1. Effects of Microglial
NF-κB Inhibition
in the ATXN1[82Q]
Mouse Model of
Spinocerebellar Ataxia
Type 1
Daniel Svedberg, Dr. Marija Cvetanovic
University of Minnesota Department of Neuroscience
Minneapolis MN 55455
May 6, 2016
6,064 Words

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Effects of Microglial NF-κB Inhibition in the
ATXN1[82Q] Mouse Model of Spinocerebellar
Ataxia Type 1
Daniel Svedberg, Dr. Marija Cvetanovic
University of Minnesota Department of Neuroscience
Minneapolis MN 55455
May 6, 2016
Abstract
Understanding the role of glial cells in neurodegenerative diseases may help us
develop successful therapies for patients suffering from neurodegenerative disorders.
We sought to characterize the role of microglia in Spinocerebellar Ataxia Type-1
(SCA1), a deadly inherited neurodegenerative disease that affects the cerebellum. We
genetically inhibited microglial NF-κB activity in ATXN1[82Q] mouse models of SCA1,
and demonstrated evidence of successful inhibition of microglial activation, but further
work with a larger number of animals is needed to conclusively demonstrate an effect
(or lack of it) on SCA1 pathology. However, partial inhibition of microglial NF-κB was
shown to significantly worsen SCA1 pathology. Finally, our data suggest that inhibiting
microglial NF-κB in wild-type mice may cause deficient synaptic pruning in the
cerebellum, which is associated with the recruitment of microglia, and may confound
future studies.
Review of SCA1
Spinocerebellar Ataxia Type-1 (SCA1) is a dominantly inherited
neurodegenerative disease primarily affecting the cerebellum. Clinical features of SCA1

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typically emerge between the third and fourth decades of life and include cerebellar
atrophy and progressively worsening motor coordination deficits in balance, speech,
limb movements, and gait. These motor deficits are thought to be the direct
consequence of cerebellar degeneration. Mild cognitive impairments are also observed
in SCA1. Patients invariably die within 10-15 years of onset, usually from breathing
complications as the result of brainstem failure.
Neuroanatomical studies of SCA1 patients show atrophy of the cerebellum,
medulla oblongata, cervical spinal cord, and associated structures. Degeneration is not
observed in the cerebrum. Neuropathological studies have demonstrated degeneration
of the Purkinje cells, dentate nucleus, and inferior olivary nuclei. Additionally, lost
neurons are replaced by enlarged Bergmann glia1
. Furthermore, cerebellar tissue
samples demonstrate nuclear inclusions.
SCA1 is caused by expansion of the CAG repeat tract in the SCA1 gene. CAG is
the trinucleotide that codes for glutamine, and the encoded Ataxin-1 (ATXN1) protein
contains the resulting expanded polyglutamine tract, which confers pathogenicity.
Nonpathogenic ATXN1 contains between 6 and 42 repeats, and those with an excess
of 21 repeats contain a CAT interruption within the CAG tract, while uninterrupted CAG
tracts between 39 and 82 repeats have been observed in SCA1 patients2
. Longer
repeats are associated with earlier onset and faster disease progression. The CAG
repeat tract in ATXN1 is unstable, and usually it further elongates in successive
generations. The expansion of CAG repeats confers a gain-of-function toxicity, SCA1
knock-out mice do not develop severe ataxia3
(ref). Furthermore, studies in mouse
models have shown that the loss of normal ATXN1 function in SCA1 may confer loss-

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of-function pathologies including decreased hippocampal neurogenesis, which
accounts for the cognitive deficits observed in SCA1 patients4
.
The SCA1 gene was first cloned by Dr. Harry Orr and Dr. Huda Zoghbi in 19935
,
and a number of mouse models have been developed to study the impact of ATXN1
protein on SCA1 pathology. The first mouse model of SCA1 developed is the
transgenic Purkinje-cell-specific ATXN1[82Q] (formerly B05) mouse. The ATXN1[82Q]
mouse has approximately 30 copies of the human SCA1 transgene expressed only in
Purkinje cells through the Purkinje cell-specific promoter: PCP2. Each transgene
contains 82 CAG repeats, the greatest repeat length observed in humans at the time.
The ATXN1[82Q] mouse model faithfully recapitulates the cerebellar atrophy and motor
coordination deficit as seen in human patients, and provided the first causal evidence
that the CAG repeat expansion is responsible for neurodegeneration in SCA16
. Another
notable SCA1 model is the endogenous knock-in Sca1154Q/2Q
mouse, which has 154
CAG repeats knocked into the endogenous SCA1 gene. Unlike the transgenic
ATXN1[82Q] mouse model, knock-in Sca1154Q/2Q
mice globally express expanded
ATXN1 protein. Sca1154Q/2Q
also recapitulate cerebellar atrophy and motor coordination
deficits, but unlike ATXN1[82Q] mice, Sca1154Q/2Q
die prematurely at around 35-45
weeks of age. Histological analyses of these knock-in mice also demonstrated that
most severely affected neurons, especially Purkinje cells, had the fewest nuclear
inclusions of ATXN1 protein, suggesting that atrophied neurons may have been unable
to sequester the expanded ATXN1 protein, and therefore are most affected by the
toxicity of the expanded protein7
.

