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University of Connecticut University of Connecticut
OpenCommons@UConn OpenCommons@UConn
Master's Theses University of Connecticut Graduate School
5-10-2020
The Necessity of Vesicle-Associated Membrane Protein The Necessity of Vesicle-Associated Membrane Protein
Expression by NG2 Glia for Maintenance of Glial and Neuronal Expression by NG2 Glia for Maintenance of Glial and Neuronal
Health Health
Robert Horning robert.horning@uconn.edu
Follow this and additional works at: https://opencommons.uconn.edu/gs_theses
Recommended Citation Recommended Citation Horning, Robert, "The Necessity of Vesicle-Associated Membrane Protein Expression by NG2 Glia for Maintenance of Glial and Neuronal Health" (2020). Master's Theses. 1504. https://opencommons.uconn.edu/gs_theses/1504
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The Necessity of Vesicle-Associated Membrane
Protein Expression by NG2 Glia for Maintenance of
Glial and Neuronal Health
Robert Zachary Horning
B.S., Wittenberg University, 2016
A Thesis
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Science
At the
University of Connecticut
2020
ii
APPROVAL PAGE
Master of Science Thesis
“The Necessity of Vesicle-Associated Membrane Protein Expression by NG2 Glia for
Maintenance of Glial and Neuronal Health”
Presented by
Robert Zachary Horning, B.S.
Major Advisor______________________________________________________________
Akiko Nishiyama
Associate Advisor___________________________________________________________
Jianjun Sun
Associate Advisor___________________________________________________________
Randall Walikonis
University of Connecticut 2020
iii
ABSTRACT
NG2 glia are vital for the myelination of axons in the brain and spinal cord. Many NG2
cells receive synaptic inputs from neurons through neurotransmitters such as glutamate and
GABA, but the consequences of this input have remained unclear. Interestingly, NG2 cells
express voltage-dependent Ca2+ channels and transcripts encoding SNARE proteins that are
necessary for vesicle docking in neurons. We therefore hypothesized that NG2 cells respond to
neuronal synaptic inputs by releasing bioactive molecules in a Ca2+-dependent manner. To test
this, we generated double transgenic mice in which a cre-inducible botulinum toxin (BoNT/B) is
constitutively expressed by NG2 cells (IBOT:NG2cre mice). In the IBOT:NG2cre line, BoNT/B
cleaves vesicle-associated membrane proteins (VAMPs) in NG2 cells and their progeny.
IBOT:NG2cre mice exhibited a robust phenotype, with severe motor defects and translucent
spinal cords suggestive of severe hypomyelination. Immunostaining for MBP supported these
findings, as IBOT:NG2cre spinal cord sections displayed pronounced hypomyelination
throughout dorsal and anterolateral funiculi, particularly in the ventral region of the dorsal
column, which notably contains the corticospinal tract. Staining for the oligodendrocyte antigen
CC1 and with NG2 also showed a significant decrease in oligodendrocyte population density, but
not in NG2 cell density, suggesting an additional defect in NG2 cell differentiation and/or
oligodendrocyte survival. Lastly, through transmission electron microscopy, we observed large,
misshapen myelinated axons around the corticospinal tract of the spinal cord. Our findings
suggest that vesicle-associated membrane proteins in NG2 cells play a key role in myelination,
NG2 cell differentiation, and/or oligodendrocyte survival, possibly mediated by exocytotic
events observable in cultured primary NG2 cells.
1
INTRODUCTION
NG2 cells, also known as polydendrocytes or oligodendrocyte progenitor cells (OPCs),
are a major glial population comprising 5-8% of glial cells in the central nervous system (CNS)
(Levine et al., 2001). NG2 cells differentiate into oligodendrocytes, the myelinating cells of the
CNS, both embryonically and throughout life (Bergles and Richardson, 2015; Murphy and
Franklin, 2017). Their role as progenitor cells is therefore essential for myelination of axons in
the brain and spinal cord, making them a highly relevant therapeutic target for the treatment of
multiple sclerosis (MS), a chronic demyelinating disease affecting millions worldwide.
