Unaffected Li-Fraumeni Syndrome Carrier Parent Demonstrates Allele-Specific mRNA Stabilization of Wild-Type TP53 Compared to Affected Offspring.

Li-Fraumeni Syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is inherited by offspring of a carrier parent. p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Unexpectedly, some mutant TP53 carriers remain unaffected, while their children develop cancer early in life. To begin unravelling this paradox, the response of dermal fibroblasts (dFb) isolated from a child with LFS was compared to those from her unaffected father after UV exposure. Phospho-Chk1[S345], a key activator of cell cycle arrest, was increased by UV induction in the LFS patient compared to their unaffected parent dFb. This result, along with previous findings of reduced CDKN1A/p21 UV induction in affected dFb, suggest that cell cycle dysregulation may contribute to cancer onset in the affected LFS subject but not the unaffected parent. Mutant p53 protein and its promoter binding affinity were also higher in dFb from the LFS patient compared to their unaffected parent. These results were as predicted based on decreased mutant TP53 allele-specific mRNA expression previously found in unaffected dFb. Investigation of the potential mechanism regulating this TP53 allele-specific expression found that, while epigenetic promoter methylation was not detectable, TP53 wild-type mRNA was specifically stabilized in the unaffected dFb. Hence, the allele-specific stabilization of wild-type TP53 mRNA may allow an unaffected parent to counteract genotoxic stress by means more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS.

GPR17 plays a role in ischemia-induced endogenous repair of immature neonatal cerebral White matter.

Whether GPR17 has the same distribution and repair mechanism in immature white matter with periventricular leukomalacia (PVL) as in the adult brain remains to be determined. This study tried to explore the expression phase and site of GPR17, and to investigate the effect of silencing GPR17 on endogenous repair mechanism of immature white matter with PVL. Ischemic PVL in vivo results showed that GPR17 gene and protein expression increased more in the PVL than in the sham group at 12 h-24 h and 72h to 7 days after PVL. NG2+/GPR17+progenitor cells at 48 h-96 h and O4+/GPR17+precursor cells at 72h to 7d were also significantly increased in the PVL compared to the sham groups. Results in vitro showed that oxygen-glucose deprivation (OGD) also induced more GPR17 gene and protein expression than control at 48 h-72 h. There were more NG2+/GPR17+progenitor cells at 24 h-48 h and O4+/GPR17+precursor cells at 48 h-72 h in the OGD groups, as well. The functional role of GPR17 in the intrinsic repair response to ischemia was tested using GPR17 gene silencing. The progenitor cells and OL precursors in the OGD+GPR17 silencing group were both significantly less than those in the control, OGD and OGD+gene silencing control groups. The apoptotic percentage of cells in OGD+GPR17 silencing group was also much higher. In summary, ischemia-induced GPR17 expression was shown to contribute to glial-derived progenitor cell proliferation and differentiation into OL precursors, which may provide a therapeutic target for immature neonatal white matter injury after ischemia.

Application of a radiosensitivity flow assay in a patient with DNA ligase 4 deficiency.

DNA ligase 4 deficiency (LIG4-SCID) causes lymphopenia (T-B-NK+) and a radiosensitive SCID (RS-SCID) phenotype. We demonstrate, for the first time, flow cytometric-based kinetic analysis of phosphorylated H2AX (γH2AX) in lymphocyte subsets, especially NK cells, for the assessment of LIG4-SCID. Measurement of phosphorylated (p) ATM, SMC1, and H2AX (γH2AX) was performed by flow cytometry to assess DNA repair defects in a 3-year-old girl. Functional assessment (phosphorylation) was measured in T and NK cells (B cells were absent) before irradiation (background control) or after low-dose (2Gy) irradiation (1 and 24 hours). We observed maximal γH2AX at 1 hour postirradiation, with dephosphorylation at 24 hours postirradiation in healthy control patients. The patient showed normal frequencies (percentage) of T cells and NK cells for γH2AX, but increased levels of γH2AX compared with control patients at 1 hour postirradiation. At 24 hours postirradiation, there was a lack of dephosphorylation in a substantial proportion of lymphocytes (with differences observed between T and NK cells) compared with healthy control patients. Although there was dephosphorylation of γH2AX at 24 hours in patient lymphocytes compared with 1 hour, the amount remained elevated at 24 hours compared with in control patients. The data from pATM and pSMC1 were uninformative. Flow-based kinetic analysis of γH2AX is a useful marker for the diagnosis of LIG4-SCID.

