The document investigates putative monoamine oxidase (MAO) genes in the nematode Caenorhabditis elegans. The researcher searched for genes containing amine oxidase domains and identified two candidate MAO genes, amx-1 and amx-2. Transgenic experiments showed amx-1 is expressed in chemosensory neurons and gut cells, while behavioral assays found differences in monoamine-dependent behaviors between wild-type and mutant worms. Further experiments aim to purify and characterize the protein products of the candidate MAO genes to better understand their roles in the worm's nervous system and behaviors.

1. Investigating putative Mono Amine Oxidase (MAO) genes
in Caenorhabditis elegans
Reetobrata Basu
PI: Dr. Janet Duerr
2013 Sigma Xi Student Research Showcase

2. Topics
• Background
• Monoamines (MA)
• Monoamine Oxidase (MAO)
• Monoamine Oxidase Inhibitors (MAOI)
• Significance
• Investigating with Caenorhabditis elegans
• Searching AO domains in the Worm
• Hypothesis, Plans and Methods
• Experiments
• Summary
• Up Next

3. Monoamines (MA) and What They Do
• MA = Neurotransmitters in the brain
• Precursor = amino acids
Examples: Serotonin, Dopamine, Norepinephrine, Epinephrine, Histamine, Tyramine,
Octopamine, Melatonin, etc
http://neurosciences.beaumont.edu/carotid-artery-stenting http://countyourculture.com/category/neurotransmitters/
These chemical molecules, called monoamine neurotransmitters, modulate
several behaviors in humans (as shown above) and other animals.

4. Monoamine-oxidase (MAO) and What They Do
= mitochondria
??
http://pearlsofprofundity.wordpress.com/2012/08/17/what-is-a-synapse/
2 of the 90 billion neurons in human
brain, communicating with each
other by electrochemical signals,
via neurotransmitters.
At the pre-synaptic neuron (left), MA neurotransmitter is synthesized from amino
acids, packaged in vesicles and released at the synapse, after which the MA binds
to specific receptors in the post-synaptic neuron (right). MAs can be removed after
release, by MA-reuptake-transporters and then MAO degrades them. MAO might
also exist extracellularly in brain.

5. Monoamine Oxidase Inhibitors (MAOI)
• Inactivates MAO = Increases level of MA at the synapse
• Prescription drugs for
Major depressive Disorder (~14.8 million adults in US only)*,
Anxiety disorder (~40 million adults in US only)*,
Obsessive Compulsive Disorder (~2.2 million adults in US only)*,
Attention Deficit Hyperactivity Disorder (~10 million young adults in US only)*,
Post Traumatic Stress Disorder (~7.7 million adults in US only)*,
Parkinson’s Disease (~1 million of adults in US only)*,
* http://www.nimh.nih.gov/health/publications/the-numbers-count-mental-disorders-in-america/index.shtml
MAOIs prescribed for the above disorders . Note that a number of them are unavailable
due to highly adverse side-effects

6. Monoamine Oxidase Inhibitors (MAOI)
MAOI  effects and side -effects

7. Significance of My Project
• MAO is a validated drug target with a potential to address a number of major
neurological disorders.
• MAO-Inhibitors, presently prescribed have strong adverse side-effects, i.e. they hit
multiple unknown non-specific targets, when administered.
• MAO and the entire MA-pathway, needs further intense research for being able to
discover molecules, which are free of unwanted side-effects and more specific.
• The entire MA-pathway is well studied in the worm – Caenorhabditis elegans. It is
an excellent model organism for the purpose.
• The existence and role of MAOs in the C. elegans MA-pathway, are yet to be
established completely

8. Investigating with Caenorhabditis elegans
Image Source: www.exploratorium.edu
Worms, expressing GFP, feeding on bacteria, on a small 10 mm diameter plate
• Elegant model in neuroscience
• 1 mm long, Transparent body, short life cycle, Easy maintenance
• First eukaryote with a completely sequenced genome
• High homology with a number of human genes
• Genetic manipulations are easy
• Total 959 somatic cells, 302 neurons; All mapped

9. MAO has amine oxidase (AO) domains
Method of Action of MAO, using FAD (Flavin adenine
dinucleotide) as a cofactor : MAO converts a MA to its
corresponding aldehyde. H2O2 is produced in the way.
Humans have two MAOs – MAO-A and MAO-B
with different substrate specificities.
• MAO enzyme has characteristic amine oxidase (AO) domains
• I looked for genes containing AO domains in C. elegans genome database

10. Searching AO domains in the Worm
Image Courtesy: Dr. Janet Duerr
• amx-2 gene had maximum ( >40%) homology with AO domain of human MAO-A
Image Courtesy: Dr. Janet Duerr
• amx-1 gene also had AO domain in addition to other domains which indicated
that it could be a histone demethylase enzyme.