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Further work on the molecular biology of SCA1 has shown that ATXN1 likely is a
transcription factor, and that domains outside of the CAG repeat tract are involved in
the pathogenicity of expanded ATXN1 protein. ATXN1 protein contains a nuclear
localization sequence, and interfering with this prevents the pathogenicity of expanded
ATXN18
. ATXN1 protein also contains an AXH domain, which has been found to be a
dimerization site, and also a binding site for a number of associated proteins. Another
notable site on the both expanded and wild-type ATXN1 proteins is residue Ser776
,
which is normally phosphorylated. Replacement of Ser776
in the ATXN1 protein with an
alanine, which cannot be phosphorylated, attenuates SCA1 pathology in spite of the
polyglutamine expansion. Phosphorylated Ser776
forms a binding site for numerous
proteins, and may be a site of protein interaction with ATXN1 which confers
pathogenicity in SCA12
.
Our understanding on the interactions of CIC and RBM17 with expanded ATXN1
has given rise to a new model on how the CAG-repeat expansion in ATXN1 may confer
both loss of function for some proteins, and gain-of-function pathogenicity for others.
CIC is stabilized by binding to the AHX domain in ATXN1. Conversely, RBM17 binds
the phosphorylated Ser776
residue. Interestingly, the CAG repeat tract expansion
causes expanded ATXN1 protein to preferentially bind RBM17 instead of CIC. In this
scenario CIC is no longer stabilized by binding to ATXN1 and loses functionality, while
the function of the RBM17-ATXN1 complex is amplified. This model may help us
understand how other protein systems may confer either loss-of-function or a gain-of-
function pathogenicity in similar fashion as described by this models9
.

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Introduction
To date, the majority of research on SCA1 pathogenesis has focused on
neuronal pathology, while the role of cells surrounding neurons (glia) have largely been
ignored. Glia are nonetheless an important consideration in disease pathogenesis as
they closely regulate neuronal function by controlling the composition of the interstitial
milieu, uptake and release neurotransmitters, regulate local blood flow, mediate
synapse maturation, and clear cellular debris. The central question of this paper is if
microglia, an immune-like glial cell, contribute to SCA1 pathology.
In any case of neuronal damage or pathogenic infiltration, glia, especially
astrocytes and microglia, mount an inflammation-like response termed “activation”,
characterized by proliferation, enlargement, and production of pro-inflammatory
cytokines10,11
. Because the activation response has evolved to combat a wide variety of
aggressive pathogens and pathologies, some of the factors secreted by activated glia
are neurotoxic. Two major questions of interest in neuroimmunology are whether
gliosis contributes to neurodegenerative disease progression, and if glial responses
can be modulated to improve disease outcomes. The answers to these questions are
highly context dependent, and are being studied in numerous neurodegenerative
diseases12
.
Gliosis has been clearly identified in human tissue studies of SCA1. Studies of
gliosis in both knock-in Sca1154Q/2Q
and transgenic ATXN1[82Q] mouse models have
identified the activation of both astrocytes and microglia early in the disease, even
before neurodegeneration occurs. These results suggest that neurons affected by

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expanded ATXN1 actively communicate with glial cells to instigate gliosis, and
demonstrate a close relationship between neuronal dysfunction and glial activation13
.
Indeed, the downregulation of glutamate and aspartate transporter (GLAST) in
astrocytes is associated with the loss of Purkinje neurons in ATXN1[82Q] mice14
.
Microglia, the resident macrophages in the brain, become activated in SCA113
.
Microglia can be crudely placed into two states; the “inactivated” M2 phenotype and
the M1 “activated” phenotype. Microglial activation is characterized by enlargement of
the cell body, shortening of processes, proliferation, and secretion of inflammatory
cytokines15
. Microglia become activated in response to detecting pro-inflammatory
cytokines secreted by pathogens or dysfunctional/necrotic neurons, signaled by a
variety of toll-like receptors on microglia. These states are not binary, but rather, they
exist on a continuum of activation, implying that the delicate balance of cytokines
secreted by microglia in response to a pro-inflammatory challenge may both positively
and negatively affect neuronal survival in neurodegenerative disease10
. Microglia also
are phagocytic, and are also responsible for neuronal remodeling in the brain,
indicating that microglia may have a direct influence on modulating circuitry16,17
and
neuronal atrophy in neurodegenerative disease10,15,18
.
We asked if inhibiting microglial influence in SCA1 could affect neuronal
pathology. We first used a genetic approach to inhibit microglial activation in mice
through the nuclear factor kappa-light-chain-enhancer of B-cells (NF-κB) classical
myeloid cell activation pathway. NF-κB is a centrally important transcription factor
which mediates the transcription of many pro-inflammatory genes. Many of these pro-
inflammatory cytokines on their own are known to exacerbate neuronal death19
, and in

