The Power of a Genetic Model System:
Using Soil Nematodes
to Discover Genes Involved in
Human Alzheimer’s Disease
Caroline Goutte
Overview
I. The Worm as a Model System
II. The Power of Genetic Analysis
III. An Example:
Research Project: Genetic Analysis of
Cell Communication Processes in C. elegans
Leads to the Identification of Genes
Important for Human Alzheimer’s Disease
Biology at the cellular/molecular level
All species share the
same fundamental
molecular components and
molecular processes
In order to understand these molecules and processes:
Use a MODEL SYSTEM
• Simplicity
• Experimentation
Model Systems
for studying fundamental questions
of biology at the genetic level
– Escherichia coli and bacteriophage
(bacteria and their viruses)
– Saccharomyces cerevisiae
(baker’s yeast)
– Caenorhabditis elegans
(nematode worm)
– Drosophila melanogaster
(fruit fly)
– Mus musculus
(mouse)
•All amenable to
traditional and molecular
genetic analysis
All have attracted a
critical mass of
investigators
•All had their genomes
fully sequenced by 2002
Sydney Brenner
early 1960s
“…nearly all the ‘classical’ problems of
molecular biology have either been solved or
will be solved in the next decade…the future
of molecular biology lies in the extension of
research to other areas of biology, notably
development and the nervous system.”
Part of the success of molecular genetics was due to the use of extremely simple organisms which could be handled in
large numbers: bacteria and bacterial viruses. The processes of genetic replication and transcription, of genetic
recombination and mutagenesis, and the synthesis of enzymes could be studied there in their most elementary
form, and, having once been discovered, their applicability to the higher forms of life could be tested
afterwards. We should like to attack the problem of cellular development in a similar fashion, choosing the
simplest possible differentiated organism and subjecting it to the analytical methods of microbial genetics.
Thus we want a multicelluar organism which has a short life cycle, can be easily cultivated, and is small enough to be
handled in large numbers, like a micro-organism. It should have relatively few cells, so that exhaustive studies of
lineage and patterns can be made, and should be amenable to genetic analysis.
We think we have a good candidate in the form of a small nematode worm…
Although the total number of cells is only about a thousand, the organism is differentiated and has an epidermis,
intestine, excretory system, nerve and muscle cells.
Caenorhabditis elegans
introduced in 1963 by Sydney Brenner
•
•
•
•
A new model system specifically chosen for its simplicity
Goal: the genetic dissection of development and behavior
Success: Today’s “Worm Community” ~15,000 scientists
3 Nobel Prizes (‘02, ‘06, ‘09)
• Features:
–
–
–
–
–
–
–
Size: 1 mm in length
Generation time: 3 days
Life Span: 2-3 weeks
~300 progeny per generation
Cultivate in lab on petri dishes
Transparent
Only 959 cells!
B. Goldstein, UNC
3 Unique Tools that made C. elegans
a powerful model system:
Complete Cell Maps
Complete Cell Lineages
Complete Genome Sequence
Tool #1: Detailed Map of Worm Anatomy
single-cell
(completed by John White, 1986)
959 somatic cells
(neurons, muscles,
intestinal cells,
epidermal cells, etc.)
Tool #2: Cell Lineage Maps
Each cell’s lineage can be traced
back to the fertilized egg
(completed by John Sulston, 1983)
1 sec = 1 hr.
B. Goldstein, UNC
fertilized
egg
AB
ABa
ABa
ABp
EMS
P2
P1
ABp
time
EMS
P2
fertilized egg
959 somatic cells in adult hermaphrodite
Tool #3: Complete Genome
Sequence
(completed by C. elegans Sequencing
Consortium, 1998)
Model System
Genome
sequence
completed
Genome
Size
(Mb) (x
106)
S. cerevisiae
(yeast)
1997
12
C. elegans
1998
D. melanogaster
(fruit fly)
Number
of genes
Gene
Density
(Kilobases
per gene)
6000
2
100
~20,000
5
2000
140
~14,000
10
M. musculus
(mouse)
2002
2500
~25,000
100
H. sapiens
2001
3000
~25,000
120
December 11, 1998
Genetic Analysis
Genes encode proteins that drive biological processes
We can learn about specific gene function by
studying the effect of gene mutations:
normal gene
defective gene
normal function
aberrant function
I
Genetic Analysis: Two different approaches
normal gene
defective gene
disrupt gene X
II
normal gene
defective gene
normal function
aberrant function
discover the
function of gene X
normal function
aberrant function
discover genes
find disrupted
responsible for function X
function X
I
Genetic Analysis: Two different approaches
normal gene
defective gene
disrupt gene X
normal function
aberrant function
discover the
function of gene X
Start with GENE of interest: What is the function of gene x ?
Use targeted mutagenesis or RNAi
* Adapt to High Throughput version
Observe effect
on phenotype
Start with PROCESS of interest: What gene products are
involved in process x?
