Neurogenesis in Human Adulthood

7/30/2019 Neurogenesis in Human Adulthood

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ADULT AND INFANT

Neurogenesis

Ins Cordn & Ingrid Meucci

Hello everyone,today Ins and I are going to talk about neurogenesis. 30years from now everyone thought that neurons were formed exclusivelyduring the embryonic development and that the only possible change duringadult life was cell death and loss of neurons.But some researchers showed in the early 80s that some parts of the brainmay function as germinal centers even in adulthood, as they have thecapacity to produce new cells which evolve to new neurons and occupyalready functional structures. This formation of new neurons is calledneurogenesis


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New neurons generatedfrom neural stemcells andprogenitors cells

Oligodendrocyte Astrocyte Neuron

Stem CellProliferation &Migration

What is Neurogenesis ?

Progenitor Cells

Neurogenesis, is not only the generation of new neurons but also otherdiferent kind of neuronal cells from the stem cells. Other than neurons,these stem cells produce oligodencrocytes and astrocytes in the brain. Thestem cell will first produce the progenitor cells which will proliferate andmigrate, and during this process the genetic and environmental factors willinduce this progenitor to produce a specific kind of neuronal cell.


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Lateral Ventricle

Hippocampus

Where does Neurogenesis

Until now, we know two structures that present neurogenesis duringadulthood. First there is the hippocampus, which generates new cellsinvolved in mechanisms such as memorization, and second, there is thesubventricular zone, a large germinal region located in the wall of the lateralventricle.


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The SVZ : SubVentricularIn Adult

Cells from this wall have been isolated, and it has been proven that theyhave the capacity to renew and diferentiate into any of the three neuronaltype of cells mentioned before. The wall of this subventricular zone isformed by three diferent layers. First, we have the epithelial layer in contactwith the liquid into the lateral ventricle. Next to these cells we find anhypocellular gap, and then there is a layer of astrocytes in close contact withmany blood vessels.


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The RMS : Rostral

As we said before, a very important characteristic of these stem cells is thecapacity they have to migrate to some specific target regions. It has beenfound a group of cells that migrates tangentially towards the OB from theSVZ forming a pathway called the rostral migratory stream.


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Evidence of Mi ration in

This route for migration is evident in rodent during infancy and adult life,asyou can see in this video


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DOES NEUROGENESISDISAPPEAR ?

It is already widely accepted that neurogenesis takes place in rodents during all their life, butwhen it comes to humans, this is not so clear. The scientific community agrees with the ideathat there is a large neurogenic process during the human embrionic states, but controversyarises when it comes to childhood and adulthood. While some scientifics think that thisneurogenesis stops just after birth, others think that there is a period of many months, until18 more or less, were this neuronal process its maintained.

In this article, the authors support this second theory were neurogenesis is kept for somemonths after birth to be then dramaticly reduced until almost disappear during the rest of theadulthood.


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Techni ue:

Stains Mark

DAPI All Cells

DCX : Doublecortin Immature neuron

PSA-NCAM Migrating neuron

Ki67 Cellular proliferation

GFAP/Vimentine Astrocytes/Glia

To prove this hypothesis, they used almost exclusively the immunohistochemical techniques. In particular they used theselabeling markers

DAPI : 4',6'-diamino-2-phenylindole. Binds strongly to A-T rich regions in DNA. DCX : Doublecortin. Neural migration protein expressed by neuronal precursor cells and immature neurons. PSA-NCAM : Polysialyted (PSA) embryonic form of the cell adhesion molecule NCAM. Marks migrating and immature neuron Vimentine : type III intermediate filament protein. Immature glial marker.

Ki67 : nuclear protein necessary for cellular proliferation GFAP : glial fibrillary acidic protein in an intermediate filament. Help to maintain astrocyte mechanical strenght. TH : Tyrosine hydroxylase convert L-Tyrosine to L-DOPA. Source of ROS : Cellular death Calretinin : involved in calcium signaling. Marks interneuron.


