1. 3
Kielian Laboratory: Tammy Kielian, Ph.D. and Juan Xiong
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 68198; tkielian@unmc.edu
INTRODUCTION
Immunohistological studies have demonstrated early
microglial activation that precedes
neurodegeneration in JNCL.
Microglia are the immune cells in the central
nervous system that participate in neurodegeneration
and neuroinflammation.
Our aim is to investigate the role of activated
microglia in JNCL. Are they beneficial or detrimental
during disease development? Can we regulate
microglial activation to delay JNCL progression?
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KEY FINDINGS
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WHAT THIS MEANS
FOR THERAPY
Acknowledgements
We thank members of the Kielian laboratory for their assistance
and helpful discussions and Dr. David Pearce for providing the
CLN3ex7/8 mice. This work is supported by the Batten Disease
Support and Research Administration (BDSRA) and an Edna
Ittner Pediatric Research Grant awarded to T.K.
N
or
m
al
C
N
S
Ne
ur
od
eg
en
er
ati
on
Conduct signals
to and from the
brain
Neurons
Resting Microglia
Astrocytes Maintain brain
homeostasis and
form the
protective blood-
brain barrier
Provide immune
protection
against CNS
infection
Release distress
signals that
induce microglial
and astrocyte
activation
Produce less
trophic factors,
increased
glutamate release
and other
inflammatory
mediators
Produce pro-
inflammatory
mediators, reactive
oxygen species, and
release glutamate
Reactive Astrocytes
Activated Microglia
Damaged Neurons
Background
Interactions between neurons, microglia,
and astrocytes during neurodegeneration
 Activated astrocytes and microglia are observed
in the brains of JNCL patients and CLN3Δex7/8
mice and predict areas where neuron loss will
occur.
 The lipid intermediate ceramide is elevated in
the JNCL brain. Ceramide has been implicated in
Alzheimer’s Disease, multiple sclerosis, and
stroke; therefore, it may be an inflammatory
trigger during JNCL.
 The pro-inflammatory cytokine IL-1β plays a
central role in the regulation of immune
responses. IL-1β expression is elevated in the
brains of JNCL patients and CLN3 knock out
mice.
 The abnormal accumulation of ceramide and
neuronal death may lead to inappropriate
microglial activation, further exacerbating
neuronal loss.
Unstimulated
LPS+ATP
C6ceramide
C6ceramide+neuronal lysate
%Caspase-1+
cells
0
20
40
60
80
100
CLN3WT
CLN3ex7/8
*
A. B.
Unstimulated
LPS+ATP
C6ceramide
C6ceramide+neuronallysate
IL-1expression(ng/ml)
0
100
200
300
400
500
*
Signal 1
TLR
Nucleus
Pro-IL-1β
Pro-IL-1β IL-1β
IL-1β
Ceramide
ROS
ATP
K+ efflux
Neuron
debris
“Danger signal”
ROS
CLN3Δex7/8 microglia

cathepsin B
NLRP3
ASC
Caspase-1
mtDNA
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 Anti-inflammatory drugs that inhibit microglial activation may have
therapeutic potential for delaying disease progression during JNCL.
 Blockade of microglial and astrocyte glutamate release may be an
effective therapeutic strategy to impair neuronal loss during JNCL.
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Figure 1. A combination of ceramide and neuron lysate
leads to abnormally high levels of caspase-1 activation (A)
and IL-1β production (B) in CLN3Δex7/8 microglia (*, p < 0.05).
Figure 2. Multiple pro-inflammatory mediators are
abnormally elevated in CLN3Δex7/8 microglia in response to
the combination of ceramide and neuron lysate (*, p <
0.05; ***, p < 0.001).
Figure 3. Possible mechanism of IL-1β production in JNCL.
Neuronal debris serves as “Signal 1” to induce the expression
of pro-IL-1β. Ceramide provides “Signal 2” for IL-1β cleavage
by the NLRP3 inflammasome. The combination of these two
signals is abnormally exaggerated in CLN3Δex7/8 microglia,
which results in elevated IL-1β levels that can be neurotoxic.
unstimulated
C6ceramide24h
C6ceramide+neuronlysate6h
C6ceramide+neuronlysate24h
Glutamaterelease(mM)
0
1
2
3
4
5
6
7
CLN3WT
CLN3ex7/8 Figure 4. CLN3Δex7/8
microglia tend to
release more
glutamate compared
to WT microglia.
Glutamate
excitotoxicity has been
implicated in neuronal
loss during JNCL.
Figure 5. CLN3Δex7/8
microglia may contribute
to neurotoxicity by
excessive glutamate
release mediated, in part,
via increased IL-1β action.
In response to elevated
glutamate, astrocyte Cx43
hemichannels open ,
causing astrocyte
dysfunction, which likely
further exacerbates
neuron damage.
Does CLN3 Mutation Lead to Aberrant Microglial Activation in Juvenile Batten Disease?
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