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					Expression of a noncoding RNA is elevated
in Alzheimer’s
disease and drives rapid feed-forward
regulation of
   Faghihi, MA et al. Nature Medicine 2008

   Presenter: Chen-Chung Lin
Main Claims

   A BACE1-antisense transcript may contribute to
    Alzheimer‘s disease by regulating BACE-1 mRNA
    and protein expression.
   BACE1-AS could be upregulated by Ab1-42 and
    stabilized the BACE1 mRNA, further express more
    BACE1 proteins and produce more Ab1-42.
   Stabilization mechanism is by forming a RNA duplex,
    and protect RNA from degradation.
   BACE1-AS contribute to AD formation.

   Alzheimer‘s disease (AD):
       Neurodegenerative disease
       Most common form of dementia
       Characterized by the progressive formation in the
        brain of insoluble amyloid plaques and vascular
        deposits consisting of the 4-kD amyloid b-peptide
   amyloid b-peptide (Ab)
       Cleavage of amyloid precursor protein (APP) by
        b-secretase-1 (BACE1)
   Ab synthesis pathway

Rate limiting

                                                  Alzheimer‘s disease (AD)

 amyloid precursor protein

                             (Adapted from wikipedia,
Dysregulation of BACE1 and Ab production in
Alzheimer's disease

          Adapted from Peter St George-Hyslop & Christian Haass Nature Medicine 14, 711 - 712 (2008)
Genomic organization of BACE1 and BACE1-

                                           It contains transcript variants

                                   It is highly conserved between species.
   (104 nts complete match)
Expression analysis of BACE1 and BACE1-AS

                       BACE1 mRNA expression is about
                       25-75% greater than BACE1-AS
BACE1-AS regulates BACE1 mRNA
expression in vitro

                   BACE1-AS siRNA knockdown also
                   see BACE1 mRNA knockdown;
                   overexpressed BACE1-AS could
                   also see BACE1 mRNA increased.
BACE1-AS regulates BACE1 protein
expression in vitro

BACE1-AS siRNA knockdown could find that BACE1 protein knockdown;
overexpressed BACE1-AS could also see BACE1 protein increased.
Knockdown of BACE1-AS reduces Ab 1–40
and Ab 1–42
Knockdown of BACE1-AS reduces BACE1
levels in vivo
      Cortex           Striatum      (No siRNA reached
                                        by perfusion)

   D. hippocampus   V. hippocampus
Cell stressors increase BACE1-AS and BACE1
BACE1-AS forms RNA duplex and increases
stability of BACE1
BACE1-AS is elevated in subjects with
Alzheimer’s disease

            In patients, BACE1-AS elevated.
APP transgenic mice have increased levels of

   BACE1-AS is a mature non coding RNA.
   BACE1-AS could protect BACE1 from degradation
    by forming a dsRNA structure, and increase BACE1
    RNA stability.
   Ab could induce both BACE1 and BACE1-AS, then
    cause AD.

   Would BACE1-AS transcript undergo NMD?
   Would RNAse H degrade BACE1 and
    BACE1-AS dsRNA?
Complex I Binding by a Virally
Encoded RNA Regulates
Mitochondria-Induced Cell
       Reeves, MB et al, Science 2007

   HCMV noncoding RNA b2.7 would
    associated with mitochondrial complex I and
    then protect cell from apoptosis.

   Human cytomegalovirus (HCMV):
     DNA virus

     Betaherpesvirus (b-HSV)

     Contains the largest DNA genome (240 kb) of

      human herpes virus.
     RNA transcripts contains coding and noncoding

     Common cause of infants infection

       Cytomegalic inclusion disease

                         (Adapted from Brooks, GF et al. Medical Microbiology, p401-405 )
Shutting down the immune response by HCMV

 Block the innate immunity of infected cell.

                                                (MICB, NK cell activator)

                                               (Adapted from Cullen, BR Science317:329, 2007 )
Mitochondria electron transport chain


                                           Cell Death
               (Adapted from wikipedia,
      HCMV protects cells from rotenone-
      induced apoptosis
                                           HMCV      -Db2.7
                                   HMCV    -Db2.7   Revertant
                                                                            (Tx b2.7 plasmid)
complex I inhibitor)

                  b2.7 transcript would protect U373 cell from retenone induced apotosis.
What are the protection mechanisms of
HCMV b2.7 RNA?

   May be to mediate protection of cell from
    apoptotic pathways activated by metabolic
    stress of complex I
   GRIM-19?
GRIM-19 (Genes associated with
retinoid/interferon-induced mortality)
   A subunit of Complex I
   Essential for Complex I assembly
   Essential for Complex I function
   Report as a cell death-regulatory gene under
    retinoid / interferon treatment
   RNA level remain steady after HCMV
HCMV prevents the rotenone-induced
relocalization of GRIM-19 in virally infected cells

   HCMV interacts with GRIM-19 in virally infected cells

IP: GRIM-19, Complex-1 or Complex-V

                RT-PCR                                                   HCMV Viral RNA

IP on biotin-labeled antisense of b2.7 or IE72

      Western blot for GRIM-19

                                                                        Indicated that b2.7 interact
                                                                        with GRIM-19

                                                 Sliver stain of gels
b2.7 expression
maintains ATP production in infected cells.
                    2 x b2.7

                                         Long term

           Short term
Toledo, but not Db2.7Tol, grows normally in
metabolically stressed cells.

                           Deletion of b2.7 gene has no significant effects on viral
                           growth in human fibroblasts (HF).
Toledo, but not Db2.7Tol, grows normally in
metabolically stressed cells.

                       Deletion of b2.7 gene has significant decrease effects on
                       viral growth in U373 cells.
                        b2.7 transcripts support viral growth

                       Deletion of b2.7 gene has significant decrease effects on
                       viral growth in glucose-depleted U373and HF cells.
                        b2.7 transcripts support viral growth under normal tissue
                       condition (w/o too much glucose)

   HCMV noncoing RNA b2.7 could binding to GRIM-
    19 and maintain the ATP production from
    mitochondrial electron transport chain; prevent
    HCMV infected cell from apoptosis.

   Other targets for b2.7 RNA?

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