The origin of neuronal addition in adult sensory ganglia ---- Do glial cells tell the truth? Henry Li Centre for Neuroscience, Department of Human Physiology, Flinders University, GPO Box 2100, Adelaide 5001, Australia.
The possibility of neurogenesis in the adult DRG?
After injury lost neurons in the DRG can be replaced:
loss of 20%–30% of DRG neurons and then recovery to nearly normal levels several months later.
Neuronal addition following maturation:
the number of neurons in adult rats is From Wikipedia: Neurogenesis (birth of neurons) is the process remarkably higher than by which neurons are created. Most active those during in pre-natal development, neurogenesis is responsible for populating neonates. the growing brain.
How is neurogenesis measured in vitro?
Neurosphere analysis/neural colonyforming cell assay: SelfFrom: Louis, S. A., et al (2008). Stem renewal/Multiple differentiation Cells 26, 988-996
eurosphere is free-floating cellular aggregate generated by neural stem cells in
Alternative approach to produce adult rat DRG neurospheres
Dissociated cells from adult rat DRG do not produce any neurospheres
New approach Explants culture: based on migration feature of stem cells, without interfering surface properties.
nt culture is for a technique usedNeurobasal-A for the isolation of cells with/without from a piece or pieces of Medium proliferation: +N2+B27 FGF2+EGF
Cells from DRG proliferate and form neurospheres
Emigrating cells from adult DRG have the ability to self-renew
Multipotency of the secondary spheres derived from adult dorsal root ganglia
NF200: neuron βIII-tubulin: neuron GFAP: glia SMA: myofibroblast DAPI: nucleus
SFM: serum-free medium Containing Containing Containing Sm Muscle Cells Neurons Glia
SCM: serumcontaining medium
eres give rise to neuron, glia, and myofibroblast multiple-lineage differentiation controlled by culture medium
DRG-Derived Neurospheres Express Progenitor-Related Genes
neural crest-specific genes (Twist1, Snail1, FoxD3) migration-related genes (CXCR4, EdnrB) self-renewal genes (Sox2, Sox10, Bmi-1), proneural basic helix-loophelix transcription factors (NeuroD, Ngn1, Hes1, Mash1, olig1) inhibitors of differentiation (Id2, Id4), Pro-myelinating genes (Sox10, Egr2,) morphogens and paired-box genes involved in the maintenance of adult NSC niche (Notch1, Wnt1, Pax6) neuronal-specific RNA binding protein gene (Msi-1) self-clearing/autophagy genes (Beclin1, MAP-LC3) NoMolecular Oct4, and basis AC133for mRNA was detected
neuronal differentiation & self-renewal
Clusters/spheres express neural crest markers & neurotrophic receptors ErbB2/ErbB4: Receptor for Glial Growth Factor-2. TrkA/TrkB/p75: Receptors for neurotrophins Nestin: marker for neural stem cells;
Can respond to growth factors
Nestin+p75NT R: Marker for neural crest
Differentiation of DRG-Derived Cells Under Growth Factors Challenge (NTs + GGF2): Neurotrophic factors promote neuritogenesis
NF200: neuron GFAP: glia NTs: Neurotrophi ns (NGF/BDNF/ NT3) GGF2: Glial growth factors 2
Neurotrophic factors promote maturation of neurons PGP9.5: and glia (NTFs + GGF2) (UCH-L1 ), marker for mature neurons Nestin: glia/stem cells S100: glia/Schwann cells; p75NTR: glia/neuron SP & CGRP: Neurotransmitt ers -
peptidergic neurons in DRG Become possible functional neurons & glia
P0: Myelin protein-zero -
What kind of cells are sphereforming cells?
Neurons? Glial cells?
Satellite glial cells (SGC) Schwann cells
Other non-neural cells?
Systemic/circulating Endothelial cells Fibroblast
Requires identification of neurogenesis in vivo
How is neurogenesis detected in vivo?
BrdU method
5-bromo-2deoxyUridine (BrdU)
DCX: immatu re neuron s
incorporates into DNA during SNeuN: mature phase of cell cycle neuron Morphological s standard: colocalization BrdU with neuronal markers Modified from: Ming G-l, Song H (2005). Annu Rev Neurosci 28:223
Positive control of BrdU labeling
Active mitotic (proliferating) cells are clearly labeled by BrdU. aSVZ: adult sub-ventricular zone SN: sciatic nerve
In vivo BrdU labelling followed by in vitro explant culture
FR: Fluororuby The cells emigrating from DRG explants probably come neuronal tracer m proliferating glial cells rather than sensory neurons
SGCs in vivo after peripheral DRG axotomy present immunochemical profiles similar to in vitro explant culture BrdU: Proliferati on PGP9.5: Neuron GFAP: glia Nestin & p75NTR: glia and/or stem cells
Proliferating cells in DRG are likely satellite glial
Conclusions I IN VITRO STUDIES:
A subpopulation of cells that emigrated out from adult rat DRG expressed nestin and p75 neurotrophin receptor and had a limited self-renewal capacity, and give rise to neurons, glia, and smooth muscle cells.
COMBINED WITH IN VIVO LABELLING AND IN VITRO CHASING:
These progenitors likely originate from satellite glial cells (SGCs).
Stem Cells (2007) 25, 2053-2065
Possible glial identity of neurogenic cells in adult sensory ganglia: in vivo
cellular identity remains unclear
Works on Trigeminal Ganglion
Emigrating cells from adult TG could also forming spheres with multiplepotential differentiation Protracted maturation of “neural crest glio-neuronal precursor cells” could be responsible for the neuronal addition found in the adult rat .
Identification of possible delayed neuronal maturation in the TG of the adult rat: Nestin+ cells in the TG in vivo
Nestin: Stem cell/glia PGP9.5: Mature neuron βIII-tubulin: late/mature neuron
Conclusions II A possible pool of immature neuroglial neural crest precursor cells could be present in the adult sensory ganglia and be responsible for the increase in sensory neurons found with age . Protracted differentiation could be a developmental strategy by adding or renewing neurons without the prerequisite of DNA synthesis or cellcycle re-entry.
Neuronal generation without proliferation in adult sensory ganglia? Neurogenesis is characterized by generation of neurons from proliferating (BrdU+) undifferentiated progenitor cells in CNS. BrdU method used for identifying NSCs in adult CNS did not reveal any BrdU+ neurons in vivo.
The increase in neuron number is not due to neuron proliferation rather than differentiation of progenitor cells (glia-
Glia-like sustentacular cells in carotid body are stem cells and can be identified using glial markers (GFAP) Cell (2007)131, 364-377
The carotid body is a neural crest-derived organ of the peripheral nervous system that senses oxygen concentration in the blood and responds to changes by regulating breathing.
Working hypothesis
Neurogenic cells exist in sensory ganglia with possible glial characteristics (glio-neuronal progenitors) which possible undergo protracted maturation/latedifferentiation
Hypothesis: : A pool of committed neural progenitors contributes to neuronal addition.
Q: what’s the cellular identity of adult neural progenitors in vivo? How to define them?
Heterogenous populations of adult sensory neurons
Histological staining: A (large) & B (small) Sense modality: proprioceptive, mechanoceptive, and nociceptive Neurotrophic receptor: TrkA, TrkB, and TrkC Neurotransmitter and channels: peptidergic & non-peptidergic (the majority bind isolectin B4, IB4); transient receptor potential channel (TRPV)
Sensory ganglion are originated from neural crest
For adult addition to occur, the sensory ganglion must maintain the appropriate progenitors niche in the peri-neuronal zone. Developmental clues?
E9.5
Embryonic neurogenesis and E11~15 E19~postnatal subtype specification in DRG From Sox10+ cell Generation: Large-size (TrkC & TrkB) neurons~before E15; Small-diameter (TrkA+) nociceptive neurons mainly generate around birth. Postnatal generation of non-peptidergic neurons: phenotype switch from TrkA+ into TrkA- nonpeptidergic neurons From: Marmigere, F. and Ernfors, P. (2007). Nat Rev Neurosci 8, 114-127
Gliogenesis by NOTCH-mediated lateral inhibition Notch signaling pathway is important for cellcell communication, control multiple cell differentiation processes during embryonic and adult life Acquisition of SGCs comes later than neuron and depends on neuronal signals
Cues from developmental studies and some insights from injury models Developmental genesis of neuron and glia in sensory ganglia: From the same progenitor, glial maturation depends on neuronal signal, postnatal transition (TrkA+ TrkA-) Injury: phenotype instability (recapitulation) of SGCs after neuronal loss? Prominent small-size neuronal loss & partial recovery.
Characteristic of SGCs
SGCs are unique (astrocytes/oligodendrocytes/microglia/Sc hwann cells) SGCs and sensory neuron envelop as a functional unit In an environment without synapses are highly plastic, can undergo mitosis.
How to define the specialized pool of
SGCs & Nestin
SGCs are nestin immunoreactive.
SGCs can give rise to neurons in vitro
Nestin+ radial glial cells in adult CNS are bona fide neural stem cells (Goldman, 2003; Anthony et al., 2004; Mori et al., 2005).
SGCs & GFAP
SGCs are GFAP+ after injury in vivo, or after migration out from explants culture in vitro.
GFAP-expressing cells as neural stem cell in postnatal and adult forebrain (Dougherty et al., 2005 ; Imura et al., 2003; Imura et al., 2005)
SGCs & NG2
NG2 proteoglycan-expressing progenitor cells in CNS are NSCs ( Aguirre et al., 2004). SGCs from DRG express NG2, can transform into multiple-lineage cells (Li et al, 2007; Svennigsen et al, 2004) in vitro. NG2 is expressed by SGCs in adult dorsal root ganglia (DRG) after injury (Rezajooi et al., 2004).