Allele-specific wild-type TP53 expression in the unaffected carrier parent of children with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is passed from parent to child. Tumor protein p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Paradoxically, some mutant TP53 carriers remain unaffected, while their children develop cancer within the first few years of life. To address this paradox, response to UV stress was compared in dermal fibroblasts (dFb) from an affected LFS patient vs. their unaffected carrier parent. UV induction of CDKN1A/p21, a regulatory target of p53, in LFS patient dFb was significantly reduced compared to the unaffected parent. UV exposure also induced significantly greater p53[Ser15]-phosphorylation in LFS patient dFb, a reported property of some mutant p53 variants. Taken together, these results suggested that unaffected parental dFb may express an increased proportion of wild-type vs. mutant p53. Indeed, a significantly increased ratio of wild-type to mutant TP53 allele-specific expression in the unaffected parent dFb was confirmed by RT-PCR-RFLP and RNA-seq analysis. Hence, allele-specific expression of wild-type TP53 may allow an unaffected parent to mount a response to genotoxic stress more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS.

Tissue factor inflammatory response regulated by promoter genotype and p38 MAPK in neonatal vs. adult microvascular endothelial cells.

OBJECTIVE AND DESIGN: Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18-basepair deletion (D) or insertion (I) at -1,208. We sought to determine the relationship between these haplotypes and interleukin-1α (IL-1α)-induced TF expression in neonatal versus adult HMVEC. RESULTS: IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed threefold higher levels of TF mRNA, twofold higher TF protein, and fourfold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates, but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. CONCLUSIONS: Upregulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants.

White matter and SVZ serve as endogenous sources of glial progenitor cells for self-repair in neonatal rats with ischemic PVL.

Mounting evidence suggests that endogenous progenitor cells may initiate cerebral WM repair. This study was designed to determine whether endogenous glial progenitor cells derived from either the subventricular zone (SVZ) or the white matter (WM) contribute to WM repair in a neonatal rat model of ischemic periventricular leukomalacia (PVL). Additionally, the role of G protein-coupled receptor 17 (GPR17), recently shown to act as a sensor for WM damage, was explored to assess its potential recruitment and activation of endogenous glial progenitor cells for such WM self-repair. Our in vivo and in vitro models consisted of five-day-old neonatal rats or cultured glial progenitor cells derived from both the SVZ and WM of these rats, randomly divided into sham/control and induced ischemic PVL/oxygen-glucose deprivation (OGD) groups. The WM of all PVL rats showed either mild or severe histopathological changes, with significantly increased in vivo apoptosis and poor myelination compared to those of the sham group. Significantly more apoptotic and necrotic cells were also detected in the OGD glial progenitor cell cultures derived from the SVZ and WM at all time intervals. The glial progenitor cells were significantly increased in both the SVZ (NG2⁺/GPR17⁻/BrdU⁺) and WM (NG2⁺/GPR17⁺/BrdU⁺) within 72 h after PVL; preOLs were also increased significantly in both the SVZ (O4⁺/GPR17⁻/BrdU⁺) and WM (O4⁺/GPR17⁺/BrdU⁺) within 7d after PVL in vivo or OGD in vitro. However, the more differentiated CNPase⁺/GPR17⁻/BrdU⁺ and MBP⁺/GPR17⁻/BrdU⁺ OLs in the SVZ and WM remained significantly less than those in the sham groups up to 14d or 21d after OGD or PVL, respectively. Hence, both the WM and SVZ were found to be potential endogenous sources of glial progenitor cells for WM repair in PVL rats. However their endogenous self-repair capacity appeared to be limited, since the more mature OLs did not completely recover from experimental ischemia, even after 14-21d.

Diphenyleneiodonium protects preoligodendrocytes against endotoxin-activated microglial NADPH oxidase-generated peroxynitrite in a neonatal rat model of periventricular leukomalacia.

The contribution of microglial activation to preoligodendroglial (preOL) damage in the central nervous system (CNS) is considered to be one of the principal causes of periventricular leukomalacia (PVL) pathogenesis. The present study explores the effect of diphenyleneiodonium (DPI), a NADPH oxidase (NOX) inhibitor, on protection of preOLs from bacterial lipopolysaccharide (LPS)-induced microglial toxicity in vivo and in vitro. In vitro, preOLs co-cultured with microglia exhibited increased preOL apoptosis, accompanied by overproduction of superoxide anion (O(2)(-)) and the formation of peroxynitrite (ONOO(-)) after LPS exposure. LPS also significantly up-regulated accumulation of activated microglial NOX subunits p67-phox and gp91-phox in the plasma membrane. Diphenyleneiodonium (DPI) (10µm) was found to significantly attenuate up-regulation of this NOX activity. In vivo, DPI was administered (1mg/kg/day) by subcutaneous injection for 3 days to two-day-old neonatal Sprague-Dawley rats subjected to intracerebral injection of LPS. Treatment with DPI within 24h of LPS injection significantly ameliorated white matter injury, decreasing preOL loss, O(2)(-) generation, and ONOO(-) formation, and inhibiting p67-phox, gp91-phox synthesis and p67phox membrane translocation in microglia. These results indicated that LPS-induced preOL apoptosis may have been mediated by microglia-derived ONOO(-). DPI prevented this LPS-induced brain injury, most likely by inhibiting ONOO(-) formation via NOX, thereby preventing preOL loss and immature white matter injury.