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Neuro enesis in :

Subventricular Zone1

Rostral Migratory Stream2

Olfactory Tract3

Lateral ventricle

Neuroblasts

Olfactory Bulb

the hypothesis is that neuroblast from the subventricular zone migratethrough the rostral migratory stream to reach the Olfactory bulb.(explication de la figure)In the first part we will see if there are immature and migratory neuronsafter birth in the subventricular zone, and then if the rostral migratorystream really exist and what is its composition. And to finish if we findimmature and migratory neurons in the olfactory bulb.


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1. Neurogenesis in the SVZ

so to begin, what is happening in the subventricular zone ?


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Horizontal Section of the SVZ

Arturo Alvarez- Buylla et al,Nature. ; 478(7369): 382386. doi:10.1038/nature10487

First, what the authors did, was to proceed doing many horizontal sectionsof the SVZ at diferent age stages.


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During the first 18 months of age

Staining

Cells

Immature Neuron

Glia

Large number of DCX(+)neurons populate the future

gap layer

Immunochemistry of the SVZ


With these horizontal cuts, they did an immunohistochimistry to find which cells can we find in the SVZ.

This is what they got. In blue we can see the DAPI staining, which marks for the cell density of the area. Then we have the red marker which stains the immature neuron or precursors. And finally we havethe green labelling which show us the glia.

As you can see, during the first 6 months after birth there is a dense area next to the ventricular wall, which correspond to immature neuron, shown by the dcx marker. After this 6 month period we seehow this neuroblast layer disappears. If we check now the glia (green marker) we appreciate the opposite order of events. During the first 6 months we cannot see any special event, but later we can seehow a dense layer of astrocytes appears.

in conclusion during the first 6 months of life there is a large number of DCX( ) neurons which populate the future gap layer. And then the glial cells will fill in the gap area.

After this immunochimistry, they quantifye the number of immature and proliferating neurons.With a DCX stain, they can remark that there is a decline in the number of immature neuron from birth to 9 months. And after 9 months, the number of cells did not change anymore.

Its the same for proliferating cells marked with Ki67.So there is a sharp decline of proliferating and immature cells during the first months of life.


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Immature neuron Proliferating subependymal cell

Staining

Sharp decline in proliferating cells and immature neuron

Quantification of Immature


This graph shows us how the decrease in the number of immature neuronsdoesnt only happen until the 18th month but it keeps like this during alladulthood. Ki67 stains proliferating cells, and as you can see they gothrough the same decrease as the immature neurons.

Taken together, these data demonstrate a sharp decline in proliferating cellsand immature migrating neurons coinciding with the emergence of thehuman SVZ gap layer.


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During infant and adult age

Conclusion

Infant Adult

Where these cells go ?

In conclusion what they saw is a dense layer of immature neurons present ininfants that disappeared after 12 months after birth.

Now the question was...whats happening with these immature neurons? arethey dying? where are they migrating? So the next step was to look at whatwas happening in the rostral migrating stream.


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2. Neurogenesis in the RMS


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At 5 months

Sagital and Coronal Reconstitution

Tributaries ofcells

Isolatedependymalislets

Out of thesagital plane

Caracterization of the RMS

Evidence of the RMS between theSVZ and the Olfactory Bulb (OB)


When they looked at the RMS what they saw is exactly the expected. With aseries of sagital and coronal sections reconstitution they showed how achain of immature neurons was formed from the SVZ until the OB,demostrating the existence of this migrating stream at 5 months of age.

We can appreciate how the proximal area of the RMS is formed thanks tosome groups of cells that come from the SVZ.