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some cases NF-κB activation in glial cells has been directly linked to neuronal death
under pathological stimuli18,20
. NF-κB normally has a basal level of expression, but is
also sequestered by the inhibitor of κB (IκB). To instigate activation, IκB kinase (IKK)
phosphorylates IkB, thus marking it for degradation and freeing NF-κB to move into
nucleus thus allowing NF-κB to mediate the production of pro-inflammatory
cytokines19
. IKKß is the critical subunit in IKK, and without functional IKKß, NF-κB
activation is inhibited.
IKKß was conditionally depleted in the microglia of mice by employing Cre-lox
recombination technology21
to create LysM-Cre IKKßflox
mice. IKKß was targeted by
“floxing” (flanking with loxP sites) exon 3 of the gene coding for IKKß (IKKßFlox
). The
loxP sites direct excision of IKKß by Cre-recombinase, and the expression of Cre is
driven by the macrophage-specific promoter LysM (LysM-Cre). Therefore, LysM-Cre
IKKßflox
mice have inhibited microglial NF-κB, and theoretically attenuated microglial
activation response (Figure 1A). Meanwhile, NF-κB activity is not altered in IKKßflox
mice
lacking the Cre gene. It is important to note that the Cre-lox system is never 100%
efficient, and that IKKß can be floxed on one (heterozygous flox, or “fl/wt”) or both
alleles (homozygous flox, or “fl/fl”), so that the depletion of IKKß in LysM-Cre IKKßfl/fl
mice is more extensive than that in LysM-Cre IKKßfl/wt
mice.
To study the effect of microglial activation in SCA1, we combined LysM-Cre
IKKßflox
mice with ATXN1[82Q] mice to generate LysM-Cre IKKßflox
ATXN1[82Q] mice.
Mice used in this experiment were 2-3 month old heterozygous and homozygous
IKKßflox
variants of control (LysM-Cre – and ATXN1[82Q] -), LysM-Cre+, ATXN1[82Q]+,
and LysM-Cre+ ATXN1[82Q]+ mice, creating a total of eight groups. It is important to

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emphasize that all non- LysM-Cre+ control mice in this experiment, including “wild
type” and ATXN1[82Q]+ control mice, contain LoxP sites flanking either one or both
copies of IKKß, and they are grouped with corresponding heterozygous or
homozygous LysM-Cre IKKßflox
mice. This was done to control for any changes in
transcription of IKKß that may result from the LoxP sites themselves. For these mice,
we assessed the motor coordination phenotype using the rotarod test, and performed
immunohistochemical studies to assess neuronal atrophy, glial activation, and
microglial recruitment (as a proxy of activation).
In addition, we questioned whether altering the activation phenotype of
microglia could independently alter synaptic pruning in the cerebellum of LysM-Cre
IKKßFlox
mice. Aberrant cerebellar circuitry can cause an ataxic phenotype, has been
implicated in numerous cerebellar pathologies, and even been observed in
ATXN1[82Q] mice22
. Microglia-mediated synaptic pruning plays a critical role in the
formation of cerebellar circuitry in the first days of a mouse’s life16,17
. Since microglial
NF-κB (a centrally important protein in microglial function) is inhibited in LysM-Cre
IKKßFlox
mice from conception, we hypothesized that this could disrupt developmental
synaptic pruning. Synaptic pruning in the cerebellum occurs in a stereotypical fashion,
and aberrant synaptic pruning often results in a histologically detectable phenotype.
From birth, Purkinje neurons are innervated by multiple climbing fibers at the cell body.
Starting around post-natal day 3, as development progresses one “winner” climbing
fiber for each Purkinje neuron is selected, and the connection between this fiber and its
respective Purkinje neuron strengthens with an increase in the number of synaptic
terminals until about P7. The precise mechanism of this competitive selection process