11. Hypothesis, Plans and Methods
Hypothesis:
The amx-2 is a monoamine oxidase gene in C. elegans, while amx-1 gene is a
possible histone demethylase with possible roles in development.
To identify the role of these two putative MAOs in C. elegans, the following methods
were adopted:
1.Transgenic analysis
2.Behavioral Assays (mutants, RNAi)
3.Heterologous Expression and Assay
4.Immunohistochemistry (not shown here)
5.In situ levels of MAs (mutant vs N2)

12. Experiments: Transgenic Analysis
• Transcriptional (A) and Translational (B) GFP-Fusion products generated (using Oliver Hobert’s Method, 2002
• Reporter protein = GFP (gfp gene)
• 5 fusion products for amx-1
• 5 fusion products for amx-2
• microinjection @ distal arm of gonad
• Transgenic experiments should tell us
when and where the genes are
expressed
3812 bp upstream 1776 bp (L2463)
amx2 start codon + first 4 aa
2071 bp upstream 5190 bp full amx2 (no stop) 1874 bp (L2463) • One transcriptional (top) and four
translational amx-2::gfp fusion products
2071 bp upstream 5190 bp full amx2 (no stop) 1776 bp (L2463) generated are shown on the left
1332 bp upstream 5190 bp full amx2 (no stop) 1776 bp (L2463)
• Similar amx-1::gfp reporter fusion
3812 bp upstream 5190 bp full amx2 (no stop) 1776 bp (L2463) products were also made (not shown here)

13. Experiments: Transgenic Analysis
• Transcriptional amx-1::gfp expressed in
most nuclei in embryo
• Transcriptional amx-1::gfp expressed in some
monoaminergic neurons of the worm nervous
system
• Transcriptional amx-2::gfp expression
was preliminarily observed in the
anterior and posterior gut in the worm
(not shown here)
• More transgenic experiments are being
done presently, to confirm the
identity of the cells and neurons, where
• Translational amx-1::gfp expressed in high amx-1 and amx-2 are expressed
levels also in cytoplasm?

14. Experiments: Behavioral Assays
• C. elegans has easily assayable MA dependant behavior –
Thrashing in liquid (M9 buffer),
Egg laying,
Movement into food,
Movement in food,
Pharyngeal pumping, etc
• These behaviors should vary in wild-type (N2) vs. amx-mutant worms
• These behaviors should be modulated by MAO-Inhibitors (MAOI)
C. Elegans laying eggs on a petriplate
(Image: Rui Lu, Louisiana State University) amx-2 and double mutants lay significantly less eggs than Wild-
type (N2) C. elegans, while amx-1 mutants lay more eggs

15. Experiments: Behavioral Assays
C. elegans thrashing in water with
centroid (red) and tail trajectories
(blue) Wild-type C. elegans thrashing decreases in presence of MAOI
http://www.youtube.com/watch?v=qDvSYxNGSNg
amx-mutants are less sensitive to MAOI (Tranylcypromine) effect
C. elegans moving in food
(bacteria) on a plate
http://www.youtube.com/watch?v=ToLYgB_bxqM
Wild-type C. elegans movement in food decreases in presence of MAOI
(Selegiline); but amx-2-mutants are less sensitive to MAOI effect

16. Experiments: Heterologous Expression & Assay
Heterologous Expression
Bacteria Yeast
(N-term 6X His tag,
C-term Myc tag)
Host: Pichia pastoris
Gateway Addgene Vector Project Date:
(N-term 6X His tag,
Vector N-term GFP tag)
October’2012 - ongoing
(C-term 6X His tag)
Host: BLR(DE3)CodonPlus
Project Date: June’2011 to September’2012
• Express amx-1 and amx-2 in a heterologous host, to purify active AMX-1 and
AMX-2 to perform biochemical assays, to prove predicted biological
functions
• General Strategy: Cloning, Expression Optimization, , Protein Purification
Biochemical Assay

17. Experiments: Heterologous Expression & Assay
Worm amx-1 and amx-2 cDNAs obtained
• amx cDNAs from Dr. Kohara (NGI, Japan)
• cDNAs completely sequenced
• I found two amx-2 cDNAs had an alternately spliced 15th exon (highlighted in red).
I named them amx-2L and amx-2S, as per size.
• I also found, amx-1 cDNAs had a 24 bp smaller seventh exon than predicted
(not shown here)

