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Sex-split analysis of pathology and motor-behavioral outcomes in a mouse model of CLN8-Batten disease reveals an increased disease burden and trajectory in female Cln8mnd mice


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Background CLN8-Batten disease (CLN8 disease) is a rare neurodegenerative disorder characterized phenotypically by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature death. Mutations in CLN8 results in characteristic Batten disease symptoms and brain-wide pathology including accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subforms of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on CLN8 disease burden and progression, we utilized a Cln8 mnd mouse model to measure the impact and progression of histopathological and behavioral outcomes between sexes. Results Several notable sex differences were observed in the presentation of brain pathology, including Cln8 mnd female mice consistently presenting with greater GFAP ⁺ astrocytosis and CD68 ⁺ microgliosis in the somatosensory cortex, ventral posteromedial/ventral posterolateral nuclei of the thalamus, striatum, and hippocampus when compared to Cln8 mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8 mnd female mice experience poorer motor performance and earlier death than their male counterparts. Cln8 mnd mice treated with an AAV9-mediated gene therapy were also examined to assess sex differences on therapeutics outcomes, which revealed no appreciable differences between the sexes when responding to the therapy. Conclusions Taken together, our results provide further evidence of biologic sex as a modifier of Batten disease progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeutic impact.
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Orphanet Journal of Rare Diseases (2022) 17:411
Sex-split analysis ofpathology
andmotor-behavioral outcomes inamouse
model ofCLN8-Batten disease reveals
anincreased disease burden andtrajectory
infemale Cln8mnd mice
Andrew D. Holmes1,2, Katherine A. White1, Melissa A. Pratt1, Tyler B. Johnson1, Shibi Likhite3,
Kathrin Meyer3,4 and Jill M. Weimer1,2*
Background: CLN8-Batten disease (CLN8 disease) is a rare neurodegenerative disorder characterized phenotypi-
cally by progressive deterioration of motor and cognitive abilities, visual symptoms, epileptic seizures, and premature
death. Mutations in CLN8 results in characteristic Batten disease symptoms and brain-wide pathology including
accumulation of lysosomal storage material, gliosis, and neurodegeneration. Recent investigations of other subforms
of Batten disease (CLN1, CLN3, CLN6) have emphasized the influence of biological sex on disease and treatment
outcomes; however, little is known about sex differences in the CLN8 subtype. To determine the impact of sex on
CLN8 disease burden and progression, we utilized a Cln8mnd mouse model to measure the impact and progression of
histopathological and behavioral outcomes between sexes.
Results: Several notable sex differences were observed in the presentation of brain pathology, including Cln8mnd
female mice consistently presenting with greater GFAP+ astrocytosis and CD68+ microgliosis in the somatosensory
cortex, ventral posteromedial/ventral posterolateral nuclei of the thalamus, striatum, and hippocampus when com-
pared to Cln8mnd male mice. Furthermore, sex differences in motor-behavioral assessments revealed Cln8mnd female
mice experience poorer motor performance and earlier death than their male counterparts. Cln8mnd mice treated
with an AAV9-mediated gene therapy were also examined to assess sex differences on therapeutics outcomes, which
revealed no appreciable differences between the sexes when responding to the therapy.
Conclusions: Taken together, our results provide further evidence of biologic sex as a modifier of Batten disease
progression and outcome, thus warranting consideration when conducting investigations and monitoring therapeu-
tic impact.
Keywords: CLN8, Batten disease, Sex differences, Lysosomal storage disorders, Disease progression, AAV9 gene
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Neuronal ceroid lipofuscinoses (NCLs) are a family of
inherited lysosomal diseases that result in neurodegen-
erative disease within pediatric and adult populations.