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is unknown. The synapses of this “winner” climbing fiber then begin to translocate after
P8, innervating the dendrites of the Purkinje neurons after the dendrites grow into the
molecular layer of the cerebellum. Excess climbing fiber synapses remaining on the cell
body that have not translocated to the dendrites are then eliminated during days P12-
P1623
. We stained LysM-Cre IKKβFlox
mice for vesicular glutamate transporter 2
(vGluT2), a protein that transports glutamate to the climbing fiber synaptic terminal,
and Calbindin, to compare the number of VGLUT2-positive synapses on Purkinje
neuron cell bodies in both LysM-Cre IKKßflox
mice and control mice, in order to
determine if aberrant synaptic pruning was present in LysM-Cre IKKßflox
mice.
Furthermore, we analyzed microglial density for paired samples. To determine if
potential LysM-Cre IKKßflox
-induced synaptic pruning abnormalities resulted in a
behavioral phenotype, I examined previously recorded behavioral data for LysM-Cre
IKKßflox
mice.
Methods
Mice
LysM-Cre and IKKβflox/flox
mouse lines were obtained from Dr. Koshnan and Dr.
Peterson at the California institute of Technology. ATXN1[82Q] mice were obtained
from Dr. Harry Orr at the University of Minnesota. LysM-Cre IKKβflox
mice were
generated by crossing LysM-Cre and IKKβflox/flox
mouse lines. ATXN1[82Q] LysM-Cre
IKKβFlox
mice were generated by crossing LysM-Cre IKKβFlox
and ATXN1[82Q] mice.
After 3 months of age, mice were subjected to rotarod behavioral analysis. Afterwards
mice were euthanized using carbon dioxide, and their brains were extracted. Brain

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samples were then cut in half; one half was immediately frozen and the other half was
fixed.
Rotarod Analysis
3-month old mice were subjected to rotarod analysis, where they were placed
on an accelerating, rotating rod for a maximum of 10 minutes, 4 times daily, for 4
consecutive days. Time to fall from the rod, or failure to run with the rod, was recorded
for each session.
Immunohistochemistry
Full-hemisphere brain samples were fixed in 4% formaldehyde and dehydrated
in sucrose solution. Fixed samples were then embedded and frozen in OCT medium.
Using a cryostat, 40-micrometer thick sagittal sections starting at the midline were
taken from embedded samples and placed in 24-well plate filled with phosphate
buffered saline (PBS, Corning). At least 6 sections from each mouse were selected and
placed into a new 24 well plate for staining. Sections were intermittently washed using
PBS between each step. Antigen retrieval was performed only on sections stained for
VGLUT2, where sections were placed in 24 well plates filled with 200 mM Sodium
Citrate buffer, steamed in a vegetable steamer for 20 minutes, and then cooled for 20
minutes before transferring sections to a fresh plate. All sections were then incubated
in a blocking buffer composed of 3% normal goat serum (NGS) and 1% Triton X-100
detergent in PBS for 1 hour at room temperature (RT). Sections were then incubated
with primary antibody at a 1:200 dilution in blocking buffer overnight at RT, and then
incubated with secondary antibody at a 1:400 dilution in blocking buffer at RT for 4
hours. Primary antibody used to stain Purkinje neurons was monoclonal anti-Calbindin-

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D-28K antibody produced in mouse (Sigma Aldrich). Primary antibody used to stain
astrocytes for quantification of astrocytic activation was monoclonal anti-Glial Fibrillary
Acidic Protein (GFAP) produced in rabbit (DAKO). Primary antibody used to stain
microglia was anti-Ionized Calcium Binding Protein 1 (IBA1) produced in rabbit (Wako
Chemical Industries). Primary antibody used to stain climbing fiber synapse terminals
was anti-vesicular glutamate transporter 2 (vGluT2) produced in mouse (Millipore),
which was co-stained with monoclonal anti-Calbindin-D-28K antibody produced in
rabbit (Sigma Aldrich). Stained sections were then mounted to slides for imaging.
Confocal imaging
At least 6 images per mouse for every stain were taken using a laser confocal
microscope (Olympus FV1000). For GFAP, Calbindin and IBA1 staining, 20 micrometer
z-stacks at 20X magnification were imaged from arbitrarily chosen lobules. For
VGLUT2+Calbindin co-staining, 10 micrometer z-stacks at 60X were imaged so that
the entire stack encompassed the cell body of at least one Purkinje neuron cell body in
the field of view.
Image analysis
IBA1 staining was quantified by counting the density of IBA1+ cells in the
cerebellar cortex and white matter using Fiji/ImageJ. Average staining intensity in the
molecular, granular layers and white matter was measured for GFAP images using
Fiji/ImageJ, and intensity values were normalized to the control averages of their
respective cohort. For calbindin images, the width of the cerebellar molecular layer was
measured in two places on each image using Fiji/ImageJ. VGLUT2/Calbindin images
were analyzed using IMARIS 3-dimensional image processing software. 3-dimensional

