γ-glutamylcysteine (ggc)-mediated upregulation of glutathione levels can ameliorate toxicity of...
TRANSCRIPT
Poster Presentations: P4P854
intracellular aggregates of hyperphosphorylated tau, and aberrant lipid me-
tabolism in the brain. Two single nucleotide polymorphisms (SNPs) within
the gene encoding ATP-binding cassette transporter, sub-family A, member
7 (ABCA7) strongly associates with the risk of late-onset AD, namely
rs3764650 within intron13 and rs3752246 within exon33. The latter results
in a glycine to alanine mutation at position 1527 of the full length protein.
ABCA7 is highly expressed in the brain and has been implicated in phago-
cytosis and the efflux of lipids. The functional implications of the two
ABCA7 SNPs were examined in this study. Methods: The SNP variants
were generated using site-directed mutagenesis of wild-type sequences. Lu-
ciferase reporter plasmids containing either wild-type or SNP variant in-
tron13 were expressed in human embryonic kidney (HEK) 293 cells.
Mevastatin treatment simulated ABCA7 expression machinery and lucifer-
ase activity was then measured. Full-length wild-type (A7wt) or exon33
SNP-containing variant (A7va) ABCA7 were expressed in Chinese hamster
ovary (CHO) cells stably expressing human APP containing the Swedish
mutation (CHO-APP Swe). The levels of Ab 1-40, Ab 1-42, APP, soluble
APPa, and b-secretase APP cleavage enzyme 1 (BACE1) were measured
in the cell and conditioned media by ELISA and immunoblot analysis, re-
spectively. Results: Luciferase activity from intron13 variant expressing
HEK293 cells was increased relative to empty vector control following me-
vastatin treatment. ELISA revealed that the levels of secreted Ab 1-42 pep-
tides were significantly increased from A7va- relative to A7wt-expressing
CHO-APP Swe cells. A7wt or A7va expression did not result in significant
changes to the cell-associated levels of APP or secreted levels of sAPPa, as
evidenced by immunoblot. A7wt or A7va expression also did not result in
protein level changes to BACE1. Conclusions: The rs3764650 SNP within
intron13 of ABCA7 gene creates a promoter enhancer capable of increasing
the expression of ABCA7. The glycine to alanine mutation caused by SNP
rs3752246 in ABCA7 increases Ab 1-42 production/secret ion without af-
fecting the levels of either APP or BACE1.
P4-332 g-GLUTAMYLCYSTEINE (GGC)-MEDIATED
UPREGULATIONOFGLUTATHIONELEVELSCAN
AMELIORATE TOXICITY OF NATURAL BETA-
AMYLOID OLIGOMERS IN PRIMARYADULT
HUMAN NEURONS
Nady Braidy1, Anne Poljak2, Martin Zarka3, Wallace Bridge3,
Perminder Sachdev4, 1School of Psychiatry, University of New South Wales,
Sydney, Australia; 2UNSW, Sydney, Australia; 3University of New South
Wales, Randwick, Australia; 4University of New South Wales,
Randwick-Sydney, Australia. Contact e-mail: [email protected]
Background: Soluble amyloid-b (Ab) oligomers can induce oxidative
stress, synaptic dysfunction and memory deficits which are present in Alz-
heimer’s disease (AD). Exogenous Ab(1-42) has been previously shown to
induce oxidative stress and deplete the levels of the important endogenous
antioxidant glutathione (GSH). However, the effect of Ab oligomers iso-
lated from natural sources such as AD brain extracts and cerebrospinal fluid,
on GSH levels in primary adult human neurons has not been previously in-
vestigated. Moreover, administration of g-glutamylcysteine (GGC) can in-
crease intracellular levels of GSH, by circumventing the regulation of GSH
biosynthesis by providing the limiting substrate. Methods: Using isolated
pure cultures of primary adult human neurons, we examined the neuropro-
tective effects of increased GSH levels by GGC against the oxidative and
cytotoxic effects of soluble Ab oligomers extracted from post-mortem
AD brains. More specifically, we assessed protein oxidation and lipid perox-
idation in vitro using standard spectrophotometric assay kits. The conforma-
tion structure of Ab oligomers in the presence of GGC was assessed using
immunoflourescence microscopy.Results:GGC treatment to neurons led to
a significant upregulation of GSH levels compared to non-treated control
cells, and protected neurons against protein, and lipid oxidation, and im-
paired mitochondrial respiration. Moreover, GGC was able to remodel the
fibrillar conformation of Ab oligomers to non-toxic forms. Conclusions:
These data provide renewed insight on the beneficial effects of increased
GSH levels by GGC in human neurons, and identify additional targets to at-
tenuate the neurotoxic effects of Ab oligomers in AD.