Open Access
1 Pediatrics and Rare Diseases Group, Sanford Research, 2301 E 60Th St N,
Sioux Falls, SD, USA
Full list of author information is available at the end of the article
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Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
Commonly known as Batten disease, NCLs have an
extensive range of phenotypic presentation, although
most forms can be clinically characterized by declining
cognitive and motor functions, ocular dysfunction, and
eventual blindness, epilepsy, and a decreased lifespan [1]
(for a recent review see [2]). Although NCLs are consid-
ered rare in nature, together they are the most prevalent
neurodegenerative disease in the pediatric population
with an estimated incidence of 2–4/100,000 births [3, 4]
and an even greater incidence within certain populations.
e etiology of NCLs is due to a mutation in one of at
least 13 currently identified ceroid lipofuscinosis neu-
ronal (CLN) genes—often encoding enzymes or regula-
tory proteins involved in proper lysosomal function [5,
6]. One of these genes, CLN8, encodes a transmembrane
endoplasmic reticulum (ER) protein (CLN8) that has
been shown to be involved in the trafficking of lysosomal-
destined enzymes between the ER and Golgi, in addition
to integral involvement with other lysosomal processes
such as biogenesis [6, 7]. Additionally, studies have dem-
onstrated neuronal-specific roles of CLN8 in neurite
maturation, differentiation, and support of various neu-
ronal populations [79]. Mutations in CLN8 results in
characteristic NCL symptoms and brain-wide pathology
including accumulation of lysosomal storage material,
gliosis, and other neurodegenerative signs [6, 10].
CLN8 Batten disease (CLN8 disease) is a variant late-
infantile form of Batten disease with an onset of symp-
toms generally between 5 and 10years old [11]. Patients
with CLN8 disease present with progressive deteriora-
tion of motor and cognitive abilities, visual symptoms,
and epileptic seizures [6]. Two classic variants arising
from mutations of CLN8 have been well described: (1)
“Northern Epilepsy” is a condition characterized by epi-
leptic seizures (tonic–clonic and/or complex partial) with
peak frequency in adolescence followed by declining cog-
nition and deteriorating motor skills due to cerebellar
atrophy [12, 13]. Hirvasniemi etal. first identified North-
ern Epilepsy within patients of Northern Finland where
patients all shared a homozygous missense mutation of
CLN8 [13], but this subtype has also been described to
result from other mutations in other populations [14,
15]; (2) Variant Late-infantile NCL (vLINCL) is a more
severe phenotype associated with CLN8 mutation first
identified in Turkish families. is variant typically pre-
sents as epileptic seizures, motor and cognitive deterio-
ration, and visual disturbances (which help distinguish it
from Northern Epilepsy clinically). Furthermore, patients
with vLINCL experience more severe disease progres-
sion with motor and cognitive deterioration occurring
within several years, as compared to Northern Epilepsy
which progresses over several decades [16]. Despite these
two well-described phenotypes of CLN8 disease within
distinct populations, cases have been described in a mul-
titude of geographic locations throughout the world with
variability in disease progression [14, 15, 1721]. As such,
clinical presentation of CLN8 disease may not always fall
into a discrete category and suspicion of the disorder
warrants further genetic and diagnostic testing [16].
Recently, greater emphasis has been placed on under-
standing and identifying sex distinctions as an impor-
tant modulator of physiology, anatomy, and pathology
in disease, including within various forms of Batten dis-
ease [2225]. A multitude of neurodegenerative diseases
demonstrate sex biases, such as greater prevalence of
Alzheimer’s disease in women and increased prevalence
of Parkinson’s disease and amyotrophic lateral sclero-
sis in men [26]. e field of Batten disease is no differ-
ent: NCLs have been shown to demonstrate sex-based
clinic and pathologic differences in patients and in ani-
mal models. Although male subjects typically experience
earlier disease onset, females with juvenile NCL (JNCL;
CLN3 Disease) suffer a more rapid disease progression
characterized by quicker cognitive decline, loss of motor
coordination, and earlier death [27, 28]. Further, Cialone
etal. [28] described female patients as having a poorer
quality of life due to greater physical impairment. Over-
all, identifying sex differences (or lack thereof) in humans
with Batten disease is exceedingly difficult due to various
mutations within the range of CLN genes and complex
interactions between their respective unique genetics
and environment.
