Mechanisms Underlying Optic Nerve Regeneration and Navigation:

Roles of Glial Cells and Cell Adhesion Molecules

Kevin K. Park and Marcio Ribeiro
Department of Neurosurgery, Miami Project To Cure Paralysis,
University of Miami Miller School of Medicine
Glaucoma Research Foundation and National Eye Institute

INTRODUCTION RESULTS
Optic neuropathies including glaucoma are characterized by damage to Regenerating retinal ganglion cell axons associate GFAP GFP Merged NCAD is expressed in astrocytes
the optic nerve which results in axonal injury and to varying degrees of closely with astrocytes in the injured optic nerve in the injured optic nerve
immediate or protracted retinal ganglion cell (RGC) death. In addition to Immunohistochemistry performed on optic
- Glial fibrillary acidic protein
the cell death, a major limitation to achieving functional recovery after (GFAP) (astrocyte marker) nerve sections. Animals were perfused 14
days after crush injury.
optic nerve damage is RGCs’ inability to regenerate axons. Recently, staining in optic nerve obtained
3 weeks after injury. The mice
significant progress has been made in promoting regeneration of injured received AAV2-CNTF injection
A genetic mouse model to induce astrocyte specific gene deletion
RGC axons in adult mice. Correct pathfinding of axons to their one week before crush. Cholera
toxin subunit B (CTB) alexa
appropriate target areas is a critical step towards wiring up the nervous fluor 555 conjugate was injected Merged GFAP
system. Recently, studies have identified major problems in axon
intravitreally three days before
perfusion to label regenerating
pathfinding in adult visual system; many regenerating RGC axons grow axons. Kim et al. Nature Neuroscience. 2014

aberrantly in the adult mouse optic nerve and brain. Thus, in addition to YFP
GLT1-eGFP mouse labels optic nerve astrocytes
promoting elongation of injured RGC axons, understanding axon NCADflox/flo
x
Exon YFP

guidance mechanisms in adult visual system is of a paramount GLT1-eGFP mouse, in which eGFP
1
GFAP

importance. During development, astroglial cells guide pioneering axons. was inserted downstream of the YFP
5 days of tamoxifen injection resulted in a high
Slc1a2 (GLT-1) promoter, display a
In adult nervous system, regenerating axons in spinal cord grow on
level of Cre recombination in astrocytes

N-cadherin
high eGFP expression and

merge
colocalization with GFAP

GFAP
astrocyte processes. Various surface and extracellular matrix molecules (astrocytic marker) positive cells in NCAD deletion in optic nerve astrocytes has only a modest effect on
including cadherins and fibronectin expressed in astrocytes have been adult optic nerve. RGC axon regeneration
Representative optic nerve
shown to mediate axon-glial cell interaction and promote axon GFP GFAP Merge sections 3 weeks after optic

regeneration and navigation. However, the extent to which astrocytes and Wild-type nerve crush. AAV-CNTF
was injected to induced
these proteins play a role in RGC axon regeneration and navigation in Astrocyte isolation from adult GLT1-eGFP mouse optic nerves

N u m b e r o f r e g e n e r a t in g a x o n s p e r s e c t io n
WT
axon regeneration. Three
40 N C A D h e te r o z y g o u s
days before perfusion the
adult animals remains largely undetermined.
N C A D hom ozygous
30
* mice received intravitreal

AAV2-CNTF
GLT1-eGFP mice received optic nerve

Tamoxifen
NCAD KO heterozygous 20
injection of CTB to trace
crush and 7 day later the optic nerve
10 the regenerating axons.
cells were isolated from the injured and
DESIGN & METHODS uninjured optic nerves. The eGFP
positive cells (astrocytes), the O1 positive
CTB
0
500 1000 1500

D is t a n c e f r o m le s io n (  M )
NCAD deletion in
astrocytes slightly decreases
the number of regenerating
cells (mature oligodendrocytes), the NCAD KO homozygous
axons near the lesion site
In Vivo Characterization of Astrocytic Gene Effects: To determine the role of CD11b positive cells
but no changes were
(microglia/macrophages) were separated
astroglial-associated cell adhesion molecules and extracellular matrix proteins in by fluorescence-activated cell sorting
observed more distal to
RGC axon regeneration/navigation, we examine regeneration in mutant mice in (FACS). The mRNA levels of cell-marker
injury site.

which gene of interest is deleted specifically in astrocytes. To this end, we use the genes (GLT1 and GFAP for astrocytes;
MOG and MAG for mature
inducible, GFAPcreERT2 mouse in which tamoxifen administration results in
U n in ju r e d O p t ic N e r v e In ju r e d O p t ic N e r v e

M AG N E G A T IV E S
M AG
N E G A T IV E S oligodendrocyte; CX3CR1 and
expression of Cre recombinase in GFAP-expressing cells (i.e. astrocytes). We
G L T 1 -G F P + G L T 1 -G F P +

ITGAM/CD11b for microglial cells)

CONCLUSIONS
MOG
C D 11b+ MOG C D 11b+
IT G A M O 1+ O 1+
IT G A M
indicate that the FACS-isolated cells are
generate GFAPcreER; Ncadf/f; Rosa26-YFP (and GFAPcreER; fibronectinf/f;
C X 3C R 1

C X 3C R 1
G LT 1
predominantly astrocytes.
Rosa26-YFP mice). Since the floxed mice are bred to Rosa26 reporter mice, Ncad- - Regenerating RGC axons use astrocytes as physical substrates.
G F AP G LT 1

0 50 100 150 0 50 100 150

- NCAD deletion in astrocytes does not change significantly the axon regeneration rate.
m R N A e x p r e s s io n

deleted cells will also express a reporter protein (i.e. YFP) and allow comprehensive
m R N A e x p r e s s io n
( % o f c o n t r o l) ( % o f c o n t r o l)

- NCAD is known to be critical as an adhesion molecule to promote axon-glia interactions during development and in
monitoring of knockout cells. Adult GFAPcre;Ncad;YFP mice received daily i.p. in vitro. However, it does not seem to have the same important role in adult mice, suggesting that the molecular
injection of tamoxifen (125 mg/kg) for five consecutive days. Two weeks after the mechanisms underlying axon-glial interaction and growth during development and in adult mice might be distinct.
Cell adhesion and guidance molecule mRNA levels in
last tamoxifen injection, animals were euthanized and optic nerve dissected out for
injured optic nerves astrocytes
immunohistochemistry. GFAPcre;GFP mice and GFAPcre;Ncad;YFP mice without
tamoxifen injection serve as control animals. These mice received intravitreal AAV- N-Cadherin Connexin-43
NEXT STEPS
ciliary neurotrophic factor (CNTF) injection (at 3 weeks after the last tamoxifen
CDH2 G JA1

U n in ju re d o p tic n e r v e U n in ju re d o p tic n e r v e

injection) and intraorbital optic nerve crush for 10 seconds using micro-forceps (at 1 These results form an important basis for future studies to further examine the roles of glial cells
O 1+
In ju r e d o p tic n e r v eO 1 + In ju r e d o p tic n e r v e

in guiding regenerating RGC axons after injury, and to define the molecular mechanisms that
C D 11b+

week after AAV-CNTF injection) and euthanized at 2-3 weeks after optic nerve crush. C D 11b+

regulate axon navigation and pathfinding in the injured adult visual system. In future studies, we
G L T 1 -G F P +

The mRNA levels of different genes

Optic nerve were cryosectioned at 10 µm thickness. N E G A T IV E S
G L T 1 -G F P +

plan to investigate how RGC axons might be able to grow properly towards the brain by using the
whose protein products are known
Astrocyte Isolation: GLT-1-eGFP mice were perfused in PBS and the optic nerves
0 5 .0  1 0 -5
1 .0  1 0 -4
1 .5  1 0 -4
2 .0  1 0 -4
0 5 .0  1 0 -3
1 .0  1 0 -2
1 .5  1 0 -2
2 .0  1 0 -2

m R N A e x p r e s s io n
(  C q )
m R N A e x p r e s s io n
(  C q ) to mediate axon-glial interaction glial cells in the optic nerve, and further examine different cell adhesion molecules that mediate
were dissected in DMEM without serum. The optic nerves were then minced and were analyzed. mRNA was obtained this process.
dissociated in 21 Units/mL of Papain and 0.25% Trypsin/EDTA during 25 min with Integrin
I T G B beta-2
2 Ephrin-B3
EFNB3 from different cell types (i.e. cells
gentle shaking. U n in ju re d o p tic n e r v e U n in ju re d o p tic n e r v e
isolated using a combination of
Quantitative RT-PCR: The total RNA from the GLT1-eGFP optic nerve cells was
O 1+

C D 11b+
O 1+
In ju r e d o p tic n e r v e

C D 11b+
In ju r e d o p tic n e r v e

FACS and flow cytometry). Ncad is ACKNOWLEDGEMENTS

highly expressed in astrocytes of
extracted using Absolutely RNA Nanoprep Kit from Agilent Technologies (USA), G L T 1 -G F P + G L T 1 -G F P +
injured optic nerve. Glaucoma Research Foundation & National Eye Institute
accordingly to the manufacturer instructions. The cDNA was synthesized using the 0 5 .0  1 0 -2
1 .0  1 0 -1
1 .5  1 0 -1
0 5 .0  1 0 -4
1 .0  1 0 -3
1 .5  1 0 -3

SuperScript III First-Strand Synthesis System for RT-PCR from Invitrogen (USA).
m R N A e x p r e s s io n m R N A e x p r e s s io n
(  C q ) (  C q )