Neuron polarization: an in vitro study with hippocampal neurons

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Neuron polarization an in vitro study with
hippocampal neurons ??? ??
?????? ---?????
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Why is polarity important?
My personal belief The really important thing is
that could be understood by layman without much
explanation.
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Neuron is polarized --- an idea dates back to 100
years ago
Santiago Ramón y Cajal (1852-1934)
Proposed information flow direction

from Cajals Nobel Lecture
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Neuron Parts
Major sites to receive input
Major sites for output
The question we are focusing on Neuron
polarization Development of axon dendrites.
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In vivo neuron polarization
Unknown !
But we have in vitro study of hippocampal neuron
polarization
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Pioneering in vitro study by Ganry Banker in
1980s-1990s
Craig AM, Banker G. Annu Rev Neurosci.
199417267-310.
Thus, the question of neuron polarization can be
simplified as the selection of one neurite growth
to become axon during the transition from stage 2
to 3 in the in vitro case of this specific type
of neuron.
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Hippocampal Neuron Polarization from Stage 2 to
Stage 3
Neuron growth cone
da Silva JS, Dotti CG. Nat Rev Neurosci. 2002
Sep3(9)694-704.
Evident from intensive axon guidance researches,
neurite growth is tightly controlled by the
structure in its tip ---growth cone. Maybe the
question of polarization can be furthur
simplified as the selection of growth cone for
growth.
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Why is the mechanism elucidated only in the very
recent period
1. The rapid expanding field of molecular
cellular neuroscience
2. The rapid progress in axon growth guidance
research.
Think about it, axon growth is tightly associated
with the axon induction in temporal sequence, and
the mechanism is very likely to be shared.
3. Polarity study in other model organisms
including yeast, C.elegans, drosophila, and
mammalian cells like Leukocytes
4. Technical breakthrough, especially neuron
transfection and RNAi.
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Molecular markers for axon or dendrite
Tau1
Axon Tau1, GAP43, synapsin, synaptotagmin
Dendrite MAP2, Glycine receptor, GABAa
receptor
MAP2
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Recent progresses in the molecular mechanism of
neuron polarization I
Actin instability in growth cone
Red actin Green microtubule
Bradke F, Dotti CG. Science. 1999 Mar
19283(5409)1931-4.
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Recent progresses in the molecular mechanism of
neuron polarization II
Microtubule stabilization by CRMP2
Red actin Green microtubule
Inagaki et al., Nat Neurosci. 2001 Aug4(8)781-2.
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Recent progresses in the molecular mechanism of
neuron polarization III
The evolutionary conserved polarity protein Par3
complex and PI3K
Shi SH, Jan LY, Jan YN. Cell. 2003 Jan
10112(1)63-75.
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Our study GSK-3 beta in neuronal polarity
Polarized inactivation of GSK-3 beta
(phosphorylation) in axon
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Inhibition of axon induction by GSK-3 beta CA
mutant --- GSK-3 beta S9A
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Induction of multiple axons from a single neuron
by inhibiting GSK-3 beta activity
Chemical inhibitors
Peptide inhibitor
RNAi
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Functionality of multiple axons induced from a
single neuron
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Conversion of preexisting dendrite to axon by
GSK-3 inhibition
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Model
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After finishing above slides, I was
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When I was seriously thinking of doing a project
(axon guidance neuron migration) in 2002

• I was facing the crucial facts
• The academic peak of axon guidance is passed.
  Many cues their receptors are identified
  signal transduction pathways are under extensive
  investigation.
• Although neuron migration is less explored. But
  it is not a new idea that guidance cues are
  shared between axon guidance and neuron
  migration. The downstream signaling pathways
  also seems likely to be the same.
• Besides Poo assay in the lab, I have no tool to
  use.

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My initial focus the cellular mechanism of
neuron migration
We want to know the relationship between
different parts of neuron during migration,
guidance cue sensing and subsequent turning
We have tools to manipulate neurons
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While probing the relationship between migrant
neuron parts, we became extremely interested in
the role microtubule in neuron migration (and
axon guidance)
microtubule
actin
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We began with chemical drugs and get some
results, but soon we are stopped by technical
difficulties
We cannot transfect neurons !!! So molecular
mechanism is far far from us !!!
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I told Yi that I have to do some tiny projects
to ensure that I can graduate.
And I chose GSK-3 beta
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Why GSK-3 beta?

1. Many papers say it regulates microtubule dynamics
   in neuron, through phosphorylating microtubule
   binding proteins.

2. GSK-3 beta is a central downstream of
Semaphorin (a guidance cue) pathway, Wnt
pathway (morphogen), growth factor/PI3K pathway.
3. We have chemical inhibitors for GSK-3 beta.
4. There are papers say GSK-3 beta regulates
polarity of some cell types.

