Novel LAMP2 mutations in Chinese patients with Danon disease cause varying degrees of clinical severity.

AIMS: Danon disease is an Xlinked dominant lysosomal glycogen storage disorder characterized by cardiomyopathy, skeletal myopathy, and mental retardation. This study described two Chinese cases of Danon disease in order to broaden the phenotypic and genetic spectrum. METHODS: Clinical data were collected and LAMP2 mutations were analyzed. RESULTS: Patient A had fluctuating limb weakness during 6 months follow-up and was diagnosed with drug-induced myopathy due to anti-hepatitis B therapy with lamivudine. However, the first muscle biopsy with large cytoplasmic vacuoles confused the diagnosis and led to the second biopsy that allowed for the final diagnosis. Patient B had severe cardiac disturbances leading to sudden death. Molecularly, patient A harbored a synonymous mutation adjacent to the exon 6-intron 6 junction; mRNA analysis provided evidence that totally abolished the donor site and caused skipping of exon 6. Patient B harbored a frame-shift deletion mutation in exon 3 (c.396delA) leading to a truncated protein. DISCUSSION: To our knowledge, this is the first report of Danon disease caused by a synonymous exon mutation that affected mRNA splicing, which indicates that a synonymous substitution may not be silent when it is in the exon sequences close to the splice sites. It is also the first description of Danon disease clinically presenting as druginduced myopathy at onset; the pathological changes might be the key point for making a differential diagnosis. *These two authors contributed equally to this work.

Chromosome 1p/19q status combined with expression of p53 protein improves the diagnostic and prognostic evaluation of oligodendrogliomas.

BACKGROUND: Our previous study confirmed that oligodendrogliomas had higher frequency of chromosome 1p/19q deletion. In order to improve the diagnostic criteria and to predict the prognosis of oligodendroglioma patients, the status of chromosome 1p/19q deletion, the methylation of O(6)-methylguanine-DNA methyltransferase (MGMT), and the expression of p53 protein were evaluated and investigated in relation to patients' outcomes. METHODS: Methylation of MGMT in 73 cases was analyzed by nested methylation-specific PCR (MSP). The levels of MGMT and p53 protein were tested with immunohistochemistry. Pearson's chi-square test and Fisher's exact test were used. Multivariate and Kaplan-Meier analysis were performed to determine patients' outcomes. RESULTS: Both oligodendrogliomas and astrocytic gliomas exhibited frequent methylation of MGMT. However, the results of MSP did not completely correspond to that of the immunohistochemical staining for MGMT. The expression of p53 protein was more frequently observed in patients without a 1p or 19q deletion in anaplastic oligodendrogliomas (P = 0.032, 0.025). In low-grade oligodendrogliomas, methylation of MGMT was more frequent in patients with 1p/19q deletion than in patients with 1p/19q intact (P = 0.038). Patients with oligodendrogliomas with 1p/19q loss of heterozygosity and p53-negative showed a longer progression-free survival. CONCLUSION: Detection of chromosome 1p/19q status combined with p53 protein immunohistochemistry might be beneficial to improve the pathological diagnosis and to determine the prognosis of patients with oligodendrogliomas.

[Loss of heterozygosity of chromosome 1p/19q and p53 protein expression in oligodendroglioma].