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images were reconstructed using the “surpass” function, a region of interest was
drawn around each well-defined Purkinje neuron cell body, and VGLUT2+ puncta
within regions of interest were detected using the “spots” function set to an optimal
spot diameter of 2.3μm. Spots detected on each Purkinje neuron cell body were then
counted.
Statistical Analysis
All datasets were handled and analyzed using R Statistics software24
. Data
handling was performed using the tidyr and base packages. Summary statistics and t-
tests were performed using the plyr, ddply, and base packages. Graphs were created
using the ggplot2 package. Analysis of variance (ANOVA) was performed on raw
datasets, pairing each dependent variable measurement to its corresponding
genotype. Statistical comparisons were derived from ANOVA using Tukey’s Honest
Statistical Differences test.
Results
Reducing NF-kB activity selectively in microglia shows trend toward rescuing
motor deficits in ATXN1[82Q] mice
The accelerating rotarod test was used to measure motor coordination of
experimental mice (Figure 1). Control IKKβfl/wt
-ATXN1[82Q] mice did not show
behavioral deficit on rotarod. ATXN1[82Q] LysM-Cre IKKβfl/wt
mice have the worst
rotarod performance of all groups, and demonstrated a 50% reduction in latency to fall
on the 4th
day as compared to control IKKβfl/wt
mice (p= 0.033) (Figure 1B). IKKβfl/fl
-
group mice did not show any statistically significant motor deficits, but ATXN1[82Q],

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LysM-Cre IKKβfl/fl
, and ATXN1[82Q] LysM-Cre IKKβfl/fl
mice demonstrated a trend
towards impairment compared to control IKKβfl/fl mice (Figure 1C). While higher
number of animals is needed (to detect a 50 second reduction in latency time to a
significance level of 5% and power of 90% we need at least 15 mice) these results
suggest that microglial NF-kB activation may be beneficial in SCA1.
Reducing NF-kB activity selectively in microglia reduces microglial recruitment in
ATXN1[82Q] mice
IBA1 staining and quantification of average microglial density was used to
examine microglial activation (Figure 2). We reproduced previously reported microglial
activation in ATXN1[82Q] mice. Notably, selective inhibition of NF-kB in microglia,
resulted in reduction of microglial density in the cortex of ATXN1[82Q] LysM-Cre
IKKβfl/fl
mice by 30% (p=0.0002) as compared to corresponding ATXN1[82Q] mice
(Figure 2A-B, D-E). Qualitative examination of IBA1 images suggests that microglial
hypertrophy is also slightly attenuated in ATXN1[82Q] LysM-Cre IKKβfl/fl
as compared
to ATXN1[82Q] mice (Figure 2F).
Microglial density in control LysM-Cre IKKβfl/wt
and LysM-Cre IKKβfl/fl
mice was
not significantly different from IKKβfl/wt
and IKKβfl/fl
controls. Microglial density in
ATXN1[82Q] LysM-Cre IKKβfl/wt
mice was not significantly different from corresponding
ATXN1[82Q] mice. These results suggest that reducing NF-kB selectively in microglia
inhibits their recruitment in SCA1.

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Reducing NF-kB activity selectively in microglia shows trend toward reduced
astrogliosis in ATXN1[82Q] mice
GFAP staining and normalized quantification of staining intensity was used to
examine activation of astrocytes (astrogliosis) (Figure 3). ATXN1[82Q] mice
demonstrate 38%(p=0.03) increased cortical GFAP intensity compared to LysM-Cre
IKKβfl/wt
mice, while LysM-Cre IKKβfl/wt
do not demonstrate significantly different GFAP
intensities as compared to IKKβfl/wt
controls. Qualitatively, GFAP images of ATXN1[82Q]
mice show increased heterogeneity and hypertrophy of astrocytic processes
characteristic of activation (Figure 3D). Similarly, GFAP intensity of white matter in
ATXN1[82Q] mice is 63% (p<0.0001) higher than in LysM-Cre IKKβfl/fl
mice. Although in
no instance was the intensity of GFAP in ATXN1[82Q] LysM-Cre IKKβfl/fl
/IKKβfl/wt
mice
significantly lower than that of corresponding ATXN1[82Q] mice, ATXN1[82Q] LysM-
Cre IKKβfl/fl
/IKKβfl/wt
frequently trended to lower intensity values than ATXN1[82Q] mice
(Figure 3 A-C, E-G). These results suggest that NF-kB activity in microglia may regulate
microglia-astrocytes crosstalk in SCA1.
Worsening of Purkinje neuron pathology may underlie exacerbated motor defects
in ATXN1[82Q] mice with reduced NF-kB signaling in microglia
Calbindin staining was used to investigate Purkinje cell atrophy, quantified by
measuring the molecular layer width. ATXN1[82Q] LysM-Cre IKKβfl/wt
mice demonstrate
significantly decreased molecular layer width compared to ATXN1[82Q] mice (-51%
reduction, p<0.0001) (Figure 4A). Qualitative examination of calbindin staining from
IKKβfl/wt
group mice shows that both ATXN1[82Q] and ATXN1[82Q] LysM-Cre IKKβfl/wt
mice exhibit visible cortical thinning, lobule disfigurement, loss of Purkinje cells, and

