CJH332H1 Lecture Notes - Lecture 18: Glial Fibrillary Acidic Protein, Rostral Migratory Stream, Neurogenesis
Lecture 18: Stem Cells Continued
Is anything new? Not really (other groups noticed that NPCs proliferate after damage)
• Several techniques have been used previously:
- X-irradiation (to kill off dividing cells/block upregulation in neurogenesis and see what happens after stroke)
- Antimitotic/ antiproliferative drugs (cytosine arabinoside/AraC)
- Conditional transgenic targeting of selected glial fibrillary acidic protein (GFAP), nestin, or homolog of
Drosophila tailless–expressing cell population with HSV-1 thymidine kinase (TK)-kill using stemcell promoter
• However, studies that use these ablation techniques to assess the functional significance of neurogenesis can be
difficult to interpret, because ablation may be accompanied by inflammatory responses with microglial
activation, alterations in microvascular anatomy, systemic toxicity or involvement of non-neuronal cell lineages
Data
• New neurons born, differentiate in SVZ → RMS (rostral migratory stream) → integrate within olfactory bulb
• Many pseudoneurons shown as they migrate (not fully neuron yet) – what we want to target after stroke
- Used different promoters (DCX or BrdU double labeling) from previous studies to make sure that only the
migrating, dividing, not yet fully differentiated cells were expressed
- When at the final destination, it will fully differentiate and express NeuN
Major hypothesis of this paper
• Injury such as stroke actually stimulates neurogenesis in the adult brain, but the role of injury-induced
neurogenesis in brain repair and recovery is uncertain
• One strategy for investigating this issue is to ablate neuronal precursors and thereby prevent neurogenesis, but
this is difficult to achieve with specificity (how do we only target neurons?)
• One way to do this is to create transgenic mice where the specific types of cells can be ablated (or lesioned) –
could we do this with cells undergoing neurogenesis?
• This paper uses transgenic mice that express herpes simplex virus thymidine kinase (virally mediated kinases
that express TK) – TK has no effect by itself, it must be stimulated
- This is under control of the promoter for DCX, a microtubule-associated protein expressed in newborn as
well as in migrating neurons (coming out of SVZ)
• Authors examine the effects of neuronal precursor cell depletion on outcome from experimental stroke in
transgenic mice that express TK under control of the DCX promoter
• DCX is more specific marker of neuronal lineage than GFAP or nestin, and because Dcx is first expressed in close
temporal relation to cell division (i.e. BrdU labeling) this makes it a more promising candidate gene target for TK
ablation-based studies of the role of adult neurogenesis in determining outcome from experimental stroke
Generation of DCX-TK Mice
• To visualize constructs, we NEED reporters (GFP, EYFP, dsRed2, etc)
• This paper used different fragments of DCX promoter to see whether they can get reporter genes to be
activated in specific cells – they were able to see expression of reporter genes using small fragment of DCX
promoter to drive the expression of reporters in both embryonic and adult neuronal tissue
- Saw that with DCX, there were co-staining of reporter with BIII-tubulin (TuJ1 neuronal specific marker) and
microtubule-associated protein 2 (MAP2)
- Astroglial/astrocytic lineage (GFAP makers) or oligodendrocytic (GalC) did not have the reporters
• This line that is marking DCX+ cells is only in the neuronal lineage (does not have any effect on other cells)
• Replaced reporter with TK in adult brain SVZ (and saw expression) – pDCX can drive TK expression
- The mice appeared healthy with normal growth – TK in DCX+ cells but nothing going on (TK not stimulated)
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Not really (other groups noticed that npcs proliferate after damage: several techniques have been used previously: X-irradiation (to kill off dividing cells/block upregulation in neurogenesis and see what happens after stroke) Conditional transgenic targeting of selected glial fibrillary acidic protein (gfap), nestin, or homolog of. Data: new neurons born, differentiate in svz rms (rostral migratory stream) integrate within olfactory bulb, many pseudoneurons shown as they migrate (not fully neuron yet) what we want to target after stroke. Used different promoters (dcx or brdu double labeling) from previous studies to make sure that only the migrating, dividing, not yet fully differentiated cells were expressed. When at the final destination, it will fully differentiate and express neun. Saw that with dcx, there were co-staining of reporter with biii-tubulin (tuj1 neuronal specific marker) and microtubule-associated protein 2 (map2) The mice appeared healthy with normal growth tk in dcx+ cells but nothing going on (tk not stimulated)
