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.

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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

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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

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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

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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

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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

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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

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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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References 1. Donato, S. Di, Mariotti, C. & Taroni, F. Spinocerebellar ataxia type 1. Handb. Clin.

Neurol. 103, 399–421 (2012). 2. Zoghbi, H. Y. & Orr, H. T. Pathogenic mechanisms of a polyglutamine-mediated

neurodegenerative disease, spinocerebellar ataxia type 1. J. Biol. Chem. 284, 7425–9 (2009).

3. Matilla, a et al. Mice lacking ataxin-1 display learning deficits and decreased hippocampal paired-pulse facilitation. J. Neurosci. 18, 5508–5516 (1998).

4. Asher, M., Johnson, A., Zecevic, B., Pease, D. & Cvetanovic, M. Ataxin-1 regulates proliferation of hippocampal neural precursors. Neuroscience 322, 54–65 (2016).

5. Orr, H. T. et al. Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat. Genet. 4, 221–6 (1993).

6. Burright, E. N. et al. SCA1 transgenic mice: A model for neurodegeneration caused by an expanded CAG trinucleotide repeat. Cell 82, 937–948 (1995).

7. Watase, K. et al. A Long CAG Repeat in the Mouse Sca1 Locus Replicates SCA1 Features and Reveals the Impact of Protein Solubility on Selective Neurodegeneration. Neuron 34, 905–919 (2002).

8. Klement, I. A. et al. Ataxin-1 Nuclear Localization and Aggregation. Cell 95, 41–53 (1998).

9. Lim, J. et al. Opposing effects of polyglutamine expansion on native protein complexes contribute to SCA1. Nature 452, 713–8 (2008).

10. Thameem Dheen, S., Kaur, C. & Ling, E.-A. Microglial Activation and its Implications in the Brain Diseases. Curr. Med. Chem. 14, 1189–1197 (2007).

11. Pekny, M. & Nilsson, M. Astrocyte activation and reactive gliosis. Glia 50, 427–34 (2005).

12. Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C. & Gage, F. H. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–34 (2010).

13. Cvetanovic, M., Ingram, M., Orr, H. & Opal, P. Early activation of microglia and astrocytes in mouse models of spinocerebellar ataxia type 1. Neuroscience 289, 289–299 (2015).

14. Cvetanovic, M. Decreased expression of glutamate transporter GLAST in bergmann glia is associated with the loss of Purkinje neurons in the spinocerebellar ataxia type 1. Cerebellum 14, 8–11 (2015).

15. Aguzzi, A., Barres, B. A. & Bennett, M. L. Microglia: scapegoat, saboteur, or something else? Science 339, 156–61 (2013).

16. Parkhurst, C. N. et al. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell 155, 1596–609 (2013).

17. Paolicelli, R. C. et al. Synaptic pruning by microglia is necessary for normal brain development. Science 333, 1456–8 (2011).

18. Frakes, A. E. et al. Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis. Neuron 81, 1009–23 (2014).

19. Lawrence, T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb. Perspect. Biol. 1, a001651 (2009).

Svedberg 2016

31

20. van Loo, G. et al. Inhibition of transcription factor NF-kappaB in the central nervous system ameliorates autoimmune encephalomyelitis in mice. Nat. Immunol. 7, 954–61 (2006).

21. Strachan, T. in Human Molecular Genetics (Wiley-Liss, 1999). at <http://www.ncbi.nlm.nih.gov/books/NBK7563/>

22. Ebner, B. A. et al. Purkinje cell ataxin-1 modulates climbing fiber synaptic input in developing and adult mouse cerebellum. J. Neurosci. 33, 5806–20 (2013).

23. Hashimoto, K. & Kano, M. Synapse elimination in the developing cerebellum. Cell. Mol. Life Sci. 70, 4667–80 (2013).

24. R Development Core Team. R: A language and environment for statistical computing. (2014). at <http://www.r-project.org/>

25. Elmore, M. R. P. et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380–97 (2014).

26. Fu, A. K. Y. et al. IL-33 ameliorates Alzheimer’s disease-like pathology and cognitive decline. Proc. Natl. Acad. Sci. U. S. A. 1604032113– (2016). doi:10.1073/pnas.1604032113

27. Dell’Orco, J. M. et al. Neuronal Atrophy Early in Degenerative Ataxia Is a Compensatory Mechanism to Regulate Membrane Excitability. J. Neurosci. 35, 11292–307 (2015).