The Mutant Hunt:
II
1)
2)
3)
4)
predict mutant phenotype
perform random mutagenesis
collect desired phenotype
identify responsible genes
normal gene
defective gene
normal function
aberrant function
discover genes
find disrupted
responsible for function X
function X
I
Genetic Analysis: Two different approaches
normal gene
defective gene
disrupt gene X
II
normal gene
defective gene
normal function
aberrant function
discover the
function of gene X
normal function
aberrant function
discover genes
find disrupted
responsible for function X
function X
An example
Research Question:
What are the molecules
that mediate cell communication?
mouth
from Z.F. Altun & D.H. Hall and from S. Mango
?
mouth
ABp
ABp
ABp
ABp
cell communication
How?
cell communication
What genes are required?
normal gene
defective gene
function
normal
How?function
aberrant
discover genes
find disrupted
responsible for
Cell communication
Cell communication
Genetic Mutant Screen
1) Random mutagenesis of C. elegans genome (20,000 genes)
2) Search through thousands of worms
3) Find mutants that have aberrant pharynx morphology
wild type:
mutants:
aph-1aph-2glp-1lag-1lag-2pen-2sel-12sup-17-
The products normally encoded by
these genes are responsible for
mediating the cell communication event
What are these products?
…turn to the gene sequence for clues
What type of protein
is encoded by each gene?
 Identify the gene in genome
 Study DNA sequence to predict gene product
 Compare to gene databases
glp-1
DNA sequence of the glp-1 gene
atgcgagttcttctaattttactcgcgttttttgcgccaatcgccagtcaacttatgggtggagaatgcggaagggaaggtgcttgctccgtcaatggaaaatgctataatg
gaaaactgattgagacatactggtgccgttgcaaaaaaggattcggaggtgctttctgtgaacgtgaatgcgatttggattgtaaacgaggcgagaagtgcatctacg
atgtttatggtgaaaatccgacgtgtatctgtcaagattgcgaagacgagactcctccaacagaacgtactcaaaaaggctgtgaagaaggctatggaggtcctgact
gcaaaactcctctattttcgggagtaaatccatgcgattcggatccttgcaacaacggactctgctatccattctatggtggatttcagtgcatatgcaacaatggatatgg
aggatcgtattgtgaagaaggaatcgatcattgtgctcaaaatgaatgcgcagaaggttcaacgtgtgtcaatagtgtatacaactattactgtgattgcccaattggaa
aatccggtcgatattgtgaacgaactgaatgtgctttgatgggaaacatttgcaatcatggaagatgtattccgaacagagatgaagacaagaacttcagatgtgtatg
cgactcgggatacgagggagaattttgcaataaggataaaaacgaatgcctcatcgaagaaacgtgtgttaacaactctacatgtttcaatttgcacggtgattttactt
gtacctgtaaacctggatacgctggaaagtattgcgaggaggctatcgacatgtgcaaggattacgtttgccaaaatgatggatactgtgcccatgactcgaatcagat
gccaatttgttattgcgaacaaggattcactggacaacgatgtgagattgagtgtccttcaggattcgggggaattcattgtgatcttccactacagagaccacactgctc
tcggagcaatggaacgtgttacaacgatggaagatgtataaatggtttctgtgtctgtgaacctgattatattggagatcgatgtgagattaataggaaagatttcaag
Predict Protein sequence of the glp-1 gene product
MRVLLILLAF FAPIASQLMG GECGREGACS VNGKCYNGKL IETYWCRCKK
GFGGAFCERE CDLDCKRGEK CIYDVYGENPTCICQDCEDE TPPTERTQKG
CEEGYGGPDC KTPLFSGVNP CDSDPCNNGL CYPFYGGFQC ICNNGYGGSY
CEEGIDHCAQNECAEGSTCV NSVYNYYCDC PIGKSGRYCE RTECALMGNI
CNHGRCIPNR DEDKNFRCVC DSGYEGEFCN KDKNECLIEETCVNNSTCFN
LHGDFTCTCK PGYAGKYCEE AIDMCKDYVC QNDGYCAHDS NQMPICYCEQ
GFTGQRCEIE CPSGFGGIHCDLPLQRPHCS RSNGTCYNDG RCINGFCVCE
glp-1 gene encodes a
cell surface receptor protein
Extracellular
cell membrane
Intracellular
glp-1 gene encodes a
cell surface receptor protein
Evolutionarily conserved
Family of “Notch” Receptors
Extracellular
cell membrane
Intracellular
C.elegans
2 Notch genes
J. Kimble et al., U. Wisconsin, 1987
J. Priess et al., MRC England, 1987
I. Greenwald et al., Columbia U., 1989
Mammals
4 Notch genes
Notch Receptor Protein
•on cell surface
•receives a signal
signaling cell
Notch Receptor Protein
•on cell surface
•receives a signal
signaling cell
wild type:
mutants:
aph-1aph-2glp-1lag-1lag-2pen-2sel-12sup-17-
The products normally encoded by
these genes are responsible for
mediating the cell communication event
What are these products?