Others markers for SGCs
S100/GS (glutamine synthetase)/Notch/Sox10/Egr2 (krox20)
Endogenous markers of immature neurons? Doublecortin (DCX),PSA–NCAM Proneural bHLH transcription factor? Ngn1,
Ngn2
Repressor for morphogenetic signals? adenomatous polyposis coli (APC) Polarization-related signalling molecules? LKB1, par3 Inhibitors for cell cycle progress? p27KIP1 Runt-related transcription factors? Runx1 *Antibodies with high quality must work well in DRG by and Runx3 immunohistochemistry Sensory neuron !!specific-transcriptional factors? Brn3a, Klf7
Heterogeneity of SGCs: location & markers expression
GS: satellite glial cell; S100: Schwann cell/SGC; Hes5: one of targets of Notch pa Brn3a: sensory neurons; Nestin+p75: glia/stem cells
ki67-p27kip1-TuJ1 Ki67: pan-cell cycle marker p27KIP1: cell cycle inhibitor protein TuJ1: clone name of antibody for βIIItubulin, neuronal marker.
A nestin+ precursor cell in the DRG of a P2w rat express a neuronal marker isolectin B4
Nestin: glia/stem cells IB4: neuron non-peptidergic
DRG p2w
Nestin: glia/stem cells βIII-tubulin: late/mature neuron
Isolated DRG cells from p2w rats and cultured for 3 days: Nestin-DCX-TuJ1 Nestin: glia/stem cells DCX: immature neuron TuJ1 (βIII-tubulin): late/mature neuron
Doublecortin (DCX) is a microtubule-associated protein expressed almost exclusively in immature neurons, Neuronal precursors begin to express DCX shortly after exiting the cell cycle, and continue to express DCX for 2-3 weeks as the cells mature into neurons, with BrdU labelling, currently a "gold standard" in measuring neurogenesis in vivo.
N52: (clonal name for NF200 antibody): mature neuron GFAP: glia TuJ1 (βIII-tubulin): late/mature neuron
DCX: immature neuron TuJ1 (βIIItubulin): late/mature neuron
peripheral glia or neural crest progenitors, also label adult neural crest stem cell
Feasibility of immunoprobes
Possible immuno-staining of the desired cells in vitro and in vivo by a set of neuronal (DCX/TuJ1/IB4) and glial markers (Nestin/GFAP/Sox10) in juvenile DRG (postnatal two weeks). A group of glia-like cells can be stained by neuronal markers in postnatal DRG
Does it work in adult DRG (>12
Preliminary data from adult rats
Samples: L5 DRG rats (p24wk): 15weeks after one-side sciatic nerve transcetion
In vivo: cryosections
In vitro: 12h after plating of cell cluster/aggregates from collagenase based semi-dissociation
Nestin: glia/stem cells GAP43: (Growth Associated Protein 43)
is a nervous tissue-specific cell membrane glycoprotein. Marker for mature neurons DCX: immature neruons
DCX mRNA in adult sensory ganglia
Nestin: glia/stem cells TrkA: neuron peptidergic IB4: neuron nonpeptidergic
Transitional phenotype: TrkA+/IB4+
Possible reduction of IB4+ neurons in axotomized DRG
Satellite glial cells (esp. clusters) can bind IB4 after injury
Do nestin+ neurons exist in adult DRG after injury
Nestin: glia/stem cells TrkA: neuron peptidergic IB4: neuron non-peptidergic
In vitro confirmation: transitional phenotype with glial/stem cells marker
Nestin: glia/stem cell
p75NTR: glia or neurons Nestin+p75:
APC (adenomatosis polyposis coli) is a gene that is classified as a tumor suppressor gene, control of beta-catenin, a critical molecule in Wnt signaling pathway. APC is also thought to be microtubules stabilizer invovling in neurite growth
Does peri-nucleus APC staining mark the progenitors?
SGCs in vivo express NG2
Nestin: glia/stem cell; GS: SGCs; NG2: glia/stem cells
NG2+ cells/clusters in vitro
Primary conclusions
SGCs in adult DGR express a set of markers for identifying of NSCs The replaced neurons are probably IB4+ after injury SGCs also bind IB4 Q: Are they from IB4+ glial cells? Ongoing works
The big question: are new added neurons functional?
Three criteria for new born cells as neurons in CNS: Morphologically they must be polarized, they must be capable of firing voltage gated action potentials and they must be able to communicate with other neurons. To examine the last two criteria requires living neurons (in vivo study in intact DRG)---. Intake tracer? Acute isolation (sorted by surface markers) for electrophysiological record?
Future directions & implications
To define the cell-fate decision in vivo. To further trace the possible developmental origin of the desired progenitors combining with knock-in or transgenic mice. To observe the cellular behavior in different physiological and pathological settings. Neuropathic pain/regeneration/peripheral neuropathy
Acknowledgeme nts
My supervisor A/Prof. Xin-Fu Zhou and his full support! Grateful to my colleagues in the Neuroregeneration Laboratory, Dr. John Wang, Dr. Tony Pollard, Dr. Ernest Aguilar, Dr. Linda Wu, Ms. Jing-Xian Mi, and Ms. Jenny Zhong for their kind help and encouragement. My gratitude to Prof. Ian Gibbins, Dr. Jennifer Clarke and Jenny Hiscock for their support with image acquisition; and Dr. Mary-Louise Rogers for gift of p75 antibody. Specific thanks to A/Prof. John Power & Dr. Jennifer Clarke for invaluable help for this presentation!
Thanks