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heterotopic organization of Purkinje cells, although these features are visibly worse in
ATXN1[82Q] LysM-Cre IKKβfl/wt
mice. Furthermore, the molecular layer in LysM-Cre
IKKβfl/wt
mice is not visibly different from control mice (Figure 4B).
ATXN1[82Q] mice failed to replicate molecular layer atrophy normally seen in
these mice most probably due to high variability and low number of examined animals.
No significant differences in molecular layer thickness among any mice were observed
(Figure 4C).
Reducing NF-kB signaling causes synaptic pruning defects in cerebellum
Calbindin and vGluT2 co-staining was used to investigate synaptic pruning,
quantified by counting the number of vGluT2+ synapses on Purkinje cell bodies only.
LysM-Cre IKKβfl/wt
mice demonstrate 73% (p<0.0001) more vGlut2+ synapses
remaining on Purkinje cell bodies than controls (Figure 6B, D). Analysis of paired
samples demonstrates that the number of vGlut2+ synapses on Purkinje cell bodies is
positively correlated with number of microglia in LysM-Cre IKKβfl/fl
and LysM-Cre
IKKβfl/wt
mice (0.82 and 0.85 respectively) while there is only a weak negative
correlation (-0.54) in control mice (Figure 6C).
For comparison with vGluT2 data, previous rotarod behavior data and IBA1
microglial density was re-examined by combining IKKβfl/fl
and IKKβfl/wt
control groups
into a single control group, since they were not significantly different from each other in
any way in either of these experiments. This new control group was then re-compared
against existing rotarod and IBA1 data for LysM-Cre IKKβfl/fl
and LysM-Cre IKKβfl/wt
mice. Motor coordination as tested by the accelerating rotarod test demonstrated that

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LysM-Cre IKKβfl/fl
and LysM-Cre IKKβfl/wt
mice are not significantly impaired, but
nonetheless trending downwards compared to control mice (Figure 5B). Reanalysis of
IBA1 data revealed that LysM-Cre IKKβfl/fl
mice had 15% (p=0.04) higher microglial
density in the cerebellar cortex as compared to controls (Figure 6A).

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Figure 1. Microglial IKKβ Depletion and Motor Behavior
(A) In LysM-Cre IKKβFlox
mice, activation of the NF-κB pathway is inhibited in
microglia by depleting IKKβ using the Cre-lox system driven by the microglia-
specific promoter LysM.
(B) Rotarod analysis of IKKβfl/wt
-group mice failed to reproduce a behavioral deficit in
ATXN1[82Q] mice. Heterozygous depletion microglial IKKβ in ATXN1[82Q] LysM-Cre
IKKβfl/wt
mice resulted in impaired rotarod performance
(C) Rotarod analysis of IKKβfl/fl
-group mice did not show any statistically significant
relationships.

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Figure 2. Microglial Activation
IBA1 staining and quantification of microglial density reveals that for IKKβfl/wt
group
mice (A-C), microglial activation in the cortex (A) and white matter (B) is increased in
both ATXN1[82Q] and ATXN1[82Q] LysM-Cre IKKβfl/wt
mice. For the IKKβfl/fl
group (D-
F), microglial activation in the cortex (D) and white matter (E) is increased in
ATXN1[82Q] mice compared to control and LysM-Cre IKKβfl/fl
mice. Notably,
microglial activation is reduced in the cortex of ATXN1[82Q] LysM-Cre IKKβfl/fl
mice
as compared to ATXN1[82Q] mice.

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Figure 3. Astrogliosis
GFAP staining and normalized quantification of intensity reveals that for the IKKβfl/wt
group (A-D), astrogliosis in the molecular layer (A) of ATXN1[82Q] mice is increased
compared to control and LysM-Cre IKKβfl/wt
mice, as well as in white matter (B)
compared to control mice. Astrogliosis is increased in all three areas of ATXN1[82Q]
LysM-Cre IKKβfl/wt
mice as compared to controls (A-C). For the IKKβfl/fl
group (E-H),
astrogliosis in ATXN1[82Q] mice was increased only in white matter (G) as compared
to control and LysM-Cre IKKβfl/fl
mice.

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Figure 4. Neuronal Atrophy
Purkinje cell atrophy seen through calbindin staining and quantified by molecular
layer width measurement reveals that for the IKKβfl/wt
group (A-B), there is significant
neuronal atrophy among LysM-Cre IKKβfl/wt
, ATXN1[82Q], and ATXN1[82Q] LysM-
Cre IKKβfl/wt
mice as compared to control mice. Notably, ATXN1[82Q] LysM-Cre
IKKβfl/wt
demonstrate the most severe Purkinje cell atrophy. For the IKKβfl/fl
group (C-
D), we fail to replicate Purkinje cell atrophy normally seen in ATXN1[82Q] mice. No
significant differences were observed.

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Figure 5. Cerebellar Synaptic Pruning and Rotarod Behavior
(A) Illustration of the developmental cerebellar synaptic pruning process
hypothesized to be disrupted in LysM-Cre IKKβflox
mice. Adapted from Hashimoto et
al. 201323
(B) Rotarod behavior of LysM-Cre IKKβfl/fl
and LysM-Cre IKKβfl/wt
mice is not
significantly impaired, but nonetheless trending downwards, compared to control
IKKβfl/fl
and IKKβfl/wt
mice grouped together.

