I glue fruit flies to tiny sticks made of tungsten, attach the free end to a magnet,
and run a program that handles a 360-degree flight simulator and controlled sprays of odor to see what they do...
NOTE: This is NOT Dr. Frye's equipment at UCLA. This is, however, an example of how to perform
behavioral tests on Drosophila.
I've signed up to do a poster presentation with my ideas for the Parkinson's Disease Symposium
on Thursday, May 21. This is an incredibly impulsive, yet exciting opportunity! My hope is that I'll
get some good feedback on my ideas and put my name and lab's name out there for the Parkinson's community
at UCLA to recognize in the future when I start writing and trying to publish.
This means more research and more notes!
Shulman JM et al: From fruit fly to bedside: Translating lessons from Drosophila models of
neurodegenerative disease.
Basic Idea:
Drosophila neurodegenerative models show promise because they use the same machinery (although less
of it than humans) and also behave in complex ways with this machinery much like humans. Their genes
are thoroughly studied, and they grow fast. Also, in PD-induced flies, there's a particular uncanny
resemblance to the human PD form. "Although still a relative newcomer to neurodegenerative disease
research, Drosophila is rapidly making signiÞcant contributions."
Beal MF: Experimental models of Parkinson's disease.
Basic Idea:
MPTP toxicity (in primates and other animals most like humans) would be the ideal animal model
for PD. It's also important to note that Beal wrote a number of points that give Drosophila the
advantage over other animal models. They are a more accurate human PD model, for example, because
mouse models so far have not proven to have PD pathology specifically in their DA neurons in their
substantia nigra. Drosophila, on the other hand, HAS had DA-neuron specificity with the
alpha-synuclein models. They're also well-characterized genetically, and only one gene is necessary
to induce PD. Rapid aging of Drosophila makes for less wait-time for adult-onset PD symptoms.
Doty et al: Olfactory dysfunction in Parkinsonism: a general deficit unrelated to neurologic
signs, disease stage, or disease duration.
Basic Idea:
81 PD patients who show minimal dementia were tested for smell detection and identification.
Relative to matched controls, the PD patients exhibited consistent and marked decrements on both
types of olfactory tests. There were no specific odors or longitudinal changes throughout the
experiment, and scores were INDEPENDENT of disease stage and duration. The data support the
hypothesis that the olfactory deficit of PD is a general and stable one which likely occurs early in
the disease process.
April 30, 2009
Some notes and ideas that I wrote up today:
Gerlach et al. Early Detection of Parkinson's Disease: Unmet Needs
Basic Idea:
This is a discussion of the importance of finding good biomakers for Parkinson's disease
(PD) to help diagnose the disorder. "No absolutely safe diagnostic test is currently available."
Some key points:
Biomarkers are important in the study of diseases because they give insight into what is normal and
what goes wrong in a given disease. Biomarkers are indicators for the diagnosis and classification of
a disease and the progression of a disease, both when a patient is deteriorating or improving.
For PD, much of the known biomarkers are neither sensitive nor specific enough to easily diagnose patients
with PD (especially in the early stages of PD). The only known fool-proof diagnosis for familial PD is
with a genetic test, but biomarker for sporadic Parkinson's disease lack adequate information on the
sensitivity and/or specificity of the tests.
Note: By testing to see whether Parkinsonian Drosophila will exhibit hyposmia/anosmia, it may prove
that this particular symptom is a universal biomarker for PD.
Feany et al. A Drosophila model of Parkinson's disease
Basic idea:
Normal and mutant forms of alpha-synuclein are expressed in Drosophila, and the mutant flies produce
adult-onset loss of dopaminergic (DA) neurons, filamentous intraneuronal inclusions (consisting of
alpha-synuclein) and locomotor dysfunction.
Some key terms to know for this paper:
Alpha-Synuclein is a protein naturally found in neural tissue, but are also the cause of Lewy Bodies
and Lewy Neurites. It is unknown how these proteins aggregate, but scientists have shown that alpha-synuclein
can exist in normal-functioning brain cells in the neocortex, hippocampus, substantia nigra, thalamus,
and cerebellum (like when upregulated in songbirds during song-acquisition), but are the known component
in the insoluble fibrils in neurodegenerative disease like Alzheimer's, Parkinson's, "dementia with Lewy
Bodies" and "multiple system atrophy."
Lewy Bodies and Lewy Neurites are aggregated clumps (called inclusions) and fibrils of alpha-synuclein,
respectively. These can be seen in brain slides under a microscope.
Dopaminergic neurons are important to study in Parkinson's research because these cells are the most
severely affected in the disease. One of the most striking pathological features of the disease is when
dopamine-producing cells of the substantia nigra die off, and the neuromelanin that should be there disappears.
(The subsequent pallor of the substantia nigra is usually the first indication of pathological confirmation.
Double et al, Influence of neuromelanin on oxidative pathways within the human substantia nigra)
A30P and A53T are two mutant proteins linked to familial PD.
What they found: Expression of human alpha-synuclein in flies show three key features of the pathology
of Parkinson's disease.
Adult Onset. PD is very rarely diagnosed in young people. The destruction of dopaminergic neurons
in the substantia nigra also start happening long before symptoms show. Tremors and other obvious PD
biomarkers begin to happen when up to 80% of the healthy DA neurons are gone.
Symptoms are restricted to the nervous system. When A30P and A53T are expressed in the mutant flies,
they eventually develop locomotor dysfunction, and don't show side effects outside of the known Parkinsonian
symptoms.
Anatomical specificity. Alpha-synuclein inclusions could be found in the brains of aged flies by
immunostaining. Just like human diffuse Lewy body disease, the brains of the transgenic flies show these
Lewy bodies and Lewy neurites, and the wildtype brains were inclusion-free. Retinal degenerationg also
occurs by day 10 when alpha-synuclein is expressed in the eye, and day 30 for transgenic flies.