• What I want to try
• Whether GSK-3 beta activity can guide in axon
  turning neuron migration. (easy work with
  chemical inhibitor)
• Test whether microtubule is the downstream of
  GSK-3beta and how it is regulated. (could be a
  Neuron/Nature Neuroscience)
• Then, test whether Wnt can be a guidance cue
  (could be a CNS paper)
• Finally test whether GSK-3beta regulates neuronal
  polarity.(could be a CNS paper)

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Preliminary result


Inhibitors for Gsk-3 beta can attract growth
cone of cerebella granule neurons.
Soon, a Nature Article paper reported that Wnt is
an axon guidance cue in drosophila, it also said
one group in Chicago had similar data with mouse
neuron.
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Then, I tested neuronal polarity.
We succeeded!
Again, Jan lab reported in Cell that Par3 complex
and PI3K is involved in neuronal polarity.
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• We are facing choices
• Microtubule regulation in axon guidance neuron
  migration.
• PI3K/GSK-3 beta in neuronal polarity.
• Both may lead to a Neuron/Nature Neuroscience
  paper.

We chosed 2, because 1. Jans didnt tell what
polarity defect is 2. GSK-3 beta can be regulated
by other pathways, like Wnt. 3. I am afraid other
labs were already working hard on microtubule
regulation in guidance.
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• Finally, we are extremely lucky
• We provided funtional test of the axonness.
• We co-submitted with Kaibucki lab. The two papers
  make a whole interesting story.

During our revision period, two papers appeared
in Neuron, reporting microtubule regulation in
axon growth cone, with one exactly describing
GSK-3 beta.
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What I think important for a PhD student
1. Interest
2. Interest
3. Interest
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What you should know before you join a lab or do
a project
Read and discuss with the PI till the following
questions are clear

1. The current status in the field. (What
   experiments are the conclusions built on How
   firm they are who did them)
2. What is the main remaining questions in the
   field.
3. Who are the major figures in the field.
4. Are they working on the remaining questions.
5. What is the major reasons that the questions
   remain unanswered or the giants not working on
   them.
6. Is it possible to find answer for some questions
   with the available techniques in this lab.

Once Prof. Poo said ideas are cheap, which is
only for those clear for the above questions.
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Work with your PI, not for your PI
Dont wait for assignment of projects!
Dont make only one idea, then be eager to try it.
If your idea failed, DONT think Aha, I tried,
but failed, now its the boss responsibility to
give me a project, then wait for assignment!!!
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Dont begin the project, if you
Didnt read through all the related papers.
Didnt discuss with your PI till nothing could be
discussed.
Didnt have a blueprint for the experiment.
Cant tell the significance of the work, and
where the paper could be published if your goals
are all achieved.
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It could be better, if you
Try to choose experiment with simple but
essential preliminary experiment.
Try to avoid doing an experiment that only could
be published in J.Neurosci/JBC if your goals are
all achieved.
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It could be of harm, if
PI gave you a project, and you worked it out.
Then you graduated. You restrict your reading in
a small issue.
It could be of great harm, if you
Joined Poo lab, Rao lab, Pei G lab, Zhu XL lab
but think my paper is safe, I just need to do
what they told me to do.
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Where are ideas?

• Know some background.
• Textbook classical reviews

2. Devoted thinking --- imaging you are the
object you are studying, and think about what you
will do when you are in its environment.
3. Interesting projects will come up when you
find some dilemmas you will face in the
enviroment, or some situations people never
thought of before.
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Let me show you one example
Drug abuse --- a 1 billion research per year
National Institute of Drug Abuse
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Reward circuitry in the brain
Eric J. Nestler TRENDS in Pharmacological
Sciences Vol.25 No.4 April 2004
How is it established?
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Is it the best way of studying addiction?
Think of addictive behavior in human
They are hunting for drugs when it is unavailable.
They will pay anything for the drug.
Neither is reflected in the animal model of drug
addiction!!! LIKE does not mean ADDICTION
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Robinson TE Science. 2004 Aug 13305(5686)951-3.
Rule 1 continued drug-seeking behavior even when
the drug is known to be unavailable
Rule 2 continued drug use even in the face of
adverse consequences
Rule 3 unusually high motivation (desire) for
the drug
Experimental result Like drug 100 41 fit
with neither rule 17 fit with 3 rules
simultaneously.
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Try to think as long as this man
A week before Crick died, Terry Sejnowski and I
went to see him at home We talked for about an
hour, most of the time about his new passion, the
claustrumAs we were leaving, Crick faltered
briefly as he got up from his chair, and then
said, with a characteristic twinkle in his eye as
we shook hands, "I can still manage to stand up
to say goodbye". he had said, quite
dispassionately, that it was unlikely he would
live through September. As ever, he was
passionate about his science and unsentimental
about what he could not control.
????Crick
Stevens CF. Nature. 2004 Aug 19430(7002)845-7.
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Now ask yourself
Am I really really interested in doing science?