…turn to the gene sequence for clues
What types of proteins do these
genes encode, and what do they do?
signaling cell
Notch
responding cell
LAG-2
more genes:
aph-1 and aph-2
SEL-12
LAG-1
target gene regulation
aph-1 is predicted to
encode a 7-pass
trans-membrane protein
Look for aph-1-like genes in
genomic databases:
Eureka!
aph-1 is a conserved gene
(Human and Drosophila genes now receive a name: “Aph1” !)
aph-2 is predicted to encode a large extracellular protein
that is anchored to the membrane
glycosylations
Look for aph-2-like genes in
genomic databases:
Eureka!
aph-2 is a conserved gene
membrane
spanning
What is the molecular role of
APH-1 and APH-2 ?
signaling cell
LAG-2
APH-2
APH-1
?
Notch
responding cell
SEL-12
LAG-1
target gene regulation
And now for something
completely different….
(or so we thought)
Studies of Alzheimer’s disease…
Alzheimer’s Disease
Neurodegenerative dementia
Amyloid Plaques in brain tissue
Abnormal accumulation of amyloid b protein (Ab )
Small % of AD patients have early onset AD
because they inherited an aberrant gene
that increases the amount of aggregating Ab
What are these genes?
1) APP (amyloid precursor protein)
2) Presenilin
•can these genes lead us to understand the
molecular mechanisms that lead to AD?
APP encodes a small membrane-anchored protein
that gets cleaved to release Ab protein
Presenilin is a membrane-bound protein that cleaves APP
amyloid plaques in
Alzheimer’s disease
amyloid
precursor
protein
APP
Cleavage II
presenilin
presenilin = human version of sel-12,
(the C. elegans protein that
cleaves Notch Receptor !)
Ab
aberrant
cleavage yields
neurotoxic Ab
Drug design?
How does it work?
?
pre-immun
presenilin
immune
How does presenilin cleave APP?
Who are its collaborators?
Protein biochemistry using presenilin as fishing bait:
200 kD
discover an interacting protein: “Nicastrin”a
b
Yu et al., 2000
presenilin Nicastrin
97 kD
55 kD
PS1-FL
36 kD
PS1-NTF
21 kD
PS1-CTF
APP
Nicastrin
presenilin
Cleavage II
Ab
1
2
What type of protein is Nicastrin?
Determine gene sequence
Look in databases for similar genes
human Nicastrin
=
C. elegans APH-2
!
Instant Lessons:
 human Nicastrin must function in
Notch-mediated cell communication
APH-2’s molecular role in cell communication
is clearer: APH-2 forms a complex with with sel-12
What is the molecular role of
APH-1 and APH-2 ?
signaling cell
LAG-2
APH-2
?
APH-2
Notch
responding cell
SEL-12
LAG-1
target gene regulation
APH-1
signaling cell
APH-1 and APH-2 interact
with presenilin/sel-12
to form a molecular machine
that cleaves cell surface proteins
(Notch Receptor, APP, etc.)
APH-2
Notch
responding cell
SEL-12
APH-1
LAG-1
target gene regulation
Model of
-Secretase
Quartet
APH-2
SEL-12
APH-1
Cell Communication
via Notch Receptor
Alzheimer’s Disease
 C. elegans model system
 hunt for mutants
 sel-12 (Levitan et al., ‘95)
 aph-2 (Goutte et al., ‘00)
 aph-1 (Goutte et al., ’02;
 pen-2
Francis et al., ‘02)
(Francis et al., ’02)
 study of inherited form
 Presenilin/SEL-12
(Sherington et al.’95;
Levy-Lahad et al., ’95;
Rogaev et al., ‘95)
 protein biochemistry
 Nicastrin/APH-2
(Yu et al., ’00)
Model of
-Secretase
Quartet
APH-2
APH-1 Nicas.
Presenilin
SEL-12
PEN-2
Useful Websites:
http://www.wormclassroom.org
designed for teachers (high school - college)
great introduction, pictures and movies,
and computer-based exercises,
http://www.wormbase.org
gateway to all C.elegans reseach tools
searchable - by author, by cell, by gene, etc.
http://elegans.swmed.edu
more simple gateway - good introductions to worms
http://www.wormatlas.org
beautiful images and explanations of all the worm cells,
tissues, major anatomical descriptions
http://www.bio.unc.edu/faculty/goldstein/lab/movies.html
great movies (single-cell divisions as well as whole worm)
1 sec = 1 hr.
An example: where is the “HSN neuron”
8000 prints from serial
section electron micrographs
Lineage of the HSN cell
HSN in adult