Ilker Y. Eyüpoglu

Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1.

Excitotoxic brain lesions initially result in the primary destruction of brain parenchyma, after which microglial cells migrate towards the sites of injury. At these sites, the cells produce large quantities of oxygen radicals and cause secondary damage that accounts for most of the loss of brain function. Here we show that this microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, regulated by the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) through the formation of a nuclear PARP–NF-κB-protein complex. Downregulation of PARP or CD11a by transfection with antisense DNA abrogated microglial migration almost completely and prevented neurons from secondary damage.

Common mutations of beta-catenin in adamantinomatous craniopharyngiomas but not in other tumours originating from the sellar region.

Dysregulation of the Wnt signalling pathway contributes to developmental abnormalities and carcinogenesis of solid tumours. Here, we examined β-catenin and adenomatous polyposis coli (APC) by mutational analysis in pituitary adenomas (n=60) and a large series of craniopharyngiomas (n=41). Furthermore, the expression pattern of β-catenin was immunohistochemically analysed in a cohort of tumours and cysts of the sellar region including pituitary adenomas (n=58), craniopharyngiomas (n=57), arachnoidal cysts (n=8), Rathke’s cleft cysts (n=10) and xanthogranulomas (n=6). Whereas APC mutations were not detectable in any tumour entity, β-catenin mutations were present in 77% of craniopharyngiomas, exclusively of the adamantinomatous subtype. All mutations affected exon 3, which encodes the degradation targeting box of β-catenin compatible with an accumulation of nuclear β-catenin protein. In addition, a novel 81-bp deletion of this exonic region was detected in one case. Immunohistochemical analysis confirmed a shift from membrane-bound to nuclear accumulation of β-catenin in 94% of the adamantinomatous tumours. Aberrant distribution patterns of β-catenin were never observed in the other tumour entities under study. We conclude that β-catenin mutations and/or nuclear accumulation serve as diagnostic hallmarks of the adamantinomatous variant, setting it apart from the papillary variant of craniopharyngioma.

Selenium deficiency increases susceptibility to glutamate-induced excitotoxicity.

Excitotoxic brain lesions, such as stroke and epilepsy, lead to increasing destruction of neurons hours after the insult. The deadly cascade of events involves detrimental actions by free radicals and the activation of proapoptotic transcription factors, which finally result in neuronal destruction. Here, we provide direct evidence that the nutritionally essential trace element selenium has a pivotal role in neuronal susceptibility to excitotoxic lesions. First, we observed in neuronal cell cultures that addition of selenium in the form of selenite within the physiological range protects against excitotoxic insults and even attenuates primary damage. The neuroprotective effect of selenium is not directly mediated via antioxidative effects of selenite but requires de novo protein synthesis. Gel shift analysis demonstrates that this effect is connected to the inhibition of glutamate‐induced NF‐κB and AP‐1 activation. Furthermore, we provide evidence that selenium deficiency in vivo results in a massive increase in susceptibility to kainate‐induced seizures and cell loss. These findings indicate the importance of selenium for prevention and therapy of excitotoxic brain damage.

Histone deacetylase inhibitors: possible implications for neurodegenerative disorders

During the past six years numerous studies identified histone deacetylase (HDAC) inhibitors as candidate drugs for the treatment of neurodegenerative disorders. Two major neuroprotective mechanisms of HDAC inhibitors have been identified, namely the transcriptional activation of disease-modifying genes and the correction of perturbations in histone acetylation homeostasis, which have been shown to be intimately involved in the neurodegenerative pathomechanisms of Huntingtons, Parkinsons and Kennedy disease, amyotropic lateral sclerosis, Rubinstein-Taybi syndrome as well as stroke. Based on the promising in vitro and in vivo analyses, clinical trials have been initiated to evaluate the safety and efficacy of HDAC inhibitors for the treatment of devastating diseases such as Huntingtons disease, amyotropic lateral sclerosis and spinal muscular atrophy. Here, the authors summarize and discuss the findings on the emerging field of epigenetic therapy strategies in neurodegenerative disorders.

In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non‐toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an α‐motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone‐rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m‐Carboxycinnamic acid bis‐Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS‐275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood–brain barrier, oral administration may allow deceleration of progressive α‐motoneurone degeneration by epigenetic SMN2 gene activation.

Small interfering RNA-mediated xCT silencing in gliomas inhibits neurodegeneration and alleviates brain edema

Neurodegeneration and brain edema are hallmarks of human malignant brain tumors. Here we show that genetic or pharmacological inhibition of the glutamate transporter xCT (Xc− system, encoded by SLC7a11) in vivo leads to abrogated neurodegeneration, attenuated perifocal edema and prolonged survival. These results show a crucial role for xCT in glioma-induced neurodegeneration and brain edema, corroborating the concept that edema formation may be in part a consequence of peritumoral cell death.