OBJECTIVE: To study the status of loss of heterozygosity (LOH) of chromosome 1p/19q and p53 protein expression in oligodendroglioma, as compared to astrocytoma. METHODS: One hundred and ninety-one cases of glioma of different histologic types and grades, including 116 cases of low-grade of oligodendroglioma (86 paraffin-embedded and 30 fresh tissues), 45 cases of anaplastic oligodendroglioma (all paraffin-embedded tissues) and 30 cases of astrocytoma of various grades (all paraffin-embedded tissues), were enrolled into the study. The LOH of chromosome 1p/19q was investigated by polymerase chain reaction (PCR)-based microsatellite analysis. The p53 protein expression was demonstrated by immunohistochemical staining. RESULTS: The rates of 1p loss, 19q loss and 1p/19q loss were 69.8%, 64%, and 57.0% respectively in the 86 paraffin-embedded low-grade oligodendroglioma samples, as compared to 71.1%, 60.0% and 55.6% respectively in the 45 paraffin-embedded anaplastic oligodendroglioma samples. There was no difference of LOH of 1p/19q between low-grade oligodendroglioma and anaplastic oligodendroglioma (P>0.05). In the 30 cases of low-grade oligodendroglioma with fresh tissues available, the rates of 1p loss, 19q loss and 1p/19q loss were 70.0%, 63.3% and 60.0% respectively. The LOH of 1p/19q between paraffin-embedded and fresh samples was not statistically significant (P>0.05). In the 30 cases of astrocytoma, the rates of 1p loss, 19q loss and 1p/19q loss were 23.3%, 33.3% and 20.0% respectively, which were significantly less than those in oligodendroglioma (P<0.05). The expression of p53 protein was significantly lower in low-grade oligodendroglioma (8.1%) than in anaplastic oligodendroglioma (31.1%, P=0.007). The expression of p53 protein in oligodendroglioma was also lower than in astrocytoma (P=0.001). Furthermore, p53 protein expression negatively correlated with 1p/19q loss in anaplastic oligodendroglioma (P<0.05). CONCLUSIONS: Both paraffin-embedded and fresh tissues are suitable for analysis of LOH of chromosome 1p/19q. Oligodendroglioma demonstrates a higher frequency of LOH of chromosome 1p/19q and lower expression of p53 protein than astrocytoma. The LOH of chromosome 1p/19q negatively correlates with the expression of p53 protein. These parameters have both diagnostic and prognostic values.

Adenosine A2a receptor induced gliosis via Akt/NF-kappaB pathway in vitro.

Gliosis is characterized by increased expression of glial fibrillary acidic protein (GFAP) and astroglial proliferation, although the mechanism underlying the process is still largely undefined. This study explores the role of the adenosine A2a receptor (A2aR) in gliosis after ischemia-like injury in a rat astrocyte cell line transfected with A2aR. A2aR transfection enhanced GFAP expression and cell proliferation. A2aR-selective antagonist Sch58261 decreased GFAP expression in a dose- and time-dependent manner with increased activation of Akt, and induced activation of NF-kappaB. An Akt inhibitor reversed the effect of Sch58261. These results suggest that the effect of A2aR on gliosis is related to the Akt/NF-kappaB signal pathway.

Effects of P2Y1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways.

OBJECTIVE: The present study aimed to explore the role of P2Y(1) receptor in glial fibrillary acidic protein (GFAP) production and glial cell line-derived neurotrophic factor (GDNF) secretion of astrocytes under ischemic insult and the related signaling pathways. METHODS: Using transient right middle cerebral artery occlusion (tMCAO) and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, enzyme linked immunosorbent assay (ELISA) were used to investigate location of P2Y(1) receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. RESULTS: Blockage of P2Y(1) receptor with the selective antagonist N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate diammonium (MRS2179) reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y(1) receptor caused elevation of phosphorylated Akt and cAMP response element binding protein (CREB), and reduction of phosphorylated Janus kinase2 (JAK2) and signal transducer and activator of transcription3 (STAT3, Ser727). After blockage of P2Y(1) receptor and deprivation of oxygen-glucose-serum, AG490 (inhibitor of JAK2) reduced phosphorylation of STAT3 (Ser727) as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase1/2 (MEK1/2) U0126, an important molecule of Ras/extracellular signal-regulated kinase (ERK) signaling pathway, decreased the phosphorylation of JAK2, STAT3 (Ser727), Akt and CREB. CONCLUSION: These results suggest that P2Y(1) receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

Involvement of MAPK/ERK kinase-ERK pathway in exogenous bFGF-induced Egr-1 binding activity enhancement in anoxia-reoxygenation injured astrocytes.