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At 5 months

Staining

Migrating neuron

Immature neuronTributaries of

cells

Isolatedependymalislets

Out of thesagital plane

Immunohistochimie of neurons

Clusters of immature and migrating


Once again, to prove that this cells were exactly immature migratingneurons and not any other kind of neurons they used theimmunohistochemical technique. And as you can see in the enlargement ofthis central segment, we can appreciate how cells are dcx and psa-ncampositive cells in this area


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At 1 week

Individual DCX(+) neuronal chains Ultrastructural analysis of a neuronal

chain cross-section

Label

Glia

Immature neuron A : Immature neuron

B : Glia

Immunohistochimie and

Evidence of a chain of immature and migrating neurons


In these figures we can see how these immature neurons are present in theRMS at 1 week of age and how they are surrounded by astrocytes and oftenmigrate along blood vessels.


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General Structure of the RMS

Here we can see a general view of whats happening in an active RMS.Immature neurons coming from the SVZ forming a chain that migrates alongthe blood vessels towards the OB surrounded by the glia. This migrationstream seems to disappear after 18 months after birth but some data hasshown how some single or pairs of cells may still be present in olderchildren and adults.


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Serial Coronal Sections of the

Zhengang Yang et al, Cell Research (2011) 21:1534-1550

Staining

Cells

Glia



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Horizontal Section of the Adult

The RMS-like pathway exists in the adult human

Zhengang Yang et al, Cell Research (2011) 21:1534-1550

Staining

Immature neuron

Glia

Cells



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3. Neurogenesis in the OlfactoryTract

As a final step, the authors of the article analysed the oltactory tract.


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At 1 day,8 months, 7 years

Cross-Sectional Reconstitutions Staining

Migrating and immature neuron

Immature neuron Cells

Immunohistochemistry in the

Decline of an active RMSwith age


As we can see in the high magnification fields i, j and k, there is aprogressive loss of immature migrating neurons also in the olfactory tract.In i there is a high concentration of red and green spots corresponding tothese dcx psa-ncam positive neurons, spots that gradually disappear withage to leave only the blue spots corresponding to the DAPI staining at 7years of age.


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Toluidine Blue Staining in a

Central chain of immature neurons surrounded by a matrixof astroc tes


Here we can see a toluidine blue staining of a longitudinal section from a 6month OT which shows a central chain of darkened immature neuronssurrounded by a matrix of lighter-coloured astrocytes.


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At 6 months

Quantification of cells in the

Proximal RMS

Distal RMS

There is a decline in the total amount of cells and inimmature neurons between the proximal and distal RMS


Tracing the infant RMS with serial coronal reconstructions the authorsnoticed a decrease in caliber of the RMS from the proximal to the distal limb.Based on quantification of DAPI nuclear staining they calculated a decline ofa 58% in total cells and a 61% decline in immature neurons. They thoughtthis could be due to immature migratory neurons taking alternative paths,so they kept doing serial coronal reconstructions and.


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Another way of neurogenesis

Hypothesis for decline in cell

Staining

Migrating and immature neuron

Immature neuron


A medial migratory stream (MMS) of immatureneurons branches from the proximal rostral migratory

what they found is exactly that. A second migration stream called MedialMigration Stream (MMS) that branched of the proximal limb of the RMS andgoes to the ventro-medial pre-frontal cortex (VMPFC). This MS was observedin human specimens of 4-6 months but not in 8-18 months specimens.

As in the RMS, they repeted the dcx and psa-ncam staining to demostratethey were immature migrating neurons, and as you can see in figures f and ethe cells were dcx and psa-ncam positive.


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Summary

Lateral ventricle

Subventricular Zone

Rostral Migratory Stream

Neuroblasts

Olfactory Tract

Olfactory Bulb


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Why RMS is no longer active in

Other pathways like MMS

Serial sections (sagital, coronal, horizontal) : DAPI

If yes : Immunohistochemistry (DCX, Hud+/Sox10-, NeuN)

Apoptosis ?

Hybridation in-situ : caspase, P53 ?

Which are the transcription factors expressed by theastrocytes ?

Transcriptome : microarray

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Neurogenesis in Human Adulthood