Download (Figure 4B) in 12 month old Cln5-/- mice. To survey effects on glial

Neuropathological and reactive changes in aged Cln5 deficient mice
Stine N Hansen1,2, Noreen A Alexander1,2, Carina von Schantz3, Outi Kopra3,
3
1,2
Anu Jalanko , Jonathan D Cooper
1Pediatric
Storage Disorders Laboratory, and 2Department of Neuroscience, Institute of Psychiatry, King's College
London, UK; 3National Public Health Institute, Department of Medical Genetics and Molecular Medicine, Biomedicum
Helsinki, Finland.
INTRODUCTION
The neuronal ceroid lipofuscinoses (NCLs) are a significant cause of progressive
intellectual and neurological deterioration during childhood. Collectively, this
group of up to nine genetically distinct disorders (CLN1-CLN9) is considered
the most common pediatric neurodegenerative encephalopathy. Clinically, the
NCLs are typified by their progressive nature, presenting with visual failure
leading to blindness, neurocognitive impairment, severe seizures and premature
death. Cases usually present during childhood and are distinguished as infantile
(INCL), late infantile (LINCL) and juvenile (JNCL). However, rare adult forms
and several variant forms are also recognized. The pathological hallmark of the
NCLs is the intralysosomal accumulation of autofluorescent storage material.
Finnish variant LINCL (vLINCLFin) is the result of mutations in the Cln5 gene.
Cln5 null mutant mice (Cln5-/-) are a useful model for studying the underlying
pathological mechanisms in this disorder. To gain a detailed series of
pathological landmarks from severely affected Cln5-/- mice, we have
undertaken a stereological analysis of neurodegenerative and reactive changes
in the CNS of 12 month old mutant mice.
REGIONAL VOLUME
Figure 1
Significantly reduced volume of the cortex,
hippocampus, striatum and thalamus in 12 month old Cln5 -/mice vs. age-matched controls (+/+) is revealed by Cavalieri
estimates. * p<0.05 one-way ANOVA
CORTICAL THICKNESS
LAMINAR THICKNESS
Figure 2
Widespread shrinkage of the cortical mantle in 12
month old Cln5-/- mice vs. age-matched controls (+/+). Cortical
subfields surveyed include primary somatosensory barrelfield
(S1BF), primary motor (M1), primary visual (V1) and lateral
entorhinal (Lent) cortex. *** p<0.001, one-way ANOVA.
ASTROCYTOSIS
NEURONAL NUMBER
Figure 3 Laminar-specific changes in cortical thickness in 12 month old Cln5-/- mice compared with age-matched
controls (+/+, A-D). Laminar thickness measurements in primary motor cortex (M1, A), primary somatosensory
barrelfield cortex (S1BF, B), primary visual cortex (V1, C), lateral entorhinal cortex (Lent, D). * p<0.05, ** p<0.01, ***
p<0.001, one-way ANOVA.
METHODS
We have examined the CNS of 12 month old Cln5-/- mice and age-matched control mice (+/+)
on the same mixed strain background. To determine the extent of neuropathological changes
at both regional and cellular levels we examined the expression of different
immunohistochemical markers of neuronal and glial phenotype, using unbiased stereological
methods and thresholding image analysis.
CONCLUSIONS
Figure 4
RESULTS
Aged Cln5-/- mice displayed atrophy of the cortical mantle and hippocampus (Figure 1).
This atrophy also extended to subcortical structures including the thalamus and striatum.
Cortical thickness measurements revealed a widespread and significant atrophy of the
cortical mantle (Figure 2). This thinning of the cortex differed in extent between regions, with
effects that extended to individual laminae (Figure 3). To explore the basis for these laminarspecific events we obtained optical fractionator estimates of neuronal number in the primary
somatosensory barrelfield (S1BF) and the ventral posterior thalamic nucleus (VPM/VPL)
which sends projections to S1BF (Figure 4).