23. Svedberg 2016
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Figure 6. Depletion of Microglial IKKβ Leads to Deficient Cerebellar Synaptic
Pruning Associated with Microglial Activation.
(A) Microglial activation is elevated in LysM-Cre IKKβfl/fl
mice.
(B,D) Calbindin and vGluT2 co-staining reveals evidence of impaired synaptic
pruning in LysM-Cre IKKβfl/wt
mice, as quantified by counting the number of vGluT2
spots on Purkinje cell bodies.
(C) Analysis of paired samples demonstrates that synaptic pruning deficiency is
positively correlated with microglial activation in LysM-Cre IKKβfl/fl
and LysM-Cre
IKKβfl/wt
mice

24. Svedberg 2016
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Discussion
Based on the numerous studies highlighting the potential toxicity of microglial
activation in neurodegenerative disease10,12
, we hypothesized that inhibiting the central
activation pathway through NF-κB would ameliorate SCA1 pathology. A very similar
study in amyotrophic lateral sclerosis (ALS) found evidence that inhibiting microglial
NF-κB ameliorated neuronal degeneration and pathology18
. It has been suggested that
the presence of microglia is not necessary for survival or even proper cognitive
function during adulthood, although they are crucial during development25
.
Nonetheless we were aware that in certain conditions microglia can play the opposite
side of the coin, and protect from neurodegeneration26
. Ultimately the outcome may be
highly context dependent.
Does genetically inhibiting the NF-κB activation pathway in microglia by
depleting IKKβ truly inhibit microglial activation? Our analysis of IBA1 staining suggests
that only homozygous depletion of microglial IKKβ in ATXN1[82Q] mice modestly
decreases the density of microglia in the cerebellar cortex and dampens the visual
activation phenotype as compared to ordinary ATXN1[82Q] mice. On its own though,
the general trend in the IBA1 experiment suggests that homozygous depletion of
microglial IKKβ inhibits microglial activation, but we cannot yet definitively conclude
that this effect is real. Finally, the results of this experiment only tell us about the
number of microglia and not whether they are neuroprotective or neurotoxic. Current
ongoing experiments will accomplish this by measuring RNA expression of microglial

25. Svedberg 2016
25
genes that are commonly associated with these functions in-vitro from isolated
microglia.
Astrogliosis is another important part of the neuroinflammatory response, and
we asked if microglial activation could modulate astrogliosis in SCA1. Attenuation of
astrogliosis following inhibition of microglial NF-κB has been previously shown in
ALS18
. Although no significant changes in astrogliosis were observed between ordinary
ATXN1[82Q] mice and ATXN1[82Q] LysM-Cre IKKβflox
mice in the GFAP experiment,
the general trend is that astrogliosis is attenuated in both homo- and heterozygous
ATXN1[82Q] LysM-Cre IKKβflox
mice. Because GFAP measurements are highly variable,
it is possible that with added subjects we will see a significant attenuation of
astrogliosis.
Most importantly, we sought to characterize SCA1 neuronal pathology in the
context of genetically inhibited microglial NF-κB. Control IKKβfl/fl
mice did not
demonstrate any significant neuronal pathology. This was echoed in behavioral
experiments. Interestingly we were not able to reproduce rotarod motor coordination
deficit in ATXN1[82Q] mice that has been previously characterized. A couple of factors
may have contributed to this: 1) the relatively early age of 12 weeks that we performed
rotarod phenotyping on these mice (although previous studies showed deficits at 5
weeks), 2) potential errors in animal identification/handling during experiments by the
behaviorists, and mis-genotyping. Despite these challenges, rotarod experiments
provided one important point of significance: ATXN1[82Q] LysM-Cre IKKβfl/wt
mice
demonstrated the worst motor coordination deficit. Likewise, ATXN1[82Q] LysM-Cre
IKKβfl/wt
mice demonstrated the worst Purkinje neuron atrophy out of any group. The