e utilization of murine models in Batten disease
research has greatly expanded the ability to investi-
gate sex differences in this family of diseases, in addi-
tion to highlighting the importance of sex as a factor to
be considered when designing and analyzing therapeu-
tic trials [29]. For instance, sex-dependent differences
in gene expression response to galactosylceramide were
found in the Cln3Δex7/8 murine model [30]. Further, Pop-
pens etal. described female Cln6nclf mice to experience
accelerated disease progression, more severe behavioral
issues and motor decline, and differences in histopatho-
logical effects [31]. A prior investigation of Cln8mnd mice
revealed sex differences in retinal vulnerability where
female retinas exhibited higher oxidation rates and cas-
pase-3 mediated apoptosis, in addition to a more severe
histopathological profile of the retina [32]. However,
the disease associated phenotypes in relationship to sex
examined in this study were limited to visual deficits in
the Cln8mnd mouse model. To add to this body of work,
we examined the influence of sex on psychomotor behav-
ioral outcomes and histopathology within thalamus and
primary somatosensory cortex of Cln8mnd mice. Addi-
tionally, Cln8mnd sexual dimorphisms in AAV9 gene ther-
apy response were also explored.
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Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
Cln8mnd mice have sex dependent dierences instorage
material accumulation
e Cln8mnd mouse model is a widely used and well-
characterized mouse model of CLN8 disease, in which
mice show disease associated histopathologic changes in
the brain as early as 2months of age, behavioral deficits
by 6months of age, and premature death by 10months
of age [6, 33]. Here, Cln8mnd mice at varying ages were
analyzed to determine whether sex differences existed in
classic Batten disease pathologies within somatosensory
thalamic nuclei (VPM/VPL) and the somatosensory cor-
tex (S1BF), as well as lesser studied regions such as the
striatum and CA3 of the hippocampus.
Autofluorescent storage material (ASM) accumulation
is a pathological characteristic of all Batten disease vari-
ants, and Cln8mnd mice had greater accumulation of ASM
compared to wild type mice within both the VPM/VPL
and S1BF at most time points studied (Fig.1A, B). While
there were generally no differences in ASM accumula-
tion between the sexes in the VPM/VPL, Cln8mnd males
showed greater ASM accumulation than female counter-
parts at 8months of age (Fig.1A). Importantly, Cln8mnd
males showed earlier and more severe ASM accumula-
tion in the S1BF than Cln8mnd females, with ASM accu-
mulation beginning at 2 months of age and showing a
larger burden at 4months of age (Fig. 1B). is male-
specific difference disappeared at later time points, which
may indicate that males have sooner pathological onset
of ASM accumulation while females have a quicker pro-
gression of accumulation after 4months of age.
Mitochondrial ATP synthase subunit c (SubC) is
one of the known constituents of the storage mate-
rial accumulated in various forms of Batten disease
[34, 35]. Cln8mnd mice had greater amounts of SubC
Fig. 1 Sex differences evident in Cln8mnd accumulation of autofluorescent storage material and ATP Synthase subunit C. Cln8mnd males demonstrate
greater ASM accumulation within the VPM/VPL at 8 months (A) and within the S1BF at 4 months of age (B). Cln8mnd females show enhanced
SubC accumulation at 8 months within the VPM/VPL (C) while no sex differences were detected in the S1BF (D). Two-way ANOVA with Fisher’s
LSD post-hoc. Mean ± SEM, n = 2–4 animals/sex/group, detailed n described in Additional file 4: Table S1. *p < 0.05, **p < 0.01, ***p < 0.001,
****p < 0.0001. ASM Scale Bar: 200 µm; SubC Scale Bar: 150 µm
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Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
accumulation relative to wild type mice at 8months
of age within both anatomic locations (Fig. 1C-D).