(A) Unbiased optical fractionator estimates of
neuronal number in individual laminae of primary
somatosensory barrelfield cortex (S1BF, A) and the ventral
posterior thalamic nucleus (VPM/VPL, B) in 12 month old Cln5/- mice and age-matched controls (+/+). Note the significant loss
of thalamic relay neurons with no loss of their cortical target
neurons.
Figure 5
Thresholding image analysis confirms the
pronounced up regulation of the astrocyte marker GFAP in the
cortex and thalamus of 12 month old Cln5-/- mice vs. control
littermates (+/+). Areas surveyed include somatosensory
barrelfield cortex (S1BF), and the ventral posterior thalamic
nucleus (VPM/VPL). *** p<0.001, one-way ANOVA.
MICROGLIAL ACTIVATION
STORAGE MATERIAL
+/+ B
A
Cln5 -/-
C
Cln5 -/-
D
Cln5 -/-
Counts of neuronal number revealed no significant loss of neurons in laminae II & III,
lamina IV or lamina V of somatosensory barrelfield cortex (Figure 4A). However, compared
to control mice there was a profound loss of VPM/VPL thalamic neurons (Figure 4B) in 12
month old Cln5-/- mice.
Rho
To survey effects on glial cell populations in 12 month old Cln5-/- mice we used
immunohistochemical staining for markers of astrocytosis (GFAP) and microglial activation
(F4/80). These markers revealed widespread astrocytosis (Figure 5) and a more localized
microglial activation (Figure 6) within individual thalamic nuclei and other subcortical
structures, together with the cortex. Quantitative thresholding image analysis confirmed
these pronounced effects on both astrocyte and microglial cell populations in the
interconnected thalamic relay nucleus VPM/VPL and primary somatosensory cortex (S1BF).
Conventional epifluorescence illumination revealed widespread intracellular accumulation
of autofluorescent storage material within neuronal soma throughout the CNS of aged Cln5
deficient mice (Figure 7).
+/+ F
E
Rho
Figure 6
Thresholding image analysis confirms the
pronounced up-regulation of the microglial marker F4/80 in the
cortex and thalamus of 12 month old Cln5-/- mice vs. control
littermates (+/+). Areas surveyed include somatosensory
barrelfield cortex (S1BF), and the ventral posterior thalamic
nucleus (VPM/VPL). *** p<0.001, one way ANOVA.
Figure 7
FITC
Rho
Cln5 -/-
Rho
G
Cln5 -/-
FITC
Merge
H
12 month old Cln5 deficient mice exhibit a severe neurodegenerative and
reactive phenotype that is more pronounced compared to results from a
study in younger Cln5-/- mice. These data emphasize the progressive
nature of the NCLs. Consistent with a mouse model of JNCL (Cln3 null
mutant), Cln5-/- mice display a profound loss of sensory relay thalamic
neurons, yet no loss of their target neurons in lamina IV of
somatosensory cortex. Our preliminary data suggest that this
vulnerability of thalamic neurons is an early event in pathogenesis. Cln5
deficient mice also exhibit pronounced glial responses within individual
thalamic nuclei, which appear to occur very early in disease progression.
It will be important to determine the relationship between these glial
responses and neuronal loss, and whether this relationship differs
between mouse models. These data provide further evidence for
neurodegenerative and reactive changes in the thalamocortical system in
mouse models of NCL and emphasize the localized nature of these events.
Cln5 -/-
Merge
Widespread intracellular accumulation of storage
material in 12 month old Cln5-/- mice vs. littermate controls (+/+).
The storage material of Cln5 deficient mice fluoresces at both
rhodamine (Rho) and FITC filter sets and appears yellow in
merged images. Representative images are coronal sections
through the primary somatosensory barrelfield cortex (A-D) and
sagittal sections through the cerebellum (E-H).
AKNOWLEDGEMENTS
This work was supported by European Commission 6th Framework Research Grant LSHMCT-2003-503051 (JDC), National Institutes of Health (NINDS) grant NS41930 (JDC), and
grants to JDC from The Natalie Fund, Batten Disease Support and Research Association,
Batten Disease Family Association, Batten Disease Research Trust and the Remy Fund.