The Identify of NG2 Cells
Despite their prevalence in the CNS, NG2 cells have only recently been recognized as a
major glial population distinct from oligodendrocytes, astrocytes, and microglia (Nishiyama et
al., 1999). However, over the decades from their discovery, much information about this fourth
major glial cell type has come to light. Their namesake is derived from their expression of
neuron-glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4). While
pericytes also express CSPG4, NG2 glia can be easily distinguished by their expression of
platelet-derived growth factor receptor alpha (PDGFRa), a receptor tyrosine kinase, as well as
OLIG2, a basic helix-loop-helix transcription factor expressed throughout the entirety of the
oligodendrocyte lineage. As NG2 cells differentiate into oligodendrocytes, NG2 and PDGFRa
expression are downregulated (Levine and Stallcup, 1987; Stallcup and Beasley, 1987), while
expression of myelin regulatory factor (MYRF) and the oligodendrocyte antigen CC1 are
upregulated (Bhat et al., 1996).
2
The Uncertain Role of NG2 Cells in the Neural Network
NG2 cells express numerous receptors for the neurotransmitters glutamate and GABA
(Lin and Bergles, 2004; Steinhäuser and Gallo, 1996), and many neurons form direct synapses
with NG2 cells (Bergles et al., 2000; Bergles et al., 2010; Ge et al., 2006). Additionally,
exogenously applied glutamate and GABA have been shown to elicit Ca2+ transients in NG2
cells (Cheli et al., 2015; Harlow et al., 2015; Sun et al., 2016). The consequences of this neuron-
glia synapses and Ca2+ transients in NG2 cells are currently unclear, and studies thus far have
focused almost entirely on one-sided communication from neurons to NG2 cells.
Despite thorough characterization of NG2 cell gene expression to the point of distinction
as a major glial population, very little is known about the actual function of NG2 cells beyond
their role as oligodendrocyte progenitors. Recent studies suggest potential trophic actions in the
normal CNS (Birey et al., 2015; Djogo et al., 2016; Fernandez-Castaneda and Gaultier, 2016),
and others have demonstrated the release of factors such as matrix metalloproteinase 9 (MMP9)
in disease states (Seo et al., 2013).
However, few studies have assayed for secreted factors, and few if any published works
have investigated the mechanism or consequences of neuroactive or autocrine factor secretion by
NG2 cells. NG2 cells express many genes and proteins associated with exocytotic machinery in
neurons and astrocytes, including synaptophysin (Hamilton et al., 2010; Wigley and Butt, 2010),
vesicular glutamate transporter VGLUT1 (Elsayed et al., 2012; Haberlandt et al., 2011), voltage-
dependent Ca2+ channels such as Cav1.2 (Barres et al., 1990; Cheli et al., 2016; Larson et al.,
2016), and vesicle-associated membrane proteins 2 and 3 (VAMP2 and 3) (Madison et al., 1999),
among many others, but the functions of these proteins in NG2 cells are still unclear. Taking all
of this information into account, we hypothesized that OPCs may be capable of Ca2+-dependent
3
exocytosis, and that blocking this process could inhibit trophic support functions usually
provided by NG2 cells.
Mouse Lines and Effect of BoNT/B Toxin
In order to investigate the potential for Ca2+-dependent exocytosis in NG2 cells, our lab
had generated a double transgenic IBOT:NG2creER line by crossing our NG2creER single
transgenic line (Jackson Labs #008538) (Zhu et al., 2011), which expresses tamoxifen-inducible
cre recombinase under the control of the Cspg4 (NG2) promoter, with the IBOT single
transgenic line (Jackson Labs #010856). The IBOT line which contains a cre-inducible
botulinum toxin that can be visualized with an internal ribosome entry site (IRES)-linked
enhanced green fluorescent protein (EGFP) (Figure 1A), has been shown to cleave vesicle-
associated membrane proteins (VAMPs) in neurons and retinal glia when crossed to specific
transgenic cre lines (Slezak et al., 2012), making it a promising candidate for application in NG2
glia given their similarities in exocytotic protein expression. Our lab also generated a double
transgenic IBOT:NG2cre line by crossing the aforementioned IBOT single transgenic line with
our NG2cre single transgenic line (Jackson Labs #008533) (Zhu et al., 2008), which expresses
constitutive cre in NG2-expressing cells and their progeny.
A
4
Figure 1. Generation of the IBOT:NG2cre and IBOT:NG2creER Lines
A) Transgenic mice were generated by crossing an IBOT line (Slezak et al., 2007), which
contains a cre-inducible botulinum toxin that can be visualized with internal ribosome entry site
(IRES)-linked enhanced green fluorescent protein (EGFP), with either NG2cre or NG2creER
single transgenic mice, which express constitutive and tamoxifen-inducible cre recombinase,
respectively. B) The toxin functions by cleaving vesicle-associated membrane proteins (VAMPs),
SNARE proteins necessary for vesicle docking during Ca2+-dependent exocytosis in neurons, but
with a currently unknown function in NG2 cells. C) Transgene specificity was confirmed by
immunostaining for colocalization of GFP (green) with Olig2 (blue) alongside either NG2 or
CC1 (red).
MATERIALS AND METHODS
Animals
B
C
GFP Olig2 CC1 GFP Olig2 CC1
5
Male and female IBOT:NG2creER double transgenic mice and NG2creER single
transgenic littermates were used between postnatal days (P) P2-8, P8-17, P30-38, or P92-101.
Male and female IBOT:NG2cre double transgenic mice and NG2cre single transgenic littermates
were used at postnatal (P) days P10, P13, P14, P16, and P21. Housing, handling, care, and
processing of animals were carried out in accordance with regulations approved by the
Institutional Animal Care and Use Committee (IACUC) at the University of Connecticut.
Tamoxifen Injections
In order to activate cre recombinase and induce transgene expression in IBOT:NG2creER
animals, we injected the estrogen analogues tamoxifen and 4-hydroxytamoxifen. Tamoxifen and
4-hydroxytamoxifen (4-OHT) were dissolved in a 19:1 mixture of canola oil and ethanol and
sonicated prior to every injection. P2 and P8 animals were injected intraperitoneally with 0.2 mg
4-hydroxytamoxifen (10 mg/mL stock), while P30 and P92 animals were injected with 60 mg/kg
tamoxifen (40 mg/mL stock). Control animals were injected with 19:1 canola oil:ethanol without
tamoxifen. All animals were injected once each day for four consecutive days, then perfused four
to six days after the final injection.
Immunohistochemistry
To validate specific expression of the IBOT transgene (marked by EGFP) and investigate
the effects of transgene expression on CNS cell populations and myelination, we immunostained
brain and spinal cord sections from IBOT:NG2creER and IBOT:NG2cre animals prior to
analysis with fluorescent and confocal microscopy.
Tissue Collection and Fixation
6
P17, P38, and P101 IBOT:NG2creER animals and P10, P13, P14, and P21 IBOT:NG2cre
animals were anesthetized with isoflurane and transcardially perfused with freshly prepared 4%
paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer. Spinal cords were extracted through
laminectomy and postfixed alongside brains in chilled 4% PFA on a rotator for 2 hours, washed
three times (10 minutes per wash) in cold 0.2 M sodium phosphate buffer, then processed for
under either free-floating or cryosectioning protocols.
Processing of Free-Floating Tissue
Following the three washes with 0.2 M sodium phosphate buffer, the tissue was left in
fresh 0.2 M sodium phosphate buffer overnight at 4°C. The next day, brains were embedded in
1% agarose and cut into 50 μm-thick sections using a Leica 1000 S vibratome before being
placed in cryostorage solution and left at -20°C until staining.
