, FoxO1 and p27
Kip1
may serve as promising target molecules for promoting timely oligodendrogenesis in neonatal DWMI.
Diffuse white matter injury (DWMI) caused by hypoxia is associated with permanent neurodevelopmental disabilities in preterm infants. The cellular and molecular mechanisms producing DWMI are poorly ...defined. Using a mouse model of neonatal hypoxia, we demonstrate a biphasic effect on oligodendrocyte development, resulting in hypomyelination. Oligodendrocyte death and oligodendrocyte progenitor cell (OPC) proliferation during the week after hypoxia were followed by delayed oligodendrocyte differentiation and abnormal myelination, as demonstrated by electron microscopy. Cdk2 activation was essential for the regenerative OPC response after hypoxia and was accompanied by reduced FoxO1-dependent p27(Kip1) expression. p27(Kip1) was also reduced in OPCs in human infant white matter lesions after hypoxia. The negative effects of hypoxia on oligodendrogenesis and myelination were more pronounced in p27(Kip1)-null mice; conversely, overexpression of FoxO1 or p27(Kip1) in OPCs after hypoxia promoted oligodendrogenesis. Our studies demonstrate for the first time that neonatal hypoxia affects the Foxo1/p27(Kip1) pathway during white matter development. We also show that molecular manipulation of this pathway enhances oligodendrocyte regeneration during a critical developmental time window after DWMI. Thus, FoxO1 and p27(Kip1) may serve as promising target molecules for promoting timely oligodendrogenesis in neonatal DWMI.
Abstract
Background
Zika virus (ZIKV) infection during pregnancy may result in severe neurologic injury to the fetus. The mechanisms by which ZIKV injures fetal brain are not fully characterized. ...Although cell culture and animal models shed valuable insight into pathogenesis, they do not fully recapitulate human disease.
Methods
To characterize the mechanism of ZIKV-induced human brain injury, we performed immunolabeling on brain tissue from a 20-week fetus with intrauterine ZIKV infection. Formalin-fixed sections of brain tissue were co-immunostained with ZIKV envelope antibody, as well as neuronal and non-neuronal lineage cell markers to assess infection within populations. Apoptosis was assessed by quantifying activated caspase 3 -positive staining cells. Minimum 3–5 random microscopic fields per brain region were photographed and quantified in an automated fashion using the ImageJ Cell Counter plug-in. GraphPad Prism and Microsoft Excel software were used for data analysis.
Results
ZIKV demonstrated a wide range of neuronal and non-neuronal tropism. However, infection rate was highest in Tbr2+ - Intermediate Progenitor Cells (IPC; 81.4±12%) and DCX+ Immature Neurons (IN; 51.5±13.9%), followed by SOX2+/ Nestin+ Neural Precursor Cells (NPC; 26.6±13.4%). NeuN+ Mature Neurons had the lowest frequency of infection (MN; 10.0±7.0 %) (Figure). Apoptosis was observed in both infected and uninfected bystander cortical neurons. A high infection frequency was also observed in non-neuronal cells (astrocytes, microglia, macrophages, lymphocytes).
Conclusion
Our study provides valuable insights into ZIKV pathogenesis in the fetus; it is the first to demonstrate differential infectivity/susceptibility of neuronal lineage cells to ZIKV, and evidence of apoptosis in and around these cells. The high frequency of ZIKV+ IPC and IN implies that that infection can be supported until the immature stage of neuronal differentiation. The resistance of mature neurons to ZIKV infection may also explain why ZIKV infection in the third trimester poses less risk of microcephaly in infants. The high infection rate of non-neuronal cells also suggests potential contribution of immune-mediated mechanisms of brain injury in the setting of congenital ZIKV infection.
Disclosures
All authors: No reported disclosures.
During the perinatal period, unique metabolic adaptations support energetic requirements for rapid growth. To gain insight into perinatal adaptations, quantitative proteomics were performed comparing ...the livers of yorkshire pigs at postnatal day seven and adult. These data revealed differences in the metabolic control of liver function including significant changes in lipid and carbohydrate metabolic pathways. Newborn livers showed an enrichment of proteins in lipid catabolism and gluconeogenesis concomitant with elevated liver carnitine and acylcarnitines levels. Sugar kinases were some of the most dramatically differentially enriched proteins comparing neonatal and adult pigs including galactokinase 1 (Galk1), ketohexokinase (KHK), hexokinase 1 (HK1) and hexokinase 4 (GCK). Interestingly, hexokinase domain containing 1 (HKDC1), a newly identified fifth hexokinase associated with glucose disturbances in pregnant women was highly enriched in the liver during the prenatal and perinatal periods and continuously declined throughout postnatal development in pigs and mice. These changes were confirmed via Western blot and mRNA expression. These data provide new insights into the developmental and metabolic adaptations in the liver during the transition from the perinatal period to adulthood in multiple mammalian species.
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