16 replies to this topic

[Mark Hamalainen]

Hello everyone!

I've just signed up recently, so allow me to introduce myself. I'm an undergraduate student from Queen's University in Ontario, Canada. I've been interested in aging research for a few years now. Like many, I was inspired by Aubrey de Grey's work.
Last summer I worked in Dr. Seymour Benzer's lab at CalTech where I tried to convince them of the merits of allotopic expression, unsuccessfully :(. This summer I have the opportunity to collaberate with a bioremediation lab and a drosophila longevity lab (http://seroudelab.biology.queensu.ca/). My project will be to identify bacteria and fungi which can catabolize lipofuscin and other aggregates extracted from old flies and other sources using simple selection experiments with minimal media. Depending on our preliminary success or lack thereof, the project could continue as a masters/PhD with the goal of identifying the enzymes and using them to extend Drosophila lifespan.
I've attached a draft of experimental procedures for my project. I'm not sure how many people here have bench experience or are currently doing research (My own bench experience isn't that extensive yet), but I'm interested in any comments/suggestions. Its just a collection of ideas so far. I'll be starting the research in May, so by then I'll need to have picked the specific experiments that I'll be starting on.

Cheers,
Osiris

Edited by osiris, 25 April 2005 - 04:02 PM.

[Mark Hamalainen]

Prometheus,

Thanks! My ambition is something I'm certainly not modest about. I'll be starting the preliminary work in May as a summer research project and continuing through both terms next year as a credited research project towards my undergraduate degree, which I will be completing next year. I will only be taking a minimal of lecture courses in the next academic year, so the research will be my primary concern. Also, my supervisors have already expressed interest in having me stay to do graduate work towards a PhD assuming results of the preliminary work are promising. Therefore, I'm interested in designing my experiments with long-term potential in mind. On the other hand, I hope to get some publishable results within the year, hence the idea of using bacteria to develop an assay for the toxicity/presence of metabolic end points. Much of the primary experiments will involve just waiting around to see whether the bacteria/fungi are growing or not, so I intend to use that time on as many side-projects as I can think of, or that are suggested to me [thumb] .

[John Schloendorn]

Hi Osiris,
Awesome project! I have also been thinking a lot about bioremediation lately. It looks like I will get the chance to do a PhD on hematopoietic stem cell therapies (outlined here, although in much less detail than yours). It would be nice if we could cooperate one day, regarding macrophage augmentation with gene and cell therapy.

I have a few questions about your project:
1. What are the grounds to expect that lipofuscin remediation would extend fly life span? Do flies have a problem with it?
2. What are your thougts on lipofuscin and plaque lipid degradation assays, when doing metagenomic screens?
3. Will you have access to effective screening hardware (a protein arrayer, basically)?
4. Are any of the organisms you speak of sequenced, which would facilitate bioinformatics? I'm saying, I'm missing Rhodococcus. (Sure you are familiar with the SENS talk by John Archer)

Best, John.

[Mark Hamalainen]

John,
There has actually been suprisingly little interest in lipofuscin in Drosophila recently. Jaime Miquel studied it briefly in the 70s, but since then the Drosophila aging research community seems to have become obsessed with mutant screening and forgot about lipofuscin.
I dug up some info though, lipofuscin definitely accumulates in a variety of Drosophila tissues, particularly the nervous system and gut:

"An electronic microscopic study was performed on the brain and visceral organs of Drosophila melanogaster ranging in age from 7 to 102 days. The subcellular localization of acid phosphatase was determined by the Gomoi technique and the level of fluorescent products was measured by spectrophotofluorometry performed on chloroform-methanol extracts of whole flies. It was found that the tissues of aged Drosophila melanogaster contain abundant lipopigments that occasionally fill up to 50% of the cytoplasmic volume... lysosomal participation in the genesis of the Drosophila pigment is suggested by the demonstration of acid phosphatase activity in the dense bodies present in the midgut epithelium." [1]

Also, glycation products accumlate:

"We found that Drosophila melanogaster maintained at 24°C accumulate significant AGE over their lifespan. Young flies (10 days old) had 44% less AGE than senescent flies (75 days old)." [3]

Of course, this doesn't prove that lipofuscin accumulation is pathogenic, or a significant factor in, the aging of Drosophila... perhaps my experiments will determine that. However, I'm only interested in Drosophila as a possible stepping stone. I intend to experiment on lipofuscin and other metabolic end points from as many sources as I can get my hands on.

As for degradation assays, do you just mean tracking the degradation of an MEP while in the presence of the potentially catabolizing bacteria?