OBJECTIVE: Intravenous administration of basic fibroblast growth factor (bFGF) is effective to reduce the volume of cerebral infract due to ischemia. This study was designed to investigate the molecular mechanism, especially the signal transduction pathways, involved in this protective role of bFGF. METHODS: Anoxia-reoxygenation treated astrocytes were used to study the role of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MAPK/ERK kinase, MEK)-ERK signaling pathway after exogenous bFGF administration by Western blot. Electrophoretic mobile shift assay was used to detect the binding activity of early growth response factor-1 (Egr-1), an important transcription factor for endogenous bFGF. RESULTS: bFGF could protect some signal transduction proteins from the oxygen-derived free radicals induced degradation. ERK1/2 was activated and involved in Egr-1 binding activity enhancement induced by exogenous bFGF. CONCLUSION: MEK-ERK MAPK cascade may be an important signal transduction pathway contributed to bFGF induced enhancement of Egr-1 binding activity in anoxia-reoxygenation injured astrocytes.

Permeability of injured blood brain barrier for exogenous bFGF and protection mechanism of bFGF in rat brain ischemia.

The study aims to explore the protection mechanism of exogenous basic fibroblast growth factor (exo-bFGF) in brain ischemia. The first part of experiment was to determine the optimal time window for the permeation of exo-bFGF through damaged blood-brain barrier in rats with permanently occluded middle cerebral arteries. 125I labeled bFGF was administered to the rats through the caudal vein. The level of gamma-rays of 125I-bFGF in the ischemic brain were found to increase at 2 h and a high level was maintained for 14 days. The morphology of the basement membrane of capillaries was observed using anti-blood-brain barrier basement membrane glycoprotein immunohistochemistry. The normal continuous linear or ribbon-like immunostain of the basement membrane became granular at 0.5 h, gradually faint and finally negative. The newly formed capillaries at the edge of the infarct still showed a negative stain after 14 days. The result suggested the optimal time window of exo-bFGF began 2 h after insult. The second part of experiment was to observe the dynamic expression of early growth response protein (Egr-1), endogenous basic fibroblast growth factor (endo-bFGF) and bFGF receptor (bFGFR) using immunohistochemistry after exo-bFGF is administered to brain. Egr-1 was more significantly enhanced in the exo-bFGF-used group than in the control group. Endo-bFGF increased gradually, reaching its peak at 7 days in the control group, while in experiment group, the endo-bFGF expression showed its first peak at 6 h, indicating that exo-bFGF could induce earlier and stronger expression of endo-bFGF. The bFGFR-group presented an early expression, reaching its maximal level at 3 h, and declining at 6 h. There were no difference in expression of bFGFR between the two groups. The infarct areas reduced from 17% to 24% in the different time intervals. The results suggested that in exo-bFGF enhanced Egr-1 protein. Egr-1 in turn might play an important role in up-regulating the expression of endo-bFGF which overlapped with the expression of bFGFR to ensure the combination of ligand and receptor to protect against brain ischemia.

Growth enhancement effect of BzATP on primary cultured astrocytes from rat brain.

OBJECTIVE: To explore whether BzATP could promote the growth of primary cultured astrocytes (AS) of rat and its possible mechanism, and whether TGF-ß1 was involved in the event. METHODS: The primary cultured ASs were derived from new born Sprague-Dawley rats. Glial fibrillary acidic protein (GFAP) immunofluorescent staining was used to check the purity of cultured AS. Morphometry was used to detect the changes of AS. The proliferation index of AS was detected by BrdU incorporation assay. Western blot was used to detect the changes of GFAP under different conditions. Changes of TGF-ß1 gene transcription were detected by RT-PCR. ELISA was utilized to detect the variation of TGF-ß1 protein in the supernate. RESULTS: The purity of primary cultured AS reached 99%. BzATP promoted the hypertrophy of AS including the elongation of AS processes and the enlargement of cell bodies, BzATP also promoted the expression of GFAP in existence of Ca²âº, but had no effect on cell proliferation. BzATP increased the transcription of TGF-ß1 mRNA and the release of TGF-ß1 protein in existence of Ca²âº. TGF-ß1 neutralizing antibody partially inhibited the expression of GFAP induced by BzATP, but had no effect on AS proliferation and cell morphology. CONCLUSION: BzATP enhanced the hypertrophy of primary cultured AS, increased the expression of GFAP partially through TGF-ß1. Mechanisms of the enhancement of AS growth induced by BzATP other than TGF-ß1 pathway remain to be elucidated.