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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

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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.

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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).

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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

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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-

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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

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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

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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

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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

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10

0 5 10 15 20

Mass (

g)

Postnatal Day

Mouse Weight Over Time

IBOT tg/+ Control

Linear (IBOT tg/+) Linear (Control)

C

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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

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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

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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

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M y e lin G -R a t io

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My

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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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