Suberoylanilide hydroxamic acid (SAHA) has potent anti-glioma properties in vitro, ex vivo and in vivo

Current treatment modalities for malignant gliomas do not allow long‐term survival. Here, we identify suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylases (HDAC), as an effective experimental anti‐glioma agent. Administration of SAHA to various glioma cell lines obtained from human, rat and mouse inhibited tumour cell growth in a range of 1–10 μm. This anti‐glioma property is associated with up‐regulation of the cell cycle control protein p21/WAF, as well as the induction of apoptosis. A novel tumour invasion model using slice cultures of rat brain corroborated the anti‐glioma properties of SAHA in the organotypic brain environment. In this model, glioma invasion compromised adjacent brain parenchyma, and this tumour‐associated cytotoxicity could be inhibited by SAHA. In addition, a 10‐fold dose escalation experiment did not challenge the viability of cultured brain slices. In vivo, a single intratumoural injection of SAHA 7 days after orthotopic implantation of glioma cells in syngeneic rats doubled their survival time. These observations identify chromatin‐modifying enzymes as possible and promising targets for the pharmacotherapy of malignant gliomas.

Survival motor neuron gene 2 silencing by DNA methylation correlates with spinal muscular atrophy disease severity and can be bypassed by histone deacetylase inhibition

Spinal muscular atrophy (SMA), a common neuromuscular disorder, is caused by homozygous absence of the survival motor neuron gene 1 (SMN1), while the disease severity is mainly influenced by the number of SMN2 gene copies. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We demonstrate that the SMN2 gene is subject to gene silencing by DNA methylation. SMN2 contains four CpG islands which present highly conserved methylation patterns and little interindividual variations in SMN1-deleted SMA patients. The comprehensive analysis of SMN2 methylation in patients suffering from severe versus mild SMA carrying identical SMN2 copy numbers revealed a correlation of CpG methylation at the positions −290 and −296 with the disease severity and the activity of the first transcriptional start site of SMN2 at position −296. These results provide first evidence that SMN2 alleles are functionally not equivalent due to differences in DNA methylation. We demonstrate that the methyl-CpG-binding protein 2, a transcriptional repressor, binds to the critical SMN2 promoter region in a methylation-dependent manner. However, inhibition of SMN2 gene silencing conferred by DNA methylation might represent a promising strategy for pharmacologic SMA therapy. We identified histone deacetylase (HDAC) inhibitors including vorinostat and romidepsin which are able to bypass SMN2 gene silencing by DNA methylation, while others such as valproic acid and phenylbutyrate do not, due to HDAC isoenzyme specificities. These findings indicate that DNA methylation is functionally important regarding SMA disease progression and pharmacological SMN2 gene activation which might have implications for future SMA therapy regimens.

Improving the extent of malignant glioma resection by dual intraoperative visualization approach.

Despite continuing debates around cytoreductive surgery in malignant gliomas, there is broad consensus that increased extent of tumor reduction improves overall survival. However, maximization of the extent of tumor resection is hampered by difficulty in intraoperative discrimination between normal and pathological tissue. In this context, two established methods for tumor visualization, fluorescence guided surgery with 5-ALA and intraoperative MRI (iMRI) with integrated functional neuronavigation were investigated as a dual intraoperative visualization (DIV) approach. Thirty seven patients presumably suffering from malignant gliomas (WHO grade III or IV) according to radiological appearance were included. Twenty-one experimental sequences showing complete resection according to the 5-ALA technique were confirmed by iMRI. Fourteen sequences showing complete resection according to the 5-ALA technique could not be confirmed by iMRI, which detected residual tumor. Further analysis revealed that these sequences could be classified as functional grade II tumors (adjacent to eloquent brain areas). The combination of fluorescence guided resection and intraoperative evaluation by high field MRI significantly increased the extent of tumor resection in this subgroup of malignant gliomas located adjacent to eloquent areas from 61.7% to 100%; 5-ALA alone proved to be insufficient in attaining gross total resection without the danger of incurring postoperative neurological deterioration. Furthermore, in the case of functional grade III gliomas, iMRI in combination with functional neuronavigation was significantly superior to the 5-ALA resection technique. The extent of resection could be increased from 57.1% to 71.2% without incurring postoperative neurological deficits.

Experimental therapy of malignant gliomas using the inhibitor of histone deacetylase MS-275

Inhibitors of histone deacetylases are promising compounds for the treatment of cancer but have not been systematically explored in malignant brain tumors. Here, we characterize the benzamide MS-275, a class I histone deacetylase inhibitor, as potent drug for experimental therapy of glioblastomas. Treatment of four glioma cell lines (U87MG, C6, F98, and SMA-560) with MS-275 significantly reduced cell growth in a concentration-dependent manner (IC90, 3.75 μmol/L). Its antiproliferative effect was corroborated using a bromodeoxyuridine proliferation assay and was mediated by G0-G1 cell cycle arrest (i.e., up-regulation of p21/WAF) and apoptotic cell death. Implantation of enhanced green fluorescent protein–transfected F98 glioma cells into slice cultures of rat brain confirmed the cytostatic effect of MS-275 without neurotoxic damage to the organotypic neuronal environment in a dose escalation up to 20 μmol/L. A single intratumoral injection of MS-275 7 days after orthotopic implantation of glioma cells in syngeneic rats confirmed the chemotherapeutic efficacy of MS-275 in vivo. Furthermore, its propensity to pass the blood-brain barrier and to increase the protein level of acetylated histone H3 in brain tissue identifies MS-275 as a promising candidate drug in the treatment of malignant gliomas. [Mol Cancer Ther 2006;5(5):1248–55]