18. Experiments: Heterologous Expression & Assay
The above cloning of amx-1 and amx-2 constructs, for heterologous expression,
were completed and confirmed by sequencing:
• I and II are in Gateway expression vectors for E. coli
• III and IV are in Addgene expression vector for E. coli
• V, VI, and VII are in yeast expression vector for yeast (Pichia pastoris)

19. Experiments: Heterologous Expression & Assay
Expression with Gateway constructs: Expression with Addgene constructs:
Western Blot of Western Blot of
AMX-2L against anti- AMX-1 against anti- Western blot with Anti-6His Ab
6His Ab 6His Ab Bacterial cultures as seen
under microscope in UV light M AMX2 AMX1 AMX1 Vector
Ctrl M P S P S Ctrl M Ind Ind UI Ind
GFP tagged AMX-1; 1000X mag
GFP tagged AMX-2; 1000X mag
M = protein marker, UI = No
Ctrl = +ve control for Western blot, M = protein
Induction, Ind = Induced with 1 mM
marker, P = Insoluble fraction, S = Soluble fraction
IPTG
• Truncated expression observed for
AMX-2; Very low intact expression • AMX-1 expression was in high amount and
(insoluble fraction only) intact (in insoluble fraction only)
• Intact expression observed for AMX-1; • AMX-2 expression too low and not detected
Not high enough for purification by Western blot
(insoluble fraction only)

20. Experiments: Heterologous Expression & Assay
Biochemical Assay: Monoamine Oxidase (MAO) function
• I decided to use an HRP-coupled assay to detect the H 2O2, to check MAO activity
• Ampliflu-Red + H2O2  Resorufin (fluorogen) Exc: 530 nm; Em: 59o nm
• Assay done with whole-cell-lysate of
AMX expressed in E. coli
• Time course for 250 ug protein shown
• High noise : signal
• Need purer fractions to get good window

21. Experiments: Heterologous Expression & Assay
Biochemical Assay: Histone Demethylase (HDM) function
• I used Epigentek (Epigentek, NY) kit for H3K4 specific HDM activity/Inhibition assay
• substrate = H3K4me2, product = H3K4me1
• Detection by antibody and fluorogenic substrate
• Fluorogen Ex: 530 nm; Em: 587 nm
• Assay done with whole-cell-lysates of AMX expressed in E. coli
[Assay done only once; hence no error bars]
• Preliminary results indicate that AMX-1 has significant HDM activity
• Need purer fractions to get good window

22. Experiments: In situ Levels of MA
• Levels of Dopamine (DA) and Serotonin (5HT) in worm bodies were measured
• Method: Glyoxylic Acid induced Fluoroscence
DA (blue) and 5HT (red) as seen in the anterior (head) part of the worm, with and without pre-soaking in DA
• Slightly higher levels of DA was seen in double mutant worms
• The levels are qualitative; not very quantifiable.

23. Summary
 Transgenic experiments indicated localization of AMX in chemosensory neurons
 Behavioral assays with mutants and MAOI treatments, show significant
differences in a number of MA-dependant behaviors.
 Cloning of amx-1 and amx-2 cDNAs for heterologous expression vectors
successfully completed
 Heterologous expression of AMX-1 observed (@ insoluble
fraction). Purifying intact, active AMX-1 is underway. AMX-2 expression in
bacteria was low, truncated, absent.
 Both MAO and HDM activity assays work. HDM assay showed (preliminary results)
significant activity for AMX-1. Pure protein necessary.
 RNAi (with dpy-15 strain) and Immunohistochemistry (with anti-H3, anti-H3K4me2
Abs) successfully attempted. Optimizations underway.

24. Work Ahead
 Complete expression in yeast and purify AMX-1 and AMX-2 for assay
 ID the chemosensory neurons in which the amx genes are expressed
 Supplemental behavior assays with mutants and knocked-down (RNAi) N2s
 The roles of AMXs in the worm nervous system and corresponding stimulus
(pH, chemical, temperature, etc) to be identified

25. Thanks
Acknowledgements
 Dr. Yuji Kohara (NGI, Japan)
 Dr. Tomohiko Sugiyama
 Dr. Mark Berryman
 Dr. Robert Colvin
Lab
 Dr. Janet Duerr (PI)
 Nanda Filkin (Lab Technician)
 Setu Kaushal
 Melissa La Bonty
Funds
 Student Enhancement Award’ 2012-13
Ohio University