While there were no differences in SubC accumula-
tion between male and female Cln8mnd mice for most
time points, Cln8mnd females had greater accumula-
tion of SubC within the thalamic nuclei and striatum
at 8months of age relative to males (Fig.1C, Addi-
tional file1: Fig. S1A). Although some differences were
observed at end-stage disease, ASM and SubC accu-
mulation show little sex-dependent differences over
the course of the disease.
Cln8mnd mice have female specic increases in astrocyte
and microglial reactivity
Glial fibrillary acidic protein (GFAP) is an intermediate
filament commonly associated with reactive astrocytes of
the central nervous system (CNS) and it can be utilized
to indicate non-specific pathological reactions [36, 37].
Cln8mnd mice displayed increased evidence of GFAP+
astrocytosis compared to wild type mice at most time
points in the VPM/VPL and S1BF (Fig.2A, B). Interest-
ingly, Cln8mnd males had increased astrocytosis within
the VPM/VPL at 6months of age (Fig.2A), yet Cln8mnd
females had greater evidence of astrocytosis within the
somatosensory cortex at 4 and 8months of age, and in
the striatum at 8months of age (Fig.2B, Additional file1:
Fig. S1B). Astrocytic activation progresses with time and
differs by sex and brain region.
Fig. 2 Female Cln8mnd mice show enhanced glial activation in brain. Male Cln8mnd mice demonstrate greater astrocyte expression (GFAP+) within
the VPM/VPL of the thalamus at 6 months of age (A) whereas female Cln8mnd mice have greater expression within the S1BF at 4 and 8 months of
age (B). Female Cln8mnd mice exhibit enhanced microglial activation (CD68+) at months 4, 6, and 8 within the VPM/VPL (C) and at month 4 within
the S1BF (D). Two-way ANOVA with Fisher’s LSD post-hoc. Mean ± SEM, n = 1–4 animals/sex/group, detailed n described in Additional file 4:
Table S1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale Bars: 150 µm
Page 5 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
Cluster of differentiation protein 68 (CD68) is a cell
surface marker for microglial activation often used in
mouse models of neurodegenerative disease [6, 31]. Akin
to the astrocyte response, Cln8mnd mice had enhanced
microglial activation relative to wild type within both
anatomic sites beginning at 4months of age (Fig.2C, D).
Overall, within both the VPM/VPL and S1BF, there was a
marked increase in reactive astrocytosis and microglio-
sis from 2 to 4months of age. When analyzing by sex,
Cln8mnd females displayed substantially greater evidence
of CD68+ microgliosis at 4, 6, and 8months of age within
the VPM/VPL (Fig.2C) and at 4months of age within
the S1BF (Fig. 2D). Additionally, female Cln8mnd mice
showed exacerbated microgliosis in the striatum and
CA3 of the hippocampus, prior to when their male coun-
terparts present with a phenotype in these regions (Addi-
tional file 1: Fig. S1C). Taken together, female Cln8mnd
mice show a consistent upregulation of astrocyte and
microglia reactivity in several regions of the brain that is
more severe than their male counterparts.
Lastly, as these are models of a neurodegenerative dis-
ease, thinning of the cortical plate was measured at two
time points to determine if cell death occurred in a sex
specific manner. From this broad experiment, Cln8mnd
mice showed no cortical thinning at 2 or 6months of age,
regardless of sex (Additional file2: Fig. S2).
Cln8mnd mice have sex dependent dierences in life span
and motor‑behavioral assessments
To determine if there were sex differences in Cln8mnd
survival and motor-behavioral performance, animals
were examined at 2, 4, 6, 8, and 10months of age for
behavioral outcomes and through 24months of age for
survival assessment. As a whole, Cln8mnd mice perished
earlier than their respective wild type counterparts, with
a median survival of 10months of age (Fig.3A). Impor-
tantly, Cln8mnd females perished significantly earlier than
their Cln8mnd male counterparts, living approximately
0.5months less compared to Cln8mnd males (Fig.3A).
