γ-glutamylcysteine (ggc)-mediated upregulation of glutathione levels can ameliorate toxicity of...

1
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 UPREGULATION OF GLUTATHIONE LEVELS CAN AMELIORATE TOXICITY OF NATURAL BETA- AMYLOID OLIGOMERS IN PRIMARYADULT HUMAN NEURONS Nady Braidy 1 , Anne Poljak 2 , Martin Zarka 3 , Wallace Bridge 3 , Perminder Sachdev 4 , 1 School of Psychiatry, University of New South Wales, Sydney, Australia; 2 UNSW, Sydney, Australia; 3 University of New South Wales, Randwick, Australia; 4 University 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-Maria 1 , Maria Eugenia Alaniz 2 , Neil Renwick 3 , Thomas Tuschl 4 , Lorraine Clark 1 , John Crary 5 , 1 Columbia University Medical Center, New York, New York, United States; 2 Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, New York, New York, United States; 3 Laboratory of RNA Molecular Biology, New York, New York, United States; 4 The Rockefeller University, New York, New York, United States; 5 Columbia 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 TAU OLIGOMERS AND ANNULAR PROTOFIBRILS IN PROGRESSIVE SUPRANUCLEAR PALSY Julia Gerson 1 , Urmi Sengupta 1 , Christian Lasagna Reeves 2 , Marcos Guerrero-Munoz 1 , Juan Troncoso 3 , George Jackson 4 , Rakez Kayed 1 , 1 University of Texas Medical Branch, Galveston, Texas, United States; 2 Baylor College of Medicine, Houston, Texas, United States; 3 Johns Hopkins School of Medicine, Baltimore, Maryland, United States; 4 UTMB, 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- Poster Presentations: P4 P854

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Page 1: γ-glutamylcysteine (GGC)-mediated upregulation of glutathione levels can ameliorate toxicity of natural beta-amyloid oligomers in primary adult human neurons

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-