Note: The adult onset of both motor dysfunction and alpha-synuclein inclusions makes it possible for
young transgenic Drosophila to be used as a good model for pre-clinical stages of PD.
The Experiments: I paid particular attention to this part because I'd like to see if my idea of using
the Frye lab equipment is within the realm of realistic testing.
Sectioning, immunostaining, and electron microscopy. Stains for DA neurons of serial tissue sections
through the entire brain of the adult fly. Just like PD, the volume of the transgenic flies' brains is
preserved. Immunostaining experiments showed age-dependent loss of dorsomedial DA neurons. Electron
microscopy showed expression of inclusions.
Climbing test. Forty flies placed in a plastic vial and tapped to the bottom. After 18 seconds,
count the number of flies that then successfully climbed to the top. Young transgenic flies showed no
physical handicaps, but began to exhibit motor dysfunction by day 23. Flies expressing A30P alpha-synuclein
lose their ability to climb earlier than their A53T-expressing and wild-type counterparts.
Bohnen et al. Selective Hyposmia in Parkinson's disease: Association with hippocampal dopamine activity
Basic idea:
Olfactory deficits do not necessarily worsen with the progression of PD, but it has been established
that hyposmia is correlated with PD pathology (including early/pre-manifest PD). This paper shows that
there is a stronger correlation between selective hyposmia and hippocampal dopamine activity as opposed
to dopamine activity in the amygdala.
Fleming et al. Olfactory deficits in mice overexpressing human wildtype alpha-synuclein
Basic idea:
Mouse models of early stage Parkinson's disease show olfactory impairments.
The Experiments: I want to know how and why they performed the experiments that they did, in hopes
to gain insight into what to do when designing an olfactory-testing experiment with animals.
Buried Pellet Test. Starved mice were put into clean cages with a randomly hidden food pellet
0.5cm underneath the bedding. Latency to pellet discovery was timed. Surface tests were also performed
by placing the randomly-placed pellet on top of the bedding instead.
Block Test. Blocks that take on the odor of each test animal are placed in a clean space,
where they are allowed to sniff and interact with the block/environment. The latency of approach
the unfamiliar block and length of time these animals sniffed were recorded as data, and they tended
to sniff unfamiliar blocks more than their own. Also, blocks with minimal unfamiliar odor were tested.
Habituation/Dishabituation Test. Three pairs of odors are presented to the mice at different ages.
Their odor differentiability was tested.
April 17-21, 2009
Iiit's my birthday weekend! It's also the weekend when Darcy comes to visit me from the East Coast,
and when I am studying for exams in my Neuroscience, O-Chem and Neurobiology of Aging classes. (I can't wait
to NOT be an undergrad anymore...)
More to come soon. Progress on "Mission: PD flies" is a bit stagnant because of legally turning
a year older and midterms. Woot!
April 9, 2009
Mark is no longer mulling.
I've discussed my options with Yan Zhu and Partha Krishnan, the two post-docs who are awesome
for offering some of their time to oversee this experiment. I don't expect to be bothering them too
much because I already have most of the techniques down, but the only hang-up with Mark was the fact
that a lowly undergrad like me can't just go AWOL with his resources and equipment.
Smooth sailing so far...
Next update: The flies and other hangups
April 7, 2009
Mark is mulling.
My biggest concern with this project at this point is whether these PD model flies will be capable
of flying in the flight arena. What if they can't fly because of the devastating effects of the aggregated
protein in their little insect brains? All the literature I could find on these flies were walking tests
and molecular studies. Nothing on flight. Is this because the researchers don't have the equipment, or
are their flies too weak to fly?
As of today, this is no longer my biggest concern! From Mel B. Feany and Welcome W. Bender's Drosophila model
of PD research publication in 2000 from Harvard Medical School: "Locomotive behavior is grossly
preserved in young flies."
Although I am aware they worsen with age, I am PSYCHED. This may not indicate a 100% guarantee that they
are capable of flight, but I think this is worth looking into further.
Next update: More research and ordering the flies!
April 5, 2009
Today I sent my awesome PI, Mark Frye, a research proposal. This means I went online and researched
a whole bunch about fruit flies and neurodegenerative disorders and discovered that my idea has never
been investigated before.
So here's my idea, simply put: I want to test whether the Drosophila melanogaster with overexpressed
alpha-synuclein-type Parkinson's Disease can smell as well as the wildtype flies.
Parkinson's Disease (PD), as you may already know, is a neurogenerative disorder where the
dopaminergic neurons inexplicably die off in the substantia nigra, a key component in the brain that
controls body movement.
There are a few ways to induce PD in fruit flies. Although there is some research done on the
causes of spontaneous PD (the latest culprit is a heavy-duty toxins used in agriculture),
about 5% of the cases are genetically triggered. These genes are what we are inserting into the Drosophila
DNA, which are essentially causing the flies to overproduce human alpha-synuclein. Alpha-synuclein is
a protein naturally found in the brain, but is also the guilty misfolded protein that causes the aggregates
in neurodegenerative disorders such as PD and Alzheimer's.
There is no cure, and we don't know exactly how the synuclein protein aggregates in the disease.
We do know, however, that early on in the disease, even before the obvious symptoms wreak havoc on the
body, PD patients start losing their sense of smell.
Research at the VU Medical University in Amsterdam showed that scientists could guess who was
going to get PD even before they had any tremors, just by testing their sense of smell.
Mark (the PI for this lab,) is the perfect guy to work under for a project like this because
his grad and post-doc lab members do behavioral experiments with equipment specially designed for fruit
flies and their response to odor.