I'm not sure if there is a protein arrayer available here, I'm guessing that neither of the labs I'm working with have them, but there may be one somewhere at the university. I'm not actually familiar with that hardware...

Thanks for the reminder, Rhodococcus is an obvious choice for my experiments! B. cepacia has been sequenced: http://www.sanger.ac.../B_cenocepacia/


[1] Miquel, J., A. L. Tappel, et al. (1974). "Fluorescent products and lysosomal components in aging Drosophila melanogaster." J Gerontol 29(6): 622-37.

[2] Herman, M. M., J. Miquel, et al. (1971). "Insect brain as a model for the study of aging. Age-related changes in drosophila melanogaster." Acta Neuropathol (Berl) 19(3): 167-83.

[3] Oudes, A. J., C. M. Herr, et al. (1998). "Age-dependent accumulation of advanced glycation end-products in adult Drosophila melanogaster." Mech Ageing Dev 100(3): 221-9.

[John Schloendorn]

Very cool thanks for the refs! The drosophila info sounds encouraging. With such levels of junk you really should see some effect if you remove some of it.

Sorry, I did not explain that assay question well. I was thinking of cell-free testing of enzymes vs atherosclerotic plaques.
When you test bacterial growth against isolated MEPs, you might not cover all MEPs (since the plaque lipid is so heterogenous), but you would cover all the catabolic activity present in the bacteria. Similarly, in a cell-free assay, enzyme vs plaque, you will cover the entire MEP spectrum, but you may miss some of the catabolic activity. Thus it looks like the two could complement each other.
Further, there is still this discussion whether the modification that clogs up the macrophages is a chemical one at all. Thus, unclogging activity might also be something not directly catabolic, such as the restoration of low pH in the lysosomes or some sort of signalling intervention. But don't get me wrong, I'm not trying to badmouth the growth assay, since I do think that is certainly the way to start. Just wondering if you have further plans.

As for metagenomics, I can't see how you would both maintain sequences from the "unculturables" and be able to identify individual species (Or can you?). Thus I understand metagenomics as to skip the species level and assay enzymatic activity from environmental DNA, i.e. in a cell free system only, which would profit from a clever assay method.

It seems to me that for the homogenous aggregates (Tau, NFT, beta-amyloid) the growth assay seems inappropriate. They're proteins after all, and everybody can eat proteins. What you do want is something that *specifically* degrades them. If such a thing exists at all, it's gonna be hell rare. So here is where the arrayer really comes in.

Again, great that you have made it into SENS research!

[Mark Hamalainen]

As for metagenomics, I can't see how you would both maintain sequences from the "unculturables" and be able to identify individual species (Or can you?). Thus I understand metagenomics as to skip the species level and assay enzymatic activity from environmental DNA, i.e. in a cell free system only, which would profit from a clever assay method. 

Right, metagenomics skips the species level. The bioremediation lab I'm working with doesn't use cell free systems though, they express the library in E.coli and screen the bacteria against different media. I agree that cell free systems to look at individual enzymes would complement the cell selection experiments, but I don't know exactly how to go about them, or if I'll have have access to the needed resources. I'll have to look into this more...

It seems to me that for the homogenous aggregates (Tau, NFT, beta-amyloid) the growth assay seems inappropriate. They're proteins after all, and everybody can eat proteins. What you do want is something that *specifically* degrades them. If such a thing exists at all, it's gonna be hell rare. So here is where the arrayer really comes in.

True, it wouldn't be much use to screen for such specific activity from normal bacteria or fungi, most anything could use them. However, if you had a bacteria which, say, couldn't utilize polyglutamine as its sole carbon source then you could do a screen by expressing a metagenomic library in it. In general you are right though, most anything can use whole proteins as a carbon source. But even if there is not much chance of success, it may be worth the try if the experiment doesn't take much effort.

A small update for anybody looking at my proposal, the volumes I've described are actually rather excessive. Many of the experiments could be done in as little as 250ul on a microtiter plate, I'll probably post an updated version later next week.

[manofsan]

Hallooo gentlemen,

Wow, it's surprising to know that lipofuscin was being investigated - even briefly - all the way back in the 1970s. Cool, I think it's great that you're trying to find enzymes that can metabolize it. Hell, there's gotta be something.

If it can even be done in microtiter amounts, I'd imagine that these lab-on-chip devices could be very applicable to this kind of work. They would help bring to bear the brute force power of the combinatorial chemistry approach.

What I'd first be curious to know though, is whether lipofuscin has been sufficiently characterized to know whether it's of uniform composition, so that the same enzyme or set of enzymes will work on it.

[John Schloendorn]

However, if you had a bacteria which, say, couldn't utilize polyglutamine as its sole carbon source then you could do a screen by expressing a metagenomic library in it.

I suppose if you use a huge number of plates and control plates, you could be getting there. Fingers crossed, that'd be awesome! How do you avoid false positives from unspecific proteases? Casein control and thus a total-protease deficient strain?

Manofsan:
Lipofuscin is very heterogenic and can vary from tissue to tissue. Only its most salient constituents/functions are known. But removing these may be hugely beneficial already and help to characterize the less salient ones.

[Dey]

Hi Osiris,
I have done some experiments on the degradability of AGEs myself. (I was simply looking for chemicals to improve degradability of AGEs by the proteasome, like carnosine, vitamins….).

In the moment I am wondering why you are starting with the complex fly extract to grow your cells on. I think it would be easier and better to start with a simple self-made AGE mix. This would avoid any faults in bacterial growth due to hormones, toxins, cytokines in the fly extract.
It is very easy to make these AGEs/ lipofusin yourself. In principle you isolate protein fractions and crosslink them with UV-light (or use your mentioned BSA-glucose AGEs). You could also take whole cell/animal extract to become a more complex lipofuscin material, including, lipids and DNA.
If you want to stay with fly extract you should at least extract the protein fraction to grow your bacteria on and maybe later the lipid-protein fraction.

If you want to quantify AGEs do not forget more specific antibodies against glycated proteins, such as CML and CEL- specific antibodies. They might give you some additional data. I would back up your fluorescence and absorbance measurement with some simple SDS and native gels to look for aggregate formation and band shifts.

As you mentioned tau, amyloid and ployglutamate. I could organise some pure tau-protein extract for you, if you are interested.

DNPH reaction: I am using a similar assay (Carbonyl-ELISA) since a very long time and could send you a good protocol (as there are some tricks you have to know). The data is very reliable and repetable.


In a later stage I could also organise a proteomic study for you when you want to look for proteins of interest, just in case the involved proteases are not known.


Many greetings,

Mondey

[Mark Hamalainen]

Hey Mondey, thanks for your suggestions and generous offers. This is exactly the sort of cooperation I was hoping for, and which I hope will become more common on ImmInst.

In the moment I am wondering why you are starting with the complex fly extract to grow your cells on. I think it would be easier and better to start with a simple self-made AGE mix. This would avoid any faults in bacterial growth due to hormones, toxins, cytokines in the fly extract.

I'll be starting the self-made AGE mixes ASAP, but they will take some time to incubate. The idea with whole fly extracts is that if the aggregates are toxic enough to kill the fly they may have a measurable effect on bacterial growth. Its just a quick and easy experiment to get started on.

It is very easy to make these AGEs/ lipofusin yourself. In principle you isolate protein fractions and crosslink them with UV-light (or use your mentioned BSA-glucose AGEs). You could also take whole cell/animal extract to become a more complex lipofuscin material, including, lipids and DNA.  If you want to stay with fly extract you should at least extract the protein fraction to grow your bacteria on and maybe later the lipid-protein fraction.

Definitely I'm up for trying UV-light crosslinking and a number of different fractionations, I'll be looking into possibilities myself but if you know any specific protocols please pass them on. Thats why I posted my project, I'm in the middle of exams right now so I don't have nearly as much time as I would like to research this myself.

If you want to quantify AGEs do not forget more specific antibodies against glycated proteins, such as CML and CEL- specific antibodies. They might give you some additional data. I would back up your fluorescence and absorbance measurement with some simple SDS and native gels to look for aggregate formation and band shifts.

Cool, I'll look into it.

As you mentioned tau, amyloid and ployglutamate. I could organise some pure tau-protein extract for you, if you are interested.

That would be awesome. I haven't completely worked out the schedule for my experiments yet, so I wouldn't want you to do any unnecessary work yet, but I will get back to you on this once things get rolling in May.

DNPH reaction: I am using a similar assay (Carbonyl-ELISA) since a very long time and could send you a good protocol (as there are some tricks you have to know). The data is very reliable and repetable.

Great, I'll be glad to benefit from your experience, anything to save time My email is 2mrh@qlink.queensu.ca

In a later stage I could also organise a proteomic study for you when you want to look for proteins of interest, just in case the involved proteases are not known.

Yeah I'm really interested in the possibilities of proteomics for this line of research, its something I need to learn more about for now. If you know any good reviews/resources let me know. Once I'm caught up on the subject we can discuss possibilities.

[henri]

By the way, have amyloid-beta and tau been observed to accumulate in lysosomes? This should be simple with antibodies and fluorescence.

