Loading presentation...

Present Remotely

Send the link below via email or IM

Copy

Present to your audience

Start remote presentation

  • Invited audience members will follow you as you navigate and present
  • People invited to a presentation do not need a Prezi account
  • This link expires 10 minutes after you close the presentation
  • A maximum of 30 users can follow your presentation
  • Learn more about this feature in our knowledge base article

Do you really want to delete this prezi?

Neither you, nor the coeditors you shared it with will be able to recover it again.

DeleteCancel

Make your likes visible on Facebook?

Connect your Facebook account to Prezi and let your likes appear on your timeline.
You can change this under Settings & Account at any time.

No, thanks

Genetic Mechanisms of Coffee Extract Protection in a C. Elegans Model of B-Amyloid Peptide Toxicity

Neuro Chem presentation
by

Sarah Abbott

on 16 November 2012

Comments (0)

Please log in to add your comment.

Report abuse

Transcript of Genetic Mechanisms of Coffee Extract Protection in a C. Elegans Model of B-Amyloid Peptide Toxicity

Vishantie Dostal, Christine Roberts, and Christopher Link Genetic Mechanims of Coffee Extract Protection in a C. Elegans Model of B-Amyloid Peptide Toxicity Conclusion Genetic Mechanism of Coffee Extract Protection in a Caenorhabditis elegans Model of B-Amyloid Peptide Toxicity Alzheimer's Disease
A form of dementia that gradually gets worse over time.
Difficulty with emotional behavior and personality, language, memory, perception, thinking, and judgement
Two types
Early onset (before 60) less common, claim to have more of a genetic cause
Late onset (after 60) more common, may run in families, role of genes is less clear
No Cure
Parkinson's Disease
Brain disorder that leads to shaking (temors), difficulty in walking, overall movement a coordination.
Runs in families
No Cure Comparisons? In all experiments, the C. elegans were exposed from hatching to the coffee extract, caffeine, or neither (control)

Coffee extract or pure caffeine was put into the agar to dissolve before the addition of bacteria
Either E. coli OP50 or E. coli HT115 with skn-1 dsRNA-expressing plasmid or vector-only control

Induced to express the B-Amyloid peptide in the third stage of larvae by increasing the temperature from 16 degrees to 25 degrees

For GFP expression eggs of CL2166 were hatched and allowed to grow to stage 4 larvae at 16 degrees
They were sorted out based upon length, optical absorbency, and integrated fluorescence intensity at 488nm (GFP). What did they do? (Prep Work) More studies are needed in order to determine whether coffee or other plant extracts (ex. tea) may be protective against Alzheimer's disease and other neuro-pathologies Vishantie Dostal, Christine Roberts, and Christopher Link Coffee vs Alzheimer's Disease (AD) and Parkinson's Disease (PD) Introduction The best supported hypothesis is that Alzheimer's Disease is caused by the accumulation of B-Amyloid peptide

The over expression of B-Amyloid peptide is found in both familial and sporadic Alzheimer's Disease

Based upon earlier studies done with mice

Caffeine administration has been reported to reverse cognitive impairment and B-amyloid levels in mice with transgenic Alzheimer's Disease



Investigated the effects of coffee extracts in C. elegans

Temperature was used to induce the expression of human AB42 in body wall muscle Caffeinated vs decaffeinated coffee vs untreated
(Starbucks House Blend)

Coffee extract vs pure caffeine vs untreated Getting the Information The worms were harvested in deionized water with a protease inhibitor cocktail added and then frozen

Worms were then boiled in sample buffer, protease inhibitor cocktail for 10 minutes, put on ice, and then centrifuged

Before loading onto electrophoresis, the samples were boiled for 5 mins with a dye

Transferred to nitrocellulose (immunoblots)

Anti-mouse (X2) and anti-rabbit IgG peroxidase conjugate was added (antibodies) Results Coffee extracts delay paralysis induced by B-Amyloid expression
Coffee extracts do not reduce B-Amyloid transgene expression
Coffee extracts induce the skn-1/Nrf2 pathway
Coffee extract protection requires the skn-1 transcription factor
Coffee exposure does not reduce feeding
Coffee extract protects against toxicity from model aggregating protein Coffee extracts delay paralysis induced by B-Amyloid expression Coffee extract do not reduce B-Amyloid transgene expression coffee in agar > delayed the onset of paralysis

both decaf and regular delayed the onset but regular had more of an effect

pure caffeine only had a moderate effect when at the regular's strength

protective elements come from other elements in coffee, not necessarily caffeine mutants were used to determine whether the effects were from coffee or caffeine
unc-54 (myosin protein in body wall muscle
determined by body thrashing
unc-22 (twitchin similar to titin(sacromere-associated protein)
percent not paralyzed