Processing of Cryosections
Following the three washes with 0.2 M sodium phosphate buffer, the tissue was left in
30% sucrose in 0.2 M sodium phosphate buffer at 4°C until all tissue sunk to the bottom of the
polyethylene vial. Spinal cords were then sectioned into ~3 mm segments and embedded in
optimal cutting temperature compound (OCT, Sakura) before being frozen using a mixture of dry
ice and methanol and stored at -80°C until cryosectioning. 20 μm-thick transverse sections were
cut using a Leica 3050S Cryostat, collected on Fisherbrand Superfrost Plus Microscope Slides,
and stored at -20°C until staining.
Primary and Secondary Antibodies
Primary antibodies used included rabbit IgG anti-NG2 (1:500, Millipore), chicken IgY
anti-GFP (1:1000, Aves), mouse IgG2b anti-APC/CC1 (1:200, Calbiochem), rabbit IgG anti-
7
OLIG2 (1:500, Abcam), rabbit IgG anti-OLIG2 (1:500, Novus), mouse IgG2a anti-OLIG2
(1:500, Millipore), goat IgG anti-mouse PDGFRa (1:500, R&D Systems), mouse IgG2b anti-
SMI99 (1:2000, Biolegend), rabbit pAb anti-Aldh1l1 (1:1000, Abcam), rabbit IgG anti-NeuN
(1:300, Cell Signaling Technology), goat IgG anti-IBA1 (1:500, Thermo Fisher Scientific),
rabbit pAb anti-GST-π (1:200, MBL International Corporation), rabbit pAb anti-MBP (1:200,
Chemicon). Secondary antibodies used included Cy3 anti-rabbit IgG (1:500, Jackson), Cy3 anti-
mouse IgG1 (1:500, Jackson), Cy3 anti-mouse IgG2a (1:500, Jackson), Cy3 anti-mouse IgG2b
(1:500, Jackson) Cy3 anti-goat IgG (1:500, Jackson), AlexaFluor488 anti-chicken IgY (1:1000,
Aves), Alexa633 anti-rabbit IgG (1:200, Jackson), Alexa633 anti-mouse IgG1 (1:200, Jackson),
Alexa633 anti-mouse IgG2a (1:200, Jackson), Alexa 647 anti-mouse IgG2b (1:200, Jackson),
Alexa647 anti-rabbit IgG (1:200, Jackson), Alexa647 anti-mouse IgG1 (1:200, Jackson),
Alexa647 anti-mouse IgG2a (1:200, Jackson), and Alexa647 anti-mouse IgG2b (1:200, Jackson).
Primary Antibody Staining (Free-Floating)
Brain sections from different animals were placed in separate wells of 12-well plates
filled with 0.1% Triton X-100 in PBS and washed three times (10 minutes per wash) on a rocker.
They were then blocked for 1 hour in 5% normal goat serum (NGS) in 0.1% Triton X-100 in
PBS, incubated overnight at 4°C in primary antibody solution consisting of three of the above
antibodies, 5% NGS, and 0.1% Triton X-100 in PBS.
Secondary Antibody Staining and Mounting (Free-Floating)
Slides were washed three times (10 minutes per wash) with 1X PBS, blocked for 1 hour
in 5% normal goat serum (NGS) in 1% Triton X-100 in PBS, and incubated overnight at room
temperature (RT) in a humid staining chamber in primary antibody solution consisting of three of
8
the above antibodies, 5% NGS, and 1% Triton X-100 in PBS. Sections were then washed three
times (5 minutes per wash) with 0.1% Triton X-100 in PBS and incubated for 1 hour at RT in
secondary antibody solution consisting of three of the above antibodies, 5% NGS, and 0.1%
Triton X-100 in PBS. Finally, sections were washed three times (5 minutes per wash), rinsed
briefly with ddH2O, mounted on glass slides, let dry before being coverslipped with Vectashield
containing 4’6-diamidino-2-phenylindole (DAPI) (Vector Laboratories), left to dry for ~15
minutes, and had their edges sealed with nail polish before being stored at -20°C.
Primary Antibody Staining (Cryosections)
Slides were left to thaw ~10 minutes, then tissue was outlined using a pap pen and left to
dry ~10 minutes before beginning staining protocol. Slides were washed three times (10 minutes
per wash) with 1X PBS, incubated for 1 hour in 5% normal goat serum (NGS) in 1% Triton X-
100 in PBS, and incubated overnight at room temperature (RT) in a humid staining chamber in
primary antibody solution consisting of two to three of the above antibodies, 5% NGS, and 1%
Triton X-100 in PBS.
Secondary Antibody Staining and Mounting (Cryosections)
The next day, slides were washed three times (10 minutes per wash) with 1X PBS before
being incubated for 1 hour in secondary antibody solution consisting of two to three of the above
antibodies, 5% NGS, and 0.3% Triton in PBS. Slides were then washed three times (10 minutes
per wash) with 1X PBS and one time with ddH2O (5 minutes). Slides were left to dry before
being coverslipped with Vectashield containing 4’6-diamidino-2-phenylindole (DAPI) (Vector
Laboratories), left to dry for ~15 minutes, and had their edges sealed with nail polish before
being stored at -20°C.
9
Imaging and Quantification
Stained slides were imaged using either a Zeiss Axiovert 200M fluorescent microscope
with an Orca ER camera (Hamamatsu) and Apotome grid confocal system (Zeiss) or a Leica SP8
confocal microscope. NG2+OLIG2+ or PDGFRa+OLIG2+ cells were considered NG2 cells,
CC1+OLIG2+ or GST-π+OLIG2+ cells were considered mature oligodendrocytes,
ALDH1L1+OLIG2- cells were considered astrocytes, IBA1+OLIG2- cells were considered
microglia, and NEUN+OLIG2- cells were considered neurons. For quantification, the dorsal
column of transverse spinal cord section used was outlined in Axiovision or ImageJ software,
and cell density was derived from the number of cells over the area times the thickness of the Z-
stack.
Transmission Electron Microscopy (TEM)
While light microscopy is ideal for quantifying cell populations and overarching structure
in fixed tissue, it is limited in its potential magnification and resolution. Thus, in order to
investigate the morphology of individual axons and myelin sheaths, we processed tissue for
analysis through transmission electron microscopy (TEM).
Tissue Collection and Fixation
P16 animals were transcardially perfused with 4% PFA and 2% glutaraldehyde (GA) in
0.1 M sodium cacodylate buffer with 2 mM CaCl2 and 4 mM MgCl. Spinal cords were extracted
through laminectomy, cut into ~1 mm-thick segments, and postfixed overnight at 4°C in the
same perfusion fixative.
Osmium Staining
10
Tissue was placed in a basket with mesh-wells and washed three times (15 minutes per
wash) with 0.1 M sodium cacodylate buffer. The tissue was incubated for 1 hour in 1% osmium
tetroxide and 0.8% potassium ferricyanide (KFe) solution in 0.1 M sodium cacodylate buffer,
washed five times (5 minutes per wash) in 0.1 M sodium cacodylate buffer, incubated for 1 hour
in 1% osmium tetroxide without KFe in 0.1 M sodium cacodylate buffer, and washed five times
(5 minutes per wash) with 0.1 M sodium cacodylate buffer.
Dehydration
The tissue was rinsed twice (30 seconds each) with ddH2O, then underwent serial
dilutions for 10 minutes each as follows: 50% ethanol in ddH2O, 70% ethanol in ddH2O, 90%
ethanol in ddH2O, 100% ethanol, 1:1 ethanol:acetone, 1:2 ethanol:acetone, 100% acetone, 100%
acetone, and 100% acetone).
Infiltration
Epon (WPE 145) resin was prepared fresh from a mixture of LX112 resin, dodecenyl
succinic anhydride (DDSA), and nadic methyl anhydride (NMA). Tissue was incubated for 1
hour in a 1:1 mixture of acetone:Epon, then overnight in a 1:4 mixture of acetone:Epon.
Embedding
The tissue was transferred to a dish of pure Epon for 1 hour, then to coffin molds
containing pure Epon. The molds were placed at 60°C for 48 hours.
Sectioning and Staining
Ultrathin (~70 nm) sections were cut from each sample using a Leica Ultracut UCT
ultramicrotome & collected on 200-mesh grids (Ted-Pella). Grids were left to dry overnight, then
11
stained for 8 minutes in 4% uranyl acetate, rinsed for 30 seconds with ddH2O, left to dry for 20
min, stained for 4 minutes in 2.5% lead citrate, rinsed for 30 seconds with ddH2O, & stored at
RT until the time of imaging.
Imaging and Quantification
Sections were imaged using a Thermo Fisher Scientific Technai T12 Transmission
Electron Microscope. 9300x magnification images of dorsal column white matter were imported
into Reconstruct (Synapseweb), and areas were derived from traces of myelinated axons with
and without their myelin sheaths.
RESULTS
IBOT:NG2creER Mice
To validate specific expression of the IBOT transgene (marked by EGFP) and investigate
the effects of transgene expression on CNS cell populations and myelination, we immunostained
brain and spinal cord sections from IBOT:NG2creER and IBOT:NG2cre animals prior to
analysis with fluorescent and confocal microscopy. As expected, GFP+Olig2+ cells were only
observed in tamoxifen-injected IBOT:NG2creER double transgenic animals and not in single
transgenic or vehicle-injected animals. However, the animals we used initially, aged P92-101,
exhibited extremely low induction efficiency. Only ~4% of NG2+ cells in the corpus callosum
and ~2% of NG2+ cells in the cortex were also GFP+ (Figure 2). In contrast, animals injected
with 4-OHT from P8-11 and perfused at P17 displayed ~30% colocalization between NG2 and
GFP. This could be attributed to a combination of the increased efficacy of 4-OHT and
significantly shorter cell cycles of NG2 cells in young animals compared to adults (Psachoulia et
al., 2009). In an attempt to produce an induction efficiency above 50%, we next attempted to
12
injected P2 animals with 4-OHT. However, none of the animals survived after more than three
days of injections. Thus, we shifted our focus toward the IBOT:NG2cre constitutive cre line, in
which all NG2-expressing cells and their progeny should be vulnerable to the transgene as early
as embryonic day 14, when NG2 is first detectable in the CNS (Dawson et al., 2000; Nishiyama
et al., 1996).
Figure 2. Low transgene induction efficiency in IBOT:NG2creER animals
While EGFP (green) staining indicative of transgene expression colocalized with Olig2 (blue)
and NG2 (red) staining, overall induction efficiency was extremely low in adult
IBOT:NG2creER animals, with only 4% of NG2+Olig2+ cells in the corpus callosum (CC, top)
and 2% of NG2+Olig2+ cells in the cortex (bottom).
Physical Phenotype of IBOT:NG2cre Mice
IBOT:NG2cre, which differ from the IBOT:NG2creER mice above in their constitutive
rather than tamoxifen-activated cre activity, exhibited severe motor defects and growth
retardation first noticeable at around postnatal day (P) P7, followed by death by around P21.
CC
C
ort
ex
13
Figure 3. Phenotype of IBOT:NG2cre mice
Compared to WT mice (A), IBOT mice (B) exhibited severe symptoms of limb weakness and lack
of motor coordination, and were unable to right themselves when placed on their backs. C) IBOT
mice (orange) experienced growth retardation compared to WT littermates (blue), with
symptoms becoming apparent by ~P7 and growing increasingly severe until their deaths by
~P21.
Hypomyelination of the Spinal Cord
In order to investigate the cause of the motor deficits seen in the IBOT:NG2cre line, we
collected brains and spinal cords from P13 and P21 animals. While the brains appeared relatively
0
2
4
6
8
10
0 5 10 15 20
Mass (
g)
Postnatal Day
Mouse Weight Over Time
IBOT tg/+ Control
Linear (IBOT tg/+) Linear (Control)
C
14
normal in size and color, there was a striking difference in the appearance of the spinal cords.
Compared to the solid-white WT spinal cords, IBOT spinal cords were nearly transparent (Figure
4A), which led us to look specifically at myelination. Upon immunostaining with an antibody
against myelin basic protein, we observed sparse, patchy myelination around the dorsal, ventral,
and lateral funiculi, with a particularly noticeable lack of MBP in the ventral portion of the
dorsal column (Figure 4B). In mice, this region contains the corticospinal tract (CST), a
descending tract from the motor cortex necessary for control of the limbs (Steward et al., 2008).
The observed myelination strongly suggested defects in myelinating oligodendrocytes as a result
of transgene expression.
Figure 4. Hypomyelination of the spinal cord in IBOT mice
A) By P13, IBOT spinal cords (right) were myelin-deficient to the point of near-transparency
compared to WT spinal cords (left). Immunostaining against myelin basic protein revealed
sparse, patchy myelination throughout the anterior and dorsolateral funiculi of IBOT spinal
cords (B).
A B
15
Decreased Density of Oligodendrocytes but Not NG2 Cells
To identify the cause of the hypomyelination, we first investigated oligodendrocyte
lineage cell population density, as the most straightforward cause of hypomyelination would be a
deficiency in myelinating oligodendrocytes. We perfused IBOT:NG2cre double transgenic
(IBOT) mice and NG2cre single transgenic (control) littermates at P14 and P21 and stained
transverse spinal cord sections with antibodies against NG2, CC1, GFP, and Olig2. We imaged
sections on a Zeiss Axiovert 200M fluorescent microscope and quantified oligodendrocyte
lineage cell population density in the dorsal column by counting NG2+Olig2+ cells and
CC1+Olig2+ cells and dividing by the volume of the dorsal column. As expected, we observed
significantly lower CC1+Olig2+ populations in IBOT:NG2cre mice compared to control
littermates at both P14 (Welch’s t-test, p = 0.0102) and P21 (Welch’s t-test, p = 0.0052) (Figure
5). Interestingly, however, NG2 cell population density did not differ significantly between
IBOT and control mice at P14 (Welch’s t-test, p = 0.4619) nor at P21 (Welch’s t-test, p =
0.3807), and while not statistically significant, NG2 cell population density was actually greater
in IBOT mice than in control littermates, suggesting an inability of NG2 cells to mature and
differentiate.
16
A B
C
17
Figure 5. Quantification of oligodendrocyte and NG2 cell populations
A-C. Immunostaining for the oligodendrocyte antigen CC1 in the dorsal column of WT (A) and
IBOT (B) spinal cords. The density of CC1+ oligodendrocytes was severely reduced in IBOT
dorsal columns at P14 (control n≥4, *** p<0.01) D-F) Immunostaining for NG2 in the dorsal
column of the spinal cord of WT (C) and IBOT (D) mice at P14. F. The density of NG2 cells was
slightly higher in IBOT animals, but not significantly different.
D E
F
18
Differences in Axon Area but not Myelin G-Ratio
In order to better characterize our established hypomyelinating phenotype and to
investigate possible axonal defects in IBOT animals, we processed P16 spinal cord tissue for
transmission electron microscopy (TEM). TEM imaging showed a significantly lower density of
both myelinated and unmyelinated axons in IBOT animals compared to WT animals.
Interestingly, while more scarce, IBOT axons were significantly greater in area than WT axons
(Figure 6C). Despite this difference, however, the myelin G-ratio, defined as the diameter of the
axon to the diameter of the axon plus its myelin sheath, did not differ significantly between
IBOT and WT animals (Figure 6D). Thus, it appears that while fewer axons were myelinated,
their myelin sheaths were formed normally.
A B
19
Figure 6. Differences in axon area but not myelin G-ratio
While the number of myelinated dorsal column axons was far greater in WT mice, average axon
area was significantly greater in IBOT mice (Mann-Whitney U = 266, n1 = 49, n2 = 46, p <
0.0001, two-tailed). However, the myelin G-ratio, defined as the diameter of the axon divided by
the diameter of the axon plus its myelin sheath) did not differ significantly between genotypes
(Mann-Whitney U = 1031, n1 = 49, n2 = 46, p = .4787, two-tailed).
DISCUSSION
Interestingly, we observed a significant decrease in oligodendrocyte density, but no
change in NG2 cell density in the spinal cord dorsal columns of IBOT:NG2cre mutants. This
suggests a disruption during oligodendrocyte lineage cell maturation. There are a few
possibilities, but the most direct are: (A) NG2 cells are maintaining a static population due to an
inability to differentiate, (B) NG2 cells are dying before differentiation but rapidly proliferating
to maintain a normal population density, or (C) NG2 cells are differentiating normally, but dying
WT
IBO
T
0
2
4
6
8
A x o n A re a
G e n o ty p e
Ax
on
Are
a
(µm
2)
C D
WT
IBO
T
0 .4
0 .5
0 .6
0 .7
0 .8
0 .9
M y e lin G -R a t io
G e n o ty p e
My
eli
n
G-R
ati
o
20
after becoming oligodendrocytes. Our lab is currently working on EDU pulse chase and
TUNEL/Caspase3 staining to identify the nature of this maturation defect.
The axonal swelling observed here through electron microscopy fits with other groups’
observations of Plp null mutant mice. Plp null mutants exhibited axonal swelling specifically in
fully myelinated axons (Griffiths et al., 1999), and transplantation of Plp mutant
oligodendrocytes into shiverer mice resulted in similar axonal swelling (Edgar et al., 2004).
This suggests that in our model, the dysfunctional oligodendrocytes that survive to begin
myelinating either fail to produce myelin properly or, after myelinating axons, die and fail to
provide trophic support necessary for axonal health. As Nave & Trapp (2008) suggest, the
myelin sheaths surrounding the axons may prevent access to outside nutrients, effectively
dooming myelinated axons without proper glial support.
CONCLUSIONS
Cleavage of VAMP2 and VAMP3 in NG2 cells and their progeny resulted in growth
retardation, pronounced motor defects, early death, severe hypomyelination of the spinal cord,
significantly lower oligodendrocyte density but not NG2 cell density, a decrease in the density of
spinal cord axons, and the development of large, abnomally-shaped axons among those that
remained. My immunostaining experiments showed that while NG2 cell population density was
unaffected by expression of the BoNT/B toxin, oligodendrocyte density was significantly lower
in IBOT animals, indicating a deficit in the differentiation and/or survival of developing
oligodendrocytes as a result of VAMP cleavage in NG2 cells. While this alone could suggest
simply an autocrine function for VAMPs in developing oligodendrocytes, preliminary TEM
analysis suggested otherwise. The total number of dorsal column axons in IBOT mice was
21
significantly lower than that observed in WT animals, and many of the remaining axons
appeared swollen and misshapen, suggesting declining neuronal health.
Despite the observation of this striking phenotype, it should be noted that my work thus
far is quite limited in scope. While the initial goal of the study was to investigate the potential for
calcium-dependent exocytosis in NG2 cells, my experiments thus far only highlight the
importance of VAMPs in NG2 cells and their progeny. At present, we have no direct evidence of
calcium-dependent exocytosis in NG2 cells, much less the involvement of VAMPs in this
hypothetical process. We also have yet to identify where along the oligodendrocyte lineage the
mutant cells are malfunctioning and the precise mechanism by which VAMP cleavage leads to
our phenotype. Taken together, our findings highlight a seemingly critical role of VAMP
expression in NG2 cells not only in their own survival and differentiation, but also in axonal
health. However, much work remains to be done in order to properly characterize the specific
developmental functions of VAMPs in oligodendrocyte lineage cells.
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