26. Svedberg 2016
26
severely worsened pathology in these mice, as echoed by both studies, provides
compelling evidence suggesting that heterozygous depletion of IKKβ in ATXN1[82Q]
mice truly worsens disease pathology.
How can we explain the deleterious effect of heterozygous IKKβ depletion, and
the null effect of homozygous IKKβ depletion in ATXN1[82Q] mice, on motor
coordination and neuronal pathology? One considered hypothesis to explain the
behavioral phenotype was based on a paper published by Dr. Vikram Shakkotai, which
argued that Purkinje cell atrophy in SCA1 was an adaptive response to improve
neuronal function by increasing the density of under-produced potassium-channel
proteins, in order to preserve resting membrane potential and firing behavior of
Purkinje cells27
. Theoretically, if inhibiting NF-κB inhibited phagocytosis, then this
adaptive atrophy may be inhibited as well. While our data does not preclude this
atrophy-centric hypothesis from explaining the phenotype in ATXN1[82Q] LysM-Cre
IKKβfl/fl
mice, it does not support the phenotype well either. Moreover, we think this is
unlikely the case for ATXN1[82Q] LysM-Cre IKKβfl/wt
mice, which demonstrate the worst
neuronal atrophy and non-attenuated microglial activation.
A different hypothesis was that the LysM-Cre IKKβflox
modification may
independently lead to deficiencies in synaptic pruning. Theoretically, NF-κB inhibition
may also inhibit phagocytosis of synapses by microglia during development. The
LysM-Cre IKKβflox
system is active from conception, and thus will have developmental
effects. Deficiencies in cerebellar synaptic pruning can cause ataxia due to disruption
of proper cerebellar circuitry. We demonstrated preliminary evidence that LysM-Cre
IKKβflox
mice do indeed exhibit deficient synaptic pruning. Interestingly, LysM-Cre

27. Svedberg 2016
27
IKKβfl/wt
mice exhibit more severe deficiency. The severity of this deficiency is positively
correlated with the density of microglia in the cortex. A closer examination of
corresponding IBA1 images of LysM-Cre IKKβflox
shows that microglia in these mice do
not exhibit hypertrophy typical of activation, suggesting that these microglia may not
really be activated, but are rather just recruited. One hypothesis posited to explain this
was that chemical signaling of microglial recruitment for synaptic pruning occurs
continuously without negative feedback. Because the microglia may be unable to
prune synapses, the recruitment signal would continue unabated. A likely candidate for
this recruitment signal is fractalkine (CX3CL1), which is produced by synapses ready to
be pruned. Microglia are the predominant expressers of the fractalkine receptor
(CX3CR1) in the brain, and CX3CL1 signaling to initiate synaptic pruning has been
shown to recruit microglia16
. Finding elevated levels of CX3CL1 protein and RNA in the
cerebella of LysM-Cre IKKβflox
mice would support this hypothesis. Further experiments
that could be done to further characterize synaptic pruning in LysM-Cre IKKβflox
mice
would be to inject dye into the inferior olivary nucleus to stain individual climbing fibers
and then co-stain with calbindin, in order to investigate if Purkinje neurons are
innervated by multiple climbing fibers. An experiment that could be done to
characterize phagocytic activity of microglia in LysM-Cre IKKβflox
mice would be to
inject fluorescent beads into the brains of these mice, sacrifice the mice, and then stain
for IBA1 to count how many beads have been taken up by microglia in the time after
the beads were injected. While characterizing synaptic pruning in LysM-Cre IKKβflox
mice may not explain the worsened atrophy observed in ATXN1[82Q] LysM-Cre

28. Svedberg 2016
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IKKβfl/wt
mice, it may explain the behavioral deficits that we may more clearly see in
LysM-Cre IKKβflox
mice going forward.
Conclusion
The role of the inflammatory response in neurodegeneration is extremely
complicated and nuanced. Not only will the role be context dependent, but the
observed role is also methodology-dependent. NF-κB is just one of many factors which
modulate the activation response of microglia. Furthermore, LysM-Cre IKKβflox
mice
experience IKKβ depletion form conception, which may have important developmental
consequences. Nonetheless, we have shown that inhibiting NF-κB selectively in
microglia attenuates microglial recruitment, worsen behavior deficits and worsens
neuronal pathology in SCA1 mice. Many further analyses as outlined above will be
performed to further unveil the role of NF-κB in microglial activation and SCA1.
Contributions
Research was conceptualized and managed by Dr. Marija Cvetanovic
Rota-rod experiments were performed by Andrea Johnson, Nadia Copeland, and
Timothy Sundem
Immunohistochemistry, microscopy, and analysis was performed by Daniel Svedberg,
Megan Rhyner, Katherine Schwister, and Dr. Marija Cvetanovic
Data and statistical analysis was performed by Daniel Svedberg and Dr. Marija
Cvetanovic
Figures were created and illustrated by Daniel Svedberg

29. Svedberg 2016
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Writing the paper was performed by Daniel Svedberg, with intellectual contributions
and editing by Dr. Marija Cvetanovic
A special thanks to Emily Wozniak for helping me conceptualize the vGlut2 staining
experiment and offering guidance during the experiment.
Acknowledgements
Research was supported by startup funds for Dr. Marija Cvetanovic by the University of
Minnesota Department of Neuroscience, Institute of Translational Neuroscience, and
the Minnesota Medical Foundation
Funding for this project was provided by University of Minnesota North Star STEM
Alliance (NSSA) Louis-Stokes Alliance for Minority Participation in STEM (LSAMP)
Funding for this project was provided by the University of Minnesota Office of
Undergraduate Research (UROP)

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