Periventricular leukomalacia long-term prognosis may be improved by treatment with UDP-glucose, GDNF, and memantine in neonatal rats.

The therapeutic effects of UDP-glucose (UDPG), an endogenous agonist of GPR17 that may promote the self-repair of white matter, glial cell line-derived neurotrophic factor (GDNF), a neurotrophic factor correlated with the growth and survival of nerve cells, and memantine, an antagonist of NMDA receptors, were evaluated for functional improvement of neonatal rats with experimental periventricular leukomalacia (PVL). Five day-old neonatal rat pups were subjected to an ischemia-induced model of PVL. The pups were then randomly divided into sham, PVL, PVL plus UDPG, PVL plus GDNF, and PVL plus memantine groups. All pups were weighed and the age at first eye opening recorded. Pathological changes and myelin sheath formation in the white matter were assessed under both light and electron microscopy on day 7 and 21 after induction of PVL. Values of escape latency (EL) and swimming distance (SD) in Morris water maze test, and the modified inclined plane scores in Rivlin inclined plane test were recorded for rats on day 26. Pups in the PVL group were found to be significantly lower in weight (p<0.05), delayed in age at first eye opening (p<0.01), and impaired in their inclined plane (p<0.01) and Morris water maze (p<0.01) performance compared with those in the sham, UDPG, GDNF and memantine groups. Histopathological grading of the white matter classified all pups in the PVL group with significantly more severe injury (p<0.01), and the number and thickness of their myelin sheaths were significantly less (p<0.01), compared to the UDPG, GDNF, memantine, or sham groups. These results indicate that treatment with UDPG, GDNF, and memantine may significantly improve long-term prognosis in neonatal rats with cerebral white matter injury, characteristic of PVL.

Curcumin protects pre-oligodendrocytes from activated microglia in vitro and in vivo.

Infection and inflammation leading to injury or death of pre-oligodendrocytes (preOLs) is one of the principal initiating mechanisms in the pathogenesis of preterm periventricular leukomalacia (PVL). The present study explores the possible protective effect of curcumin against the toxicity of lipopolysaccharide (LPS)-activated microglia on preOLs in vitro and in vivo. In vitro, preOLs in coculture with microglia exhibited increased apoptosis after exposure to LPS. LPS also induced significantly increased expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase (NOX) subunits, p67-phox and gp91-phox in microglia. Our results suggest that iNOS and NOX contribute to the apoptosis of preOLs by activated microglia. The potential anti-inflammatory effects of curcumin were tested to determine if they could help to minimize microglia-mediated damage. Curcumin (10 microg/ml) was found to significantly inhibit the apoptosis of preOL and expression of either iNOS or NOX in the LPS-activated microglia. In vivo, curcumin was administered (50 mg/kg/day, i.p.) to two-day-old neonatal Sprague-Dawley rats subjected to intracerebral injection of LPS. Treatment with curcumin either 1h before or immediately after LPS injection significantly ameliorated white matter injury and loss of preOLs, decreased activated microglia, and inhibited microglial expression of iNOS and translocation of p67phox and gp91phox to the microglial cell membranes in neonatal rat brains following LPS injection. These results suggest that curcumin has a protective effect on infection-driven white matter injury, which is associated with suppression of iNOS and NOX activation. Consequently, curcumin may have potential as a protective agent against immature white matter injury.

Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro.

To determine if megakaryocytes are targeted by immune thrombocytopenic purpura (ITP) autoantibodies, as are platelets, we have studied the effects of ITP plasma on in vitro megakaryocytopoiesis. Umbilical cord blood mononuclear cells were incubated in the presence of thrombopoietin and 10% plasma from either ITP patients (n = 53) or healthy donors. The yield of megakaryocytic cells, as determined by flow cytometry, was significantly reduced in the presence of ITP plasma containing antiplatelet glycoprotein Ib (GPIb) autoantibodies (P <.001) as compared with both the control and patient plasma with no detectable anti-GPIIb/IIIa or anti-GPIb autoantibodies. Platelet absorption of anti-GPIb autoantibodies in ITP plasmas resulted in double the megakaryocyte production of the same plasmas without absorption, whereas platelet absorption of control plasma had no effect on megakaryocyte yield. Furthermore, 2 human monoclonal autoantibodies isolated from ITP patients, 2E7, specific for human platelet glycoprotein IIb heavy chain, and 5E5, specific for a neoantigen on glycoprotein IIIa expressed on activated platelets, had significant inhibitory effects on in vitro megakaryocytopoiesis (P <.001). Taken together, these data indicate that autoantibodies against either platelet GPIb or platelet GPIIb/IIIa in ITP plasma not only are involved in platelet destruction, but may also contribute to the inhibition of platelet production.