Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor.

Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.

Arc Regulates Transcription of Genes for Plasticity, Excitability and Alzheimer's Disease.

The immediate early gene Arc is a master regulator of synaptic function and a critical determinant of memory consolidation. Here, we show that Arc interacts with dynamic chromatin and closely associates with histone markers for active enhancers and transcription in cultured rat hippocampal neurons. Both these histone modifications, H3K27Ac and H3K9Ac, have recently been shown to be upregulated in late-onset Alzheimer's disease (AD). When Arc induction by pharmacological network activation was prevented using a short hairpin RNA, the expression profile was altered for over 1900 genes, which included genes associated with synaptic function, neuronal plasticity, intrinsic excitability, and signalling pathways. Interestingly, about 100 Arc-dependent genes are associated with the pathophysiology of AD. When endogenous Arc expression was induced in HEK293T cells, the transcription of many neuronal genes was increased, suggesting that Arc can control expression in the absence of activated signalling pathways. Taken together, these data establish Arc as a master regulator of neuronal activity-dependent gene expression and suggest that it plays a significant role in the pathophysiology of AD.

The effect of Bacopa monnieri on gene expression levels in SH-SY5Y human neuroblastoma cells.

Bacopa monnieri is a plant used as a nootropic in Ayurveda, a 5000-year-old system of traditional Indian medicine. Although both animal and clinical studies supported its role as a memory enhancer, the molecular and cellular mechanism underlying Bacopa's nootropic action are not understood. In this study, we used deep sequencing (RNA-Seq) to identify the transcriptome changes upon Bacopa treatment on SH-SY5Y human neuroblastoma cells. We identified several genes whose expression levels were regulated by Bacopa. Biostatistical analysis of the RNA-Seq data identified biological pathways and molecular functions that were regulated by Bacopa, including regulation of mRNA translation and transmembrane transport, responses to oxidative stress and protein misfolding. Pathway analysis using the Ingenuity platform suggested that Bacopa may protect against brain damage and improve brain development. These newly identified molecular and cellular determinants may contribute to the nootropic action of Bacopa and open up a new direction of investigation into its mechanism of action.

A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

Chromatin modification is an important epigenetic mechanism underlying neuroplasticity. Histone methylation and acetylation have both been shown to modulate gene expression, but the machinery responsible for mediating these changes in neurons has remained elusive. Here we identify a chromatin-modifying complex containing the histone demethylase PHF8 and the acetyltransferase TIP60 as a key regulator of the activity-induced expression of Arc, an important mediator of synaptic plasticity. Clinically, mutations in PHF8 cause X-linked mental retardation while TIP60 has been implicated in the pathogenesis of Alzheimer's disease. Within minutes of increased synaptic activity, this dual function complex is rapidly recruited to the Arc promoter, where it specifically counteracts the transcriptionally repressive histone mark H3K9me2 to facilitate the formation of the transcriptionally permissive H3K9acS10P, thereby favoring transcriptional activation. Consequently, gain-of-function of the PHF8-TIP60 complex in primary rat hippocampal neurons has a positive effect on early activity-induced Arc gene expression, whereas interfering with the function of this complex abrogates it. A global proteomics screen revealed that the majority of common interactors of PHF8 and TIP60 were involved in mRNA processing, including PSF, an important molecule involved in neuronal gene regulation. Finally, we proceeded to show, using super-resolution microscopy, that PHF8 and TIP60 interact at the single molecule level with PSF, thereby situating this chromatin modifying complex at the crossroads of transcriptional activation. These findings point toward a mechanism by which an epigenetic pathway can regulate neuronal activity-dependent gene transcription, which has implications in the development of novel therapeutics for disorders of learning and memory.