Wanted to see whether decaf could reduce neuronal cell loss by a dominant mutation in the MEC-4 sodium channel this would lead to cell death by excessive ion influx, however it did not significantly reduce Figure 1 Figure 2 B-Amyloid transcription levels higher in coffee treated worms than in the controls

No difference in accumulation of B-Amyloid deposits between coffee treated and the control

A variable reduction of total B-Amyloid levels
average 25% reduction Figure 3 Coffee extracts induce the skn-1 / Nrf2 pathway worms that started out on a coffee treated petri dish were moved to a "control" dish during the third larvae stage then the temperature was increased to induce B-Amyloid peptide expression
were still significantly slowed compared to those that started out on "control" petri dish

Transgenic GFP-reporter
whether 10% decaf could induce any of the major stress response pathways identified in C. elegans - none for Phsp-16::GFP
significant induction was observed for Pgst-4::GFP
Used strain CL2166, decaf and control and GFP fluorescence was measured
Coffee exposure resulted in 77% increase

coffee led to nuclear localization
sodium azide (a known inducer)
did as well Figure 4 Table 1 Strain Genotype Pathway Responsive to Reference
CL2166 dvIs19 (Pgst-4TGFP) skn-1 Oxidative stress Link and Johnson (2002)
CL2070 dvIs70 (Phsp-16.2TGFP) hsf-1 Heat shock Link et al. (1999)
SJ4005 zcIs4 (Phsp-4TGFP) xbp-1 ER stress Calfon et al. (2002)
TJ356 zIs356 (daf-16TGFP) daf-16 Starvation, heat shock Henderson and Johnson (2001) Coffee extract protection requires the skn-1 transcription factor Treated worms with RNAi (used to down regulate or silence specific genes) (left the worms unable to have viable offspring)

skn-1 RNAi reversed the protective effects of coffee extract

Try it again but to see the genetic role
CL6222 - loss of function allele

Knockdown of skn-1 resulted in significant increase in B-Amyloid accumulation Figure 5 Coffee exposure does not reduce feeding Dietary restriction has been shown in previous experiments to reduce toxicity due to B-Amyloid accumulation
Wondered if coffee extract had its protective effect due to inhibiting C. elegans feeding
no effects were found

Also no effects were found regarding the developmental rate of C. elegans

Used zu67 (mutant) would not block coffee protection if the extract was acting directly inducing dietary restriction Figure 6 Coffee extract protects against toxicity from model aggregating protein C. elegans GFP::degron can lead to paralysis

Used CL2337 which had a inducible GFP::degron

Coffee exposure strongly blocked the paralysis induced by GFP::degron which reversed by RNAi knockdown of skn-1

Coffee exposure did not result in any qualitative change in GFP::degron deposits but did cause a small reduction in the accumulation of GFP::degron Figure 7 Discussion Exposure to aqueous coffee extract strongly reduces the paralysis resulting from induction of expression of B-Amyloid42 in transgenic C. elegans model
Coffee protection against B-Amyloid42 toxicity results from activation of the skn-1 pathway
Skn-1 is analogous to Nrf2 (humans)
Nrf2 may be protective in a range of neurogenerative conditions
The effects of coffee and tobacco have been studied in C. elegans and which have shown to be protective in fruit fly neurodegeneration References: http://www.genetics.org/content/186/3/857/suppl/DC1
http://www.nigms.nih.gov/News/Extras/RNAi/factsheet.htm
http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001767/
http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001762/ The Purpose Coffee exposure variably reduced the accumulation of B-Amyloid

Some experiments showed that coffee-induced protection against B-Amyloid toxicity without a detectable decrease in accumulation

Coffee has other components in it, other than caffeine, which that could have led to the protection of B-Amyloid accumulation The Problems skn-1 in C. elegans is restrictive to the intestines while the B-Amyloid42 accumulation occurs in the body wall muscle

Don't know whether the protective effects of coffee in C. elegans B-Amyloid toxicity is due to cell autonomous activation of the skn-1 pathway or more global physiological changes initiated by skn-1 activated intracellular signaling

Worms (C.elegans) not People
Full transcript