Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Memory consolidation is mediated by new protein synthesis. However, the transcriptional pathways induced in neurons by behavioral training that activate gene responses have yet to be fully delineated. We have previously shown that nuclear factor B (NF-B) is activated in the amygdala after fear conditioning. Here we report that fear conditioning resulted in an increase in histone acetyl-transferase activity, the association between NF-B p65 and CBP, and the increase in acetylated p65. Pretreating animals with histone deacetylase (HDAC) inhibitors prolonged the nuclear expression of acetyl-p65 and increased its DNA binding activity. Consistent with these results, HDAC inhibitors enhanced long-term but not short-term fear memory, and this effect was attenuated by B decoy DNA, whereas scrambled DNA was without effect. This study provides evidence that HDAC-mediated deacetylation functions as an intranuclear molecular switch culminating in the termination of NF-B transcriptional response that is involved in the formation of fear memory.

Previous studies have shown that biochemical changes that occur in the amygdala during fear conditioning in vivo are similar to those occur during long term potentiation (LTP) in vitro. Electrophoretic mobility shift assay of nuclear extracts from startle-potentiated rats showed a selective increase in the amygdala of nuclear factor-B (NF-B) DNA binding activity. Supershift experiments further indicated that p65 and p50 subunits but not c-Rel were involved in DNA binding. The protein levels of IB-␣ were reduced by treatments that reliably induced LTP in this area of the brain. This was accompanied by a decrease of NF-B in the cytoplasm concomitant with an increase in the nucleus. Quantitative analysis of IB kinase activity demonstrated that fear training led to an increase in kinase activity, and this effect was inhibited by thalidomide. Paralleled behavioral tests revealed that thalidomide inhibited fear-potentiated startle. Intra-amygdala administration of B decoy DNA prior to training impaired fear-potentiated startle as well as LTP induction. Similarly, NF-B inhibitors blocked IB-␣ degradation and startle response. These results provide the first evidence of a requirement of NF-B activation in the amygdala for consolidation of fear memory.It is generally believed that consolidation of long term memory in mammalian brain and long term facilitation in Aplysia require new protein synthesis (1-3). Newly synthesized proteins are thought to deposit at the synapses that have been tagged by prior activity to encode enduring changes in synaptic strength (4, 5). However, despite the importance of new protein synthesis for memory consolidation, little is known about signaling pathways leading to protein translation in neurons.NF-B, originally identified as a regulator of immunoglobulin light chain gene expression, is a DNA-binding factor that functions as a dimer. Five mammalian members of the family have been identified; p50/NF-B1, p65/RelA, c-Rel, RelB, and p52/NF-B2 (6). NF-B was localized mainly to the cytoplasm in an inactive form bound to an inhibitory protein termed IB (7). Upon stimulation by extracellular inducers, IB was rapidly phosphorylated by the IB kinase (IKK) complex on the serine residues 32 and 36. This phosphorylation led to the ubiquitination and subsequent degradation of IB by the proteasome followed by nuclear translocation of NF-B (8 -11). Once translocated to the nucleus, NF-B bound cognate DNA sequences and activated transcription of specific target genes, the majority of which encoded proteins important in immunity and inflammation (12-15). Apart from its role in hematopoiesis, accumulated evidence indicates that NF-B is involved in neuroprotection or neurodegeneration, depending on the particular system under investigation and the NF-B subunits that became activated (16 -18). Furthermore, a recent study implicated that NF-B played an important role in the synaptic plasticity, because pretreatment of hippocampal slices with B decoy DNA prevented induction of long term depression (LTD) 1 and s...

We sought to define the landscape of alternative pre-mRNA splicing in prostate cancers and the relationship of exon choice to known cancer driver alterations. To do so, we compiled a metadataset composed of 876 RNA-sequencing (RNA-Seq) samples from five publicly available sources representing a range of prostate phenotypes from normal tissue to drug-resistant metastases. We subjected these samples to exon-level analysis with rMATS-turbo, purposebuilt software designed for large-scale analyses of splicing, and identified 13,149 high-confidence cassette exon events with variable incorporation across samples. We then developed a computational framework, pathway enrichment-guided activity study of alternative splicing (PEGASAS), to correlate transcriptional signatures of 50 different cancer driver pathways with these alternative splicing events. We discovered that Myc signaling was correlated with incorporation of a set of 1,039 cassette exons enriched in genes encoding RNA binding proteins. Using a human prostate epithelial transformation assay, we confirmed the Myc regulation of 147 of these exons, many of which introduced frameshifts or encoded premature stop codons. Our results connect changes in alternative pre-mRNA splicing to oncogenic alterations common in prostate and many other cancers. We also establish a role for Myc in regulating RNA splicing by controlling the incorporation of nonsense-mediated decay-determinant exons in genes encoding RNA binding proteins.alternative splicing | prostate cancer | Myc | rMATS | PEGASAS A lternative pre-mRNA splicing is a regulated process that governs exon choice and greatly diversifies the proteome. It is an essential process that contributes to development, tissue specification, and homeostasis and is often dysregulated in disease states (1). In cancer, this includes growth signaling, epithelial-tomesenchymal transition, resistance to apoptosis, and treatment resistance (2). In prostate cancer, our area of interest, the most notable splicing change is the emergence of the ligand-independent androgen receptor ARV7 isoform in response to hormone deprivation (3). Other examples include proangiogenic splice variants of VEGFA (4), tumorigenic variants of the transcription factors ERG and KLF6 (5, 6), and antiapoptotic splicing of BCL2L2 (7, 8). However, the intersection of upstream oncogenic signaling, pre-mRNA splicing, and the biological processes affected by those splicing events has not been defined at a global level.Prostate cancers progress from hormone-responsive, localized disease to hormone-independent, metastatic disease accompanied by changes in gene expression and mutations that confer cellautonomous growth and therapeutic resistance (9). The study of disease progression from primary prostate adenocarcinoma (PrAd) to metastatic, castration-resistant prostate cancer (mCRPC) and treatment-related neuroendocrine prostate cancer (NEPC) has been aided by large-scale genomic and transcriptomic studies of patient samples representing each form of the disease (10-13). E...

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.