[John Schloendorn]

Hi Henri,
Most beta-amyloid is extracellular, and most Tau is intracellular, but outside the lysosomes. There are minor aggregates in the lysosomes and some speculate that the intracellular and extracellular deposits may be compensatory measures of the cell to keep the lysosomes working. I don't think it is yet known whether lysosomes can digest hyperphosphorylated tau. You might want to read the neurodegeneration chapter in Aubrey's medical bioremediation paper.

[henri]

You might want to read the neurodegeneration chapter in Aubrey's medical bioremediation paper.

Thanks, the chapter was informative.

[Lazarus Long]

Here is an interesting cross reference on tau that addresses it in relation to Alzheimer's and results that suggest a possible remedial treatment.

http://news.yahoo.co...h_alzheimers_dc

Mouse study suggests Alzheimer's damage reversible

By Maggie Fox, Health and Science Correspondent
Thu Jul 14, 2:03 PM ET

{excerpts}
WASHINGTON (Reuters) - Tests on mice suggest the brain damage caused by     Alzheimer's disease may be at least partly reversible, researchers reported Thursday.

Their genetically altered mice regained the ability to navigate mazes after the genes that caused their dementia were de-activated.

This suggests that the brain damage caused by Alzheimer's is not permanent, they wrote in their report, published in the journal Science.

*****

The two proteins involved are unhealthy forms of natural brain compounds called amyloid beta and tau protein.

Ashe's team worked with mice genetically engineered to develop the mutant tau, but this mutation could be stopped -- or de-activated -- with use of a drug called doxycycline.

As expected, the mice developed dementia and had brain atrophy similar to human Alzheimer's disease.

And when the engineered gene was turned off, memory loss stopped, as expected. But the mice did not merely stop getting worse. They got better.

"Even mice that had lost half the neurons that are involved in forming memories, when we removed the molecule causing the memory loss from the remaining neurons by turning off the genes, the mice were able to learn and remember new information," Ashe said in a telephone interview.

"No one suspected so many neurons would still be able to function."

*******

She noted that it is a long way from treating a mouse to treating a person.

"How are we ever going to turn off the gene in humans?" Ashe asked. What might be possible, she said, would be stopping the production of the mutant proteins. And current vaccine efforts are aimed at removing the bad proteins from the brain.

"The point that makes us hopeful is the remaining neurons were functioning," she said. "When we removed the molecules that presumably were causing the malfunction, the mice were able to perform better."

*******

"The neurofibrillary tangles, which are one of two major pathological hallmarks of Alzheimers disease, turn out not to be involved in causing memory problems, at least in mice," she said.

Some process may be going on at a microscopic level, and the tangles may be a result but not a cause of the brain damage, she said.

[John Schloendorn]

Great find Laz, this could be way cool! I have requested the original in resource sharing. Apparently it didn't fix NFT, but it did fix some of the pathology. Weird.

[Michael]

All:

henri: By the way, have amyloid-beta and tau been observed to accumulate in lysosomes? This should be simple with antibodies and fluorescence.

From a clinical, as vs theoretical, POV, we have the operationally important data that vaccinating model AD mice against Abeta -- which cures them of pathology -- causes microglia to take up Abeta (1), and that amyloid beta aggregates are in fact naturally taken up and degraded in microglia, but at a rate that is clearly inadequate for a person with existing amyloid plaque pathology: "the majority of the internalized Abeta in microaggregates was undegraded 72 h after uptake, whereas 70-80% of internalized acetylated low density lipoprotein or alpha2-macroglobulin was degraded and released from cells ... after 4 h. In the continued presence of fluorescent Abeta microaggregates for 4 days, microglia took up huge amounts of Abeta and became engorged with undigested material. These data suggest that microglia can slowly degrade limited amounts of Abeta plaque material, but the degradation mechanisms can be overwhelmed by larger amounts of Abeta." (2). This suggests that, whether or not Abeta processing is adequate for a normal person, it is not in AD -- and that it is unlikely to keep up when we are using an Abeta vaccine to clear the stuff out of AD or "normally" aging brains.

-Michael

1. Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J,
Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R,
Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M,
Yednock T, Games D, Seubert P.
Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in
the PDAPP mouse.
Nature. 1999 Jul 8;400(6740):173-7.
PMID: 10408445 [PubMed - indexed for MEDLINE]

2. Paresce DM, Chung H, Maxfield FR.
Slow degradation of aggregates of the Alzheimer's disease amyloid beta-protein
by microglial cells.
J Biol Chem. 1997 Nov 14;272(46):29390-7.
PMID: 9361021 [PubMed - indexed for MEDLINE]