P4-333 MICRORNAS THAT SILENCE TAU EXPRESSION
ARE DYSREGULATED IN ALZHEIMER’S DISEASE
Ismael Santa-Maria1, Maria Eugenia Alaniz2, Neil Renwick3,
Thomas Tuschl4, Lorraine Clark1, John Crary5, 1Columbia University
Medical Center, New York, New York, United States; 2Taub Institute for
Research on Alzheimer’s Disease and the Aging Brain, New York, New York,
United States; 3Laboratory of RNAMolecular Biology, New York, New York,
United States; 4The Rockefeller University, New York, New York, United
States; 5Columbia University Medical Center, Brooklyn, New York, United
States. Contact e-mail: [email protected]
Background: Alzheimer disease (AD) and other tauopathies are associated
with aggregates of the microtubule-associated protein tau as neurofibrillary
tangles. Currently, there is a critical need to identify strategies to treat tauo-
pathies.MicroRNAs, a class of small non-coding RNAs that bind their target
mRNA and silence expression, hold great promise as therapeutic targets and
tools. However, it is unknown whether disturbances in microRNAs contrib-
ute to neurofibrillary degeneration.Methods: Here, we profiled small RNA
levels in human brain tissue from patients with AD and tangle-predominant
dementia (TPD), a "limbic" tauopathy that shares defining clinical and neu-
ropathological features with AD. We then selected candidate tau-silencing
microRNAs and validated them in cellular models. Results:We observe al-
terations in a subset of microRNAs that are predicted to target tau, including
decreased levels of the highly-conserved and brain-specific miR-219-5p.
We further show that miR-219-5p directly binds the tau 3’ UTR and re-
presses translation. Finally, the levels of miR-219-5p inversely correlate
with tau synthesis during neurite outgrowth and over-expression of miR-
219-5p inhibits outgrowth of tau-positive neurites. Conclusions: We pro-
pose that alterations in microRNAs influence tau expression, contributing
to the accumulation of abnormal tau in AD and TPD. This hitherto unknown
mechanism of tau regulation may be useful for developing therapeutics for
tauopathies.
P4-334 TAUOLIGOMERS ANDANNULAR PROTOFIBRILS
IN PROGRESSIVE SUPRANUCLEAR PALSY
Julia Gerson1, Urmi Sengupta1, Christian Lasagna Reeves2,
Marcos Guerrero-Munoz1, Juan Troncoso3, George Jackson4,
Rakez Kayed1, 1University of Texas Medical Branch, Galveston, Texas,
United States; 2Baylor College of Medicine, Houston, Texas, United States;3Johns Hopkins School of Medicine, Baltimore, Maryland, United States;4UTMB, Galveston, Texas, United States. Contact e-mail: jegerson@utmb.
edu
Background: Progressive supranuclear palsy (PSP) is a neurodegenerative
tauopathy which is primarily defined by the deposition of tau into globose-
type neurofibrillary tangles (NFT). Tau, like other disease-associated amy-
loids, can form oligomers which may then either form fibrils or pore-like
structures, called annular protofibrils. Recent studies from our laboratory
and others have provided evidence that tau oligomers, not NFTs, are the
most toxic species in neurodegenerative tauopathies and seed the patholog-
ical spread of tau. Additionally, the amyloid pore hypothesis suggests an im-
portant link between the formation of amyloid pores and cell death.
Methods: We obtained six different PSP human brain samples and two
age-matched control samples.We used the novel anti-tau oligomer antibody,
T22, as well as additional commercial antibodies to tau to quantify tau
oligomer levels in PSP and control brains via SDS PAGE and western
blot. Phosphorylation, as seen in NFTs, was also analyzed using AT8. Im-
munohistochemistry of sectioned brain samples was utilized to observe
the presence of globose NFTs, as well as tau oligomers. Annular protofibrils
were labeled using the novel anti-annular protofibril antibody, Officer. Im-
ages were analyzed using confocal microscopy. Results: Analysis of PSP
brains via Western blot revealed the presence of phosphorylated tau typi-
cally seen in the globose NFTs common in PSP. Tau oligomers were also
detected using T22. Analysis of brain sections revealed globose-type
NFTs, as well as unphosphorylated tau oligomers. Surprisingly, sections la-
beled with the annular protofibril specific antibody, Officer, revealed pore-