MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH 2 -terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motifcontaining coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgendependent interaction with the AR NH 2 -terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted ␣-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgendependent, NH 2 -and carboxyl-terminal interaction. Nuclear receptors facilitate ligand-dependent increases of gene transcription by direct interactions with nuclear coactivators. p160 coactivators have histone acetyltransferase activity (1) and interact with nuclear receptors through their ligand binding and NH 2 -terminal regions (2-4). Additional acetyltransferases p300/cAMP response element binding protein and p300/cAMP response element binding protein associated factor interact with the p160 coactivators (5-7). Nuclear receptors also interact with multiprotein complexes referred to as thyroid hormone receptor-associated proteins (8), activator-recruited cofactor (9), or vitamin D receptor-interacting protein complex (10). The p160 coactivators and at least one of the thyroid hormone receptor-associated proteins/activator-recruited cofactor/vitamin D receptor-interacting protein subunit 205 interact in a ligand-dependent manner with activation function 2 (AF2) 1 in the ligand binding domain (LBD) of nuclear receptors through the consensus sequence LXXLL, where L is leucine and X is any amino acid (11-16). Crystal structures of nuclear receptor LBDs have shown that a hydrophobic cleft within a multilayered ␣-helical structure serves as the LXXLL coactivator binding surface AF2 (17, 18). In the estrogen receptor, agonist binding positions helix 12 over the binding cavity to complete the AF2 surface (19,20), whereas binding of an antagonist such as 4-hydroxytamoxifen displaces helix 12 (21) causing an LXXLL-like sequence in helix 12 to mimic and thereby block coactivator binding (22).It has become apparent that the AF2 region overlaps with regions that serve as the binding site for a variety of LXXLLrelated sequences as recently shown for corepressor binding (23)(24)(25). Furthermore, we demonstrated that the AR AF2 region mediates an androgen-dependent NH 2 -terminal/carboxylterminal (N/C) interaction (26). Therefore we investigated the possibility that AF2 in the ...

Activation function 2 in the ligand binding domain of nuclear receptors forms a hydrophobic cleft that binds the LXXLL motif of p160 transcriptional coactivators. Here we provide evidence that activation function 2 in the androgen receptor serves as the contact site for the androgen dependent NH 2 -and carboxyl-terminal interaction of the androgen receptor and only weakly interacts with p160 coactivators in an LXXLL-dependent manner. Mutagenesis studies indicate that it is the NH 2 -/ carboxyl-terminal interaction that is required by activation function 2 to stabilize helix 12 and slow androgen dissociation critical for androgen receptor activity in vivo. The androgen receptor recruits p160 coactivators through its NH 2 -terminal and DNA binding domains in an LXXLL motif-independent manner. The results suggest a novel function for activation function 2 and a unique mechanism of nuclear receptor transactivation.Steroid receptors interact with coactivators during the recruitment of active transcription initiation complexes required for hormone-regulated gene transcription (1). Transcriptional activation domains in the steroid receptors that may mediate these interactions include activation function 1 in the NH 2 -terminal domain and activation function 2 (AF2) 1 in the ligand binding domain (LBD). Recent studies have focused on a family of p160 coactivators that interact with the AF2 region that include steroid receptor coactivator 1 (SRC1) (2) and the human transcriptional intermediary factor 2 (TIF2) (3). SRC1 and TIF2 contain distinct nuclear receptor interaction domains in the central and/or carboxyl-terminal regions (3, 4). Mutagenesis studies demonstrated a functional link between AF2 activity in the LBD and the binding of p160 coactivators (5, 6). The p160 coactivators interact with the AF2 hydrophobic surface of the LBD through conserved LXXLL motifs that form amphipathic ␣ helices (7, 8). Recent co-crystal structures of nuclear receptor LBDs and LXXLL motif fragments confirm that AF2 recruits TIF2 and SRC1 through their LXXLL motifs (6, 9 -11). A multistep mechanism for transcriptional activation by nuclear receptors involves hormone-dependent recruitment and association through these LXXLL binding motifs of histone acetyltransferase activity associated with the p160 coactivator family, CREB-binding protein/p300, and p300/CREB-binding protein-associated factor, resulting in chromatin remodeling (12, 13) and the formation of a transcriptionally competent Srb/mediator coactivator complex (thyroid hormone receptorassociated protein/vitamin D receptor-interacting protein) complex (14). However, androgen receptor (AR) AF2 activity is not detected in a variety of mammalian cell lines (15-18) despite homology of the region with other nuclear receptors. We therefore investigated the mechanism whereby AR recruits p160 coactivators and the role of AF2 in AR function. It is demonstrated that weak interactions between the AR LBD and SRC1 and TIF2 correspond with weak AR AF2 activity. The AF2 surface in the AR LBD ins...

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