Increased matrix metalloproteinases expression in tuberous sclerosis complex: modulation by microRNA 146a and 147b in vitro.

AIM: Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) control proteolysis within the extracellular matrix (ECM) of the brain. Dysfunction of this enzymatic system due to brain inflammation can disrupt the blood-brain barrier (BBB) and has been implicated in the pathogenesis of epilepsy. However, this has not been extensively studied in the epileptogenic human brain. METHODS: We investigated the expression and cellular localization of major MMPs (MMP2, MMP3, MMP9 and MMP14) and TIMPs (TIMP1, TIMP2, TIMP3 and TIMP4) using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in resected epileptogenic brain tissue from patients with tuberous sclerosis complex (TSC), a severe neurodevelopmental disorder characterized by intractable epilepsy and prominent neuroinflammation. Furthermore, we determined whether anti-inflammatory microRNAs, miR146a and miR147b, which can regulate gene expression at the transcriptional level, could attenuate dysregulated MMP and TIMP expression in TSC tuber-derived astroglial cultures. RESULTS: We demonstrated higher mRNA and protein expression of MMPs and TIMPs in TSC tubers compared to control and perituberal brain tissue, particularly in dysmorphic neurons and giant cells, as well as in reactive astrocytes, which was associated with BBB dysfunction. More importantly, IL-1ß-induced dysregulation of MMP3, TIMP2, TIMP3 and TIMP4 could be rescued by miR146a and miR147b in tuber-derived TSC cultures. CONCLUSIONS: This study provides evidence of dysregulation of the MMP/TIMP proteolytic system in TSC, which is associated with BBB dysfunction. As dysregulated MMP and TIMP expression can be ameliorated in vitro by miR146a and miR147b, these miRNAs deserve further investigation as a novel therapeutic approach.

Effects of rapamycin and curcumin on inflammation and oxidative stress in vitro and in vivo - in search of potential anti-epileptogenic strategies for temporal lobe epilepsy.

BACKGROUND: Previous studies in various rodent epilepsy models have suggested that mammalian target of rapamycin (mTOR) inhibition with rapamycin has anti-epileptogenic potential. Since treatment with rapamycin produces unwanted side effects, there is growing interest to study alternatives to rapamycin as anti-epileptogenic drugs. Therefore, we investigated curcumin, the main component of the natural spice turmeric. Curcumin is known to have anti-inflammatory and anti-oxidant effects and has been reported to inhibit the mTOR pathway. These properties make it a potential anti-epileptogenic compound and an alternative for rapamycin. METHODS: To study the anti-epileptogenic potential of curcumin compared to rapamycin, we first studied the effects of both compounds on mTOR activation, inflammation, and oxidative stress in vitro, using cell cultures of human fetal astrocytes and the neuronal cell line SH-SY5Y. Next, we investigated the effects of rapamycin and intracerebrally applied curcumin on status epilepticus (SE)-induced inflammation and oxidative stress in hippocampal tissue, during early stages of epileptogenesis in the post-electrical SE rat model for temporal lobe epilepsy (TLE). RESULTS: Rapamycin, but not curcumin, suppressed mTOR activation in cultured astrocytes. Instead, curcumin suppressed the mitogen-activated protein kinase (MAPK) pathway. Quantitative real-time PCR analysis revealed that curcumin, but not rapamycin, reduced the levels of inflammatory markers IL-6 and COX-2 in cultured astrocytes that were challenged with IL-1ß. In SH-SY5Y cells, curcumin reduced reactive oxygen species (ROS) levels, suggesting anti-oxidant effects. In the post-SE rat model, however, treatment with rapamycin or curcumin did not suppress the expression of inflammatory and oxidative stress markers 1 week after SE. CONCLUSIONS: These results indicate anti-inflammatory and anti-oxidant properties of curcumin, but not rapamycin, in vitro. Intracerebrally applied curcumin modified the MAPK pathway in vivo at 1 week after SE but failed to produce anti-inflammatory or anti-oxidant effects. Future studies should be directed to increasing the bioavailability of curcumin (or related compounds) in the brain to assess its anti-epileptogenic potential in vivo.

Dysregulation of the (immuno)proteasome pathway in malformations of cortical development.

