Neuron polarization: an in vitro study with hippocampal neurons

神经所 蒋辉

第一作者论坛 --- 发现的历程

Why is polarity important?

My personal belief:

The really important thing is that could be understood by layman without much explanation.

Neuron is polarized --- an idea dates back to 100 years ago

Proposed information flow direction

Santiago Ramón y Cajal

(1852-1934)

from Cajal’s Nobel Lecture

Neuron Parts:Major sites to receive input

Major sites for output

The question we are focusing on:

Neuron polarization: Development of axon & dendrites.

In vivo neuron polarization:

Unknown !

But we have in vitro study of hippocampal neuron polarization

Pioneering in vitro study by Ganry Banker in 1980’s-1990’s

Craig AM, Banker G. Annu Rev Neurosci. 1994;17:267-310.

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.

Hippocampal Neuron Polarization from Stage 2 to Stage 3

da Silva JS, Dotti CG. Nat Rev Neurosci. 2002 Sep;3(9):694-704.

Neuron growth cone

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.

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.

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.

Think about it, axon growth is tightly associated with the axon induction in temporal sequence, and the mechanism is very likely to be shared.

Molecular markers for axon or dendrite

Axon: Tau1, GAP43, synapsin, synaptotagmin …

Dendrite: MAP2, Glycine receptor, GABAa receptor …

Tau1

MAP2

Recent progresses in the molecular mechanism of neuron polarization I:

Red: actinGreen: microtubule

Actin instability in growth cone:

Bradke F, Dotti CG. Science. 1999 Mar 19;283(5409):1931-4.

Red: actin

Green: microtubule

Recent progresses in the molecular mechanism of neuron polarization II:

Microtubule stabilization by CRMP2

Inagaki et al., Nat Neurosci. 2001 Aug;4(8):781-2.

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 10;112(1):63-75.

Our study: GSK-3 beta in neuronal polarity

Polarized inactivation of GSK-3 beta (phosphorylation) in axon

Inhibition of axon induction by GSK-3 beta CA mutant --- GSK-3 beta S9A

Induction of multiple axons from a single neuron by inhibiting GSK-3 beta activity

Chemical inhibitors

Peptide inhibitor RNAi

Functionality of multiple axons induced from a single neuron

Conversion of preexisting dendrite to axon by GSK-3 inhibition

Model

After finishing above slides, I was …

Nobody is coming to listen to my scientific story. Can I tell something of universal interest and suggestive?

When I was seriously thinking of doing a project (axon guidance & neuron migration) in 2002:

I was facing the crucial facts:

1. The academic peak of axon guidance is passed. Many cues & their receptors are identified; signal transduction pathways are under extensive investigation.

2. 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.

3. Besides Poo assay in the lab, I have no tool to use.

My initial focus: the cellular mechanism of neuron migration

We have tools to manipulate neurons:Local perfusionGuidance cue application

We have an in vitro model:

We want to know the relationship between different parts of neuron during migration, guidance cue sensing and subsequent turning

While probing the relationship between migrant neuron parts, we became extremely interested in the role microtubule in neuron migration (and axon guidance):

actin

microtubule

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 !!!

I told Yi that I have to do some “tiny projects” to ensure that I can graduate.

And I chose GSK-3 beta

Why GSK-3 beta?1. Many papers say it regulates microtubule dynamics in neuron, th

rough 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:

1. Whether GSK-3 beta activity can guide in axon turning & neuron migration. (easy work with chemical inhibitor)

2. Test whether microtubule is the downstream of GSK-3beta and how it is regulated. (could be a Neuron/Nature Neuroscience)

3. Then, test whether Wnt can be a guidance cue (could be a CNS paper)

4. Finally test whether GSK-3beta regulates neuronal polarity.(could be a CNS paper)

Preliminary result

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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.

They killed my

Nature paper!!!

Then, I tested neuronal polarity.

We succeeded!

Again, Jan lab reported in Cell that Par3 complex and PI3K

is involved in neuronal polarity.

We are facing choices:

1. Microtubule regulation in axon guidance & neuron migration.

2. PI3K/GSK-3 beta in neuronal polarity.

Both may lead to a Neuron/Nature Neuroscience paper.

We chosed 2, because

1. Jan’s didn’t 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.

Finally, we are extremely lucky:

1. We provided funtional test of the “axonness”.

2. 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.

What I think important for a PhD student

1. Interest

2. Interest

3. Interest

What you should know before you join a lab or do a project

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.

Read and discuss with the PI till the following questions are clear:

Once Prof. Poo said “ ideas are cheap”, which is only for those clear for the above questions.

Work with your PI, not for your PI

Don’t wait for assignment of projects!

If your idea failed, DON’T think Aha, I tried, but failed, now it’s the boss’ responsibility to give me a project, then wait for assignment!!!

Don’t make only one idea, then be eager to try it.

Don’t begin the project, if you

Didn’t read through all the related papers.

Didn’t discuss with your PI till nothing could be discussed.

Didn’t have a blueprint for the experiment.

Can’t tell the significance of the work, and where the paper could be published if your goals are all achieved.

Try to avoid doing an experiment that only could be published in J.Neurosci/JBC if your goals are all achieved.

Try to choose experiment with simple but essential preliminary experiment.

It could be better, if you

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”.

Where are ideas?

1. 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.

Remember tradictions (or established models or rules) are not always right!!!

Thinking can reshape the world (at least the field)

&

Let me show you one example:

Drug abuse --- a 1 billion $ research per year

National Institute of Drug Abuse

Reward circuitry in the brain:

How is it established?Eric J. Nestler TRENDS in Pharmacological Sciences Vol.25 No.4 April 2004

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

Robinson TE Science. 2004 Aug 13;305(5686):951-3.

Rule 1: continued drug-seeking behavior even when the drug is known to be unavailableRule 2: continued drug use even in the face of adverse consequencesRule 3: unusually high motivation (desire) for the drug

Experimental result: Like drug: 100%; 41% fit with neither rule; 17% fit with 3 rules simultaneously.

Try to think as long as this man:

白眉鹰王 Crick

“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 claustrum……As 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.”

Stevens CF. Nature. 2004 Aug 19;430(7002):845-7.

Now ask yourself:

Am I really really interested in doing science?