1) The document summarizes a research project assessing the effects of MPTP treatment on the nigrostriatal dopaminergic system in mutant ND6 mice.
2) The experiment involved treating wild-type and mutant ND6 mice with sub-acute doses of MPTP over 5 days and analyzing TH+ neuron counts in the substantia nigra pars compacta (SNpc) 21 days later using stereology.
3) The results showed little difference in TH+ neuron loss between wild-type and mutant ND6 mice, suggesting these particular ND6 mutations did not play a role in MPTP-induced dopaminergic cell death.
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Jonathan's Summer Internship Presentation.pptx FINAL copy
1. Candidate: Jonathan J. Nkangabwa
Department of Pathology and Cell Biology
Lab advisor: Serge Przedborski, MD/PhD
ASSESSMENT OF THE NIGROSTRIATAL DOPAMINERGIC
SYSTEM IN MUTANT ND6 MICE TREATED WITH MPTP
2. A LITTLE ABOUT MYSELF
Jonathan Nkangabwa, 19 years of age from Boston, MA (Currently live in
Medford, MA)
Rising Sophomore at George Washington University, Washington, D.C.
Plan to go to Medical school ( Possible specialties: Anesthesiology, Family
Practitioner, or General Physician)
First time participating in a Research Lab Internship
3. PARKINSON’S DISEASE
Common
About 60,000 new/yr
Approx. 106 cases in the US
Age of onset 55-75 yr
Dopamine deficiency
Brain stem destruction
Clinical hallmarks (TRAP)
Tremor
Rigidity
Akinesia
Postural Instability
Fahn & Przedborski, Merritt’s Neurology
5. Theories as to why DA neurons die in PD
calcium overload
mitochondrial dysfunction (lab worked on)
oxidative stress
enzyme abnormalities
environmental factors
6. Working Hypothesis
Complex 1 is impaired in PD (complex 1 has 46 subunits)
Decreases/impairment of complex 1 of the mitochondrial electron transport
chain free radical (i.e. superoxide) production no electrons traveling
down the chain no ATP production energy crisis in the neuron
death of the neuron
Assessed the effects of ND6 mitochondrial point mutations in mice
ND6 mice more susceptible to MPTP ?
7. MPTP is a blocker of
mitochondrial complex 1
MPTP kills specifically
dopaminergic neurons free
radical production
MPTP: How it Works
ND6
8. Sub-Acute Dosing Schedule
• Multiple dosing regiments for MPTP (not clear on exact course of nigral
degeneration)
• 2007 “Protocol for the MPTP mouse model of Parkinson’s disease.”
• Sub-acute dosing or chronic intoxication regimen is one injection per day for
several days/weeks (consecutive)
• Sub-acute makes more sense since it isn’t sudden onset/rapid change like
acute, and not having virtually little to no change like chronic dosing; in the
middle like PD
9. The Experiment: ND6 Gene
To do this, I learned the following:
ND6 mutant mice treated with MPTP sub-acute dosing of 30 mg/kg
x 5 days
perfusion (after 21 days after last dose of MPTP)
post fixation (PF overnight )& cryoprotection (30% sucrose
aritfacts)
froze in -40 degrees isopentane
Mounted brains on the chucks
B6ME
1A1B6ME
B6MGR
1A1B6MGR
4 groups of
Mice used
10. Collection of tissue
cut at 30 microns, Charlie Chaplin’s feet for the Substantia Nigra (stereology)
for the Striatum, I used crosshairs (an X) as landmark (collect 6 , throw away 8)
collected each section of SN individually in a 48 well plate (stereology)
stained for TH (explain it is the best to assess the dopaminergic striatal system)
Pontine nuclei
Triangular
septum
13. Stereology
Method of counting neurons in the brain
Counting neurons in 3D using 2-D images (TH+ neurons)
StereoInvestigator (computer-assisted image analysis method): an
unbiased method of counting which is not affected by volume of
SNpc or the size of neurons
Used during duration of stay at MNC (weeks of counting)
16. Nigral Striatal Dopaminergic System in Mice
Nigral striatal system
(Substantia Nigral connection
to Striatum)
K. Tieu Cold Spring Harb Perspect Med. 2011
18. Conclusion/Perspective
Conclusion:
-From results, seems that these particular ND6 mutations played little or no
role in the MPTP induced dopaminergic death in SN (in process of analyzing
DA terminals in the striatum)
Future Studies:
-I would like to investigate if other mutations in Complex 1 are in play, or a
different dosing schedule would effective with the mutations that we have
here
19. SPECIAL THANKS TO:
Serge Przedborski
Vernice Jackosn-Lewis Javier Blesa
Sudarshan Phani Kristin Politi
Radhika Pradhan Rosa De Vries
Diane B. Re Norma Romero
James Caicedo Virginia Le Verche
& the many helpful members at the MNC !
Editor's Notes
Signs aren’t shown until 80% of the dopaminergic neurons are lost
Nigrostriatal pathway degeneration
Loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc)
Loss of dopaminergic nerve terminals in the striatum
Lewy body (intraneuronal inclusion)
Free radicals (oxidants that become very harmful when there is a massive buildup
Number of theiries to the DA death in PD
None are nutually exclusive, most cases, it prob. Involves a number of these
GDNF, BDNF
Calcium overload, promotes degeneration
Oxidative stress
Thought to be in the ideology, none are mutually exclusive
Could combinations , but we do not know
Complex 1 has 46 subunits, which is the largest complex; which damaged subunit does contribute to the PD picture
I learned how to assess Dopaminergic neurons in the Nigra straital
Assesment involves,
Counting neurons in SN, and looking at the number of the remaining TH fibers in the striatal
One of the best ways to assess the system is to look at Tyrosine Hydroxylase (then brief explanation of what it is)
TO do this, I learned how to perfuse brains, cut the brains, collect the tissues, stain the brains for TH, and count the TH positive nuerons
MPTP is able to bypass, very lypophylic, so fat tissues can carry it
Gets into astrocytes, and gets turned into MPP+, OC3 transporter takes it up and takes up dopaminergic pathways
DA is dopamine and the MPP is able to go to dopaminergic cells
MPTP is able to bypass, is catylized by glial cells, inhibition of complex one in the mitochondria of dopaminergic neurons
My experiment involved ND6 mice that had point mutations in the complex 1 gene (after 21 days because the lesion has to become stable)
ND6 is a subunit of complex 1; and there are point mutations in this subunit of ND6 subunit of complex 1
-also lead to complex I defects, altered Ca++ regulation in the mitochondria, Lewy bodies and neurological signaling
SLIDE To do this, I learned the following:
-4 different groups of mice; point mutations in mitochondria
-mouse brains from the ND6 mutant mice that were treated with MPTP (talk about treatment) sub-accute dosing of 30 mg/kg x 5 days
Perfusion- (21 days after last dose of MPTP)
Post fixation & Cryoprotection- (brains immersed overnight in PF; cryo-protected and then switched in viscous solution of 30%Sucrose to prevent artifacts)
froze in dry ice in isopentane (-40 degrees)
-Mounted brains on the chucks with OCT
-Cut on the cryostat
Cut at 30 microns, as a landmark to start I used, what we call Charlie Chaplin’s feet for the Substantia Nigra; landmark to end was the 3rd ventricle) striatum, is where the neuron terminals are connected to the SN
For the Striatum, I used crosshairs (an X), to be a landmark until I
Collected each 30 micron sections of SN individually in a 48 well plate (stereology)
Stained for TH (explain it is the best to assess the dopaminergic striatal system)
Talk about Collection of striatum for TH (collect 6 , throw away 8; total representation of the striatum; assess it by staining for TH, but looking at optical density in terms of density
~RATE-LIMITING FACTOR in the production of Dopamine **
Catecholamines- major forces that act as transmitters, hormones, and regulators for motor function and behavior in mammalian and non-mammalian
~Synthesized through several enzyme, including
~TH : tyrosine-3-mono-oxygenase is the rate-limiting enzyme in dopamine
~Catalyzes the conversion of L-tyrosine
~from birth, catecholamines increase, and reach plateau when adult stage is hit. When aging, the number decreases and TH is the only rate-limiting enzyme in this synthetic pathway
~TH basically turns L-tyrosine into DOPA (dihydroxy-phenylalanine) and turned into dopamine (popular neurotransmitter, neurohormone)
SN TH positive neurons
Striatum TH positive terminals
In the lab, Serge, one of the theories we are working on is mitochondrial involvement
One of the findings, is that there is a decrease in complex 1 in the mitochondrial chain
Decrease in complex 1, produces free radicals and interferes with energy production in the cell
To mimic parkinson’s disease, we used the neurotoxin called MPTP (SHORT story of MPTP synthesis)
Found in patients. Decrease in complex 1; interference in ATP production, because mitochondria are the powerhouses
Involvement of complex 1, by blocking it
SLIDE Defects in complex 1, so how does this effect cell behavior
Needed to asses nigral strialtal system, dopaminergic system in mice, using a complex one blocker, which is MPTP
OC3 transporter, takes MPP+ and DA transporter
Nigra striatal photo
Mitochondria are the power house of the cells, because they produce the energy , which involves ATP production
40% loss of the cells in SN, and 60% of the terminals would hav been lost in Striatum. Looking at this, you see the 1A1 and M6GR, there is some neuronal loss
In all 3 groups there is neuronal loss within the mice treated with MPTP
Indidcation from nisal staining that there is actual neuronal loss
These results were not augmented like we would have expected
Sometimes with this treatement , down reg. of TH
Maybe because the dosing was not enough, or these mutations don’t play a role into MPTP
In view of the fact, we don’t see 40%
In looking at percent loss of dopaminergic neurons in the SN, we did not achieve the numbers in loss that have been found in normal animals, so we can conclude that the ND6 mutation did not effect dopaminergic neuron loss in these mice
With these results, I would like to investigate, compounds/drugs that can offer protection to both dopaminergic neurons and terminals within the brain
Talk about still having to assess the terminals in these mice (dopaminergic terminals from the striatum of these mice)
From this, I conclude, these mutations can affect responses to certain drugs,
Further experiments:
What I would like to do for further looking into this. Like to see maybe what has happened to the rest of the mitochondria chain or proteting dopaminergic neurons by using the complex 1
Thanks, any questions, etc
Change up design of this slide. Special thanks to serge and vernice