BACKGROUND: The proteasome is a multisubunit enzyme complex involved in protein degradation, which is essential for many cellular processes. During inflammation, the constitutive subunits are replaced by their inducible counterparts, resulting in the formation of the immunoproteasome. METHODS: We investigated the expression pattern of constitutive (ß1, ß5) and immunoproteasome (ß1i, ß5i) subunits using immunohistochemistry in malformations of cortical development (MCD; focal cortical dysplasia (FCD) IIa and b, cortical tubers from patients with tuberous sclerosis complex (TSC), and mild MCD (mMCD)). Glial cells in culture were used to elucidate the mechanisms regulating immunoproteasome subunit expression. RESULTS: Increased expression was observed in both FCD II and TSC; ß1, ß1i, ß5, and ß5i were detected (within cytosol and nucleus) in dysmorphic neurons, balloon/giant cells, and reactive astrocytes. Glial and neuronal nuclear expression positively correlated with seizure frequency. Positive correlation was also observed between the glial expression of constitutive and immunoproteasome subunits and IL-1ß. Accordingly, the proteasome subunit expression was modulated by IL-1ß in human astrocytes in vitro. Expression of both constitutive and immunoproteasome subunits in FCD II-derived astroglial cultures was negatively regulated by treatment with the immunomodulatory drug rapamycin (inhibitor of the mammalian target of rapamycin (mTOR) pathway, which is activated in both TSC and FCD II). CONCLUSIONS: These observations support the dysregulation of the proteasome system in both FCD and TSC and provide new insights on the mechanism of regulation the (immuno)proteasome in astrocytes and the molecular links between inflammation, mTOR activation, and epilepsy.

Promoter-Specific Hypomethylation Correlates with IL-1ß Overexpression in Tuberous Sclerosis Complex (TSC).

In tuberous sclerosis complex (TSC), overexpression of numerous genes associated with inflammation has been observed. Among different proinflammatory cytokines, interleukin-1ß (IL-1ß) has been shown to be significantly involved in epileptogenesis and maintenance of seizures. Recent evidence indicates that IL-1ß gene expression can be regulated by DNA methylation of its promoter. In the present study, we hypothesized that hypomethylation in the promoter region of the IL-1ß gene may underlie its overexpression observed in TSC brain tissue. Bisulfite sequencing was used to study the methylation status of the promoter region of the IL-1ß gene in TSC and control samples. We identified hypomethylation in the promoter region of the IL-1ß gene in TSC samples. IL-1ß is overexpressed in tubers, and gene expression is correlated with promoter hypomethylation at CpG and non-CpG sites. Our results provide the first evidence of epigenetic modulation of the IL-1ß signaling in TSC. Thus, strategies that target epigenetic alterations could offer new therapeutic avenues to control the persistent activation of interleukin-1ß-mediated inflammatory signaling in TSC brain.

Expression of microRNAs miR21, miR146a, and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA-derived cell cultures.

Tuberous sclerosis complex (TSC) is a genetic disease presenting with multiple neurological symptoms including epilepsy, mental retardation, and autism. Abnormal activation of various inflammatory pathways has been observed in astrocytes in brain lesions associated with TSC. Increasing evidence supports the involvement of microRNAs in the regulation of astrocyte-mediated inflammatory response. To study the role of inflammation-related microRNAs in TSC, we employed real-time PCR and in situ hybridization to characterize the expression of miR21, miR146a, and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). We observed an increased expression of miR21, miR146a, and miR155 in TSC tubers compared with control and perituberal brain tissue. Expression was localized in dysmorphic neurons, giant cells, and reactive astrocytes and positively correlated with IL-1ß expression. In addition, cultured human astrocytes and SEGA-derived cell cultures were used to study the regulation of the expression of these miRNAs in response to the proinflammatory cytokine IL-1ß and to evaluate the effects of overexpression or knockdown of miR21, miR146a, and miR155 on inflammatory signaling. IL-1ß stimulation of cultured glial cells strongly induced intracellular miR21, miR146a, and miR155 expression, as well as miR146a extracellular release. IL-1ß signaling was differentially modulated by overexpression of miR155 or miR146a, which resulted in pro- or anti-inflammatory effects, respectively. This study provides supportive evidence that inflammation-related microRNAs play a role in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte-mediated inflammatory response, with miR146a as most interesting anti-inflammatory therapeutic candidate.

Differential expression and clinical significance of three inflammation-related microRNAs in gangliogliomas.

PURPOSE: miR21, miR146, and miR155 represent a trio of microRNAs which has been shown to play a key role in the regulation of immune and inflammatory responses. In the present study, we investigated the differential expression and clinical significance of these three miRNAs in glioneuronal tumors (gangliogliomas, GGs) which are characterized by prominent activation of the innate immune response. METHODS: The expression levels of miR21, miR146, and miR155 were evaluated using Taqman PCR in 34 GGs, including 15 cases with sufficient amount of perilesional cortex. Their expression was correlated with the tumor features and the clinical history of epilepsy. In addition, in situ hybridization was used to evaluate their cellular distribution in both tumor and peritumoral cortex. RESULTS: Increased expression of miR146a was observed in both tumor and peritumoral cortex compared to control samples. miR146a was detected in both neuronal and astroglial cells. Tumor and peritumoral miR146a expression was negatively correlated with frequency of seizures and the density of activated microglial cells. Neuronal and astroglial expression was observed for both miR21 and miR155 with increased expression of miR21 within the tumor and miR155 in the peritumoral region. Negative correlations were observed between the miRNA levels and the expression of putative targets within the astroglial component of the tumor. CONCLUSION: We report a differential regulation of three miRNAs, known to be related to inflammation, in both tumor and peritumoral cortex of patients with GG. Moreover, our findings suggest a functional relationship between miR146a expression and epilepsy, either directly in epileptogenesis or as modulation of seizure activity.

Metabotropic glutamate receptor 5 in Down's syndrome hippocampus during development: increased expression in astrocytes.

Metabotropic glutamate receptor 5 (mGluR5) is highly expressed throughout the forebrain and hippocampus. Several lines of evidence support the role of this receptor in brain development and developmental disorders, as well as in neurodegenerative disorders like Alzheimer's disease (AD). In the present study, the expression pattern of mGluR5 was investigated by immunocytochemistry in the developing hippocampus from patients with Down's syndrome (DS) and in adults with DS and AD. mGluR5 was expressed in developing human hippocampus from the earliest stages tested (9 gestational weeks), with strong expression in the ventricular/subventricular zones. We observed a consistent similar temporal and spatial neuronal pattern of expression in DS hippocampus. However, in DS we detected increased prenatal mGluR5 expression in white matter astrocytes, which persisted postnatally. In addition, in adult DS patients with widespread ADassociated neurodegeneration (DS-AD) increased mGluR5 expression was detected in astrocytes around amyloid plaque. In vitro data confirm the existence of a modulatory crosstalk between amyloid-ß and mGluR5 in human astrocytes. These findings demonstrate a developmental regulation of mGluR5 in human hippocampus and suggest a role for this receptor in astrocytes during early development in DS hippocampus, as well as a potential contribution to the pathogenesis of ADassociated pathology.

Linking pathways in the developing and aging brain with neurodegeneration.

The molecular and cellular mechanisms, which coordinate the critical stages of brain development to reach a normal structural organization with appropriate networks, are progressively being elucidated. Experimental and clinical studies provide evidence of the occurrence of developmental alterations induced by genetic or environmental factors leading to the formation of aberrant networks associated with learning disabilities. Moreover, evidence is accumulating that suggests that also late-onset neurological disorders, even Alzheimer's disease, might be considered disorders of aberrant neural development with pathological changes that are set up at early stages of development before the appearance of the symptoms. Thus, evaluating proteins and pathways that are important in age-related neurodegeneration in the developing brain together with the characterization of mechanisms important during brain development with relevance to brain aging are of crucial importance. In the present review we focus on (1) aspects of neurogenesis with relevance to aging; (2) neurodegenerative disease (NDD)-associated proteins/pathways in the developing brain; and (3) further pathways of the developing or neurodegenerating brains that show commonalities. Elucidation of complex pathogenetic routes characterizing the earliest stage of the detrimental processes that result in pathological aging represents an essential first step toward a therapeutic intervention which is able to reverse these pathological processes and prevent the onset of the disease. Based on the shared features between pathways, we conclude that prevention of NDDs of the elderly might begin during the fetal and childhood life by providing the mothers and their children a healthy environment for the fetal and childhood development.