Mice were examined in a Morris Water Maze (MWM)
in which they were trained to find a hidden platform
in a pool of water to assess vision, memory, and spatial
learning. Cln8mnd mice took significantly longer to com-
plete the task compared to wild type mice, with Cln8mnd
females showing poor performance at 6 and 8 months
of age and Cln8mnd males showing poor performance at
8months of age (Fig.3B). Cln8mnd female mice performed
worse at an earlier stage than their male comparisons
during MWM assessments. Specifically, Cln8mnd males
completed the MWM in a significantly shorter time com-
pared to Cln8mnd females at 2 and 6months. Accounting
for swim speed did not impact these results, indicating
that Cln8mnd females have greater MWM deficiencies
than males of the same age (Fig.3C). At 8months of age,
Cln8mnd females and Cln8mnd males had no observed dif-
ference. A reverse MWM assessment, where the hidden
platform was moved to a novel location, was conducted
when the mice were at 6months of age, which demon-
strated that Cln8mnd females took significantly more
time to complete the assessment than their Cln8mnd male
counterparts despite similar swim speed (Fig.3D, E).
Animals were also measured for general locomotor
ability and tremor presence using a force plate actim-
eter. Cln8mnd males began losing weight at 6months of
age while their female counterparts generally did not,
though all Cln8mnd animals were within healthy weight
ranges for their sex (Fig.4A). While there were some dif-
ferences between genotypes and sexes in general activ-
ity (Fig.4B–D; distance travelled, bouts of low mobility,
and area covered), Cln8mnd males consistently exhibited
a greater number of focused stereotypies (i.e., rearing)
as compared to Cln8mnd females at 2, 4, 6, and 8months
of age (Fig.4E). e same pattern was seen in wild type
mice from 4, 6, 8, and 10months of age, indicating this is
likely related to male behavior as a whole. When assess-
ing tremor presence, Cln8mnd females showed increased
tremor scores significantly earlier than their male coun-
terparts at several frequencies, displaying increased
tremors as early as 4 months of age while Cln8mnd
males showed tremors beginning at 8–10months of age
(Fig. 4FI). Several other motor-behavioral tests were
conducted, including an accelerating rotarod and vertical
pole climb, and no sex dependent differences in Cln8mnd
mice were observed (Additional file 3: Fig. S3). Taken
together, Cln8mnd females consistently show a signifi-
cantly faster and more severe disease progression than
their male counterparts, including an earlier presence
of tremors, earlier MWM deficits that are indicative of
memory, learning, or visual deficits, and an earlier death.
AAV9 gene therapy ameliorates disease pathogenesis
andoverall sex discrepancies
We recently published an investigation of a virally-deliv-
ered gene therapy vector (scAAV9.pT-MecP2.CLN8;
AAV9-CLN8’) in Cln8mnd mice that demonstrated this
therapeutic agent can improve lifespan and treat patho-
logical and behavioral abnormalities in Cln8mnd mice
when delivered at postnatal day 1 via intracerebroven-
tricular injection at 5.0 × 1010 vg/animal [6]. However,
comparisons between sexes in response to therapy were
not previously examined. erefore, to determine if sex
had an impact on AAV9-CLN8 treatment response,
immunohistochemistry and behavioral data was exam-
ined across sexes in AAV9-CLN8 treated animals from 2
to 24months of age.
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Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
Fig. 3 Sex dependent differences in Cln8mnd life span and motor-behavioral assays. Cln8mnd females have a decreased life span compared to
Cln8mnd males, with a median age of 9.5 months and 10 months respectively (A). Morris Water Maze (MWM) demonstrating Cln8mnd females taking
significantly longer to complete the task at 2 and 6 months of age when compared to Cln8mnd males (B), of which was not accounted for by
differing swim speed (C). Cln8mnd females completed the reverse MWM in greater time compared to their male counterparts despite no difference
in swim speeds (D, E). Comparisons of wild type males versus wild type females^, Cln8mnd males versus Cln8mnd females*, Cln8mnd males versus
wild type males#, and Cln8mnd females versus wild type females#. Survival curve: log-rank (Mantel–Cox); n = 13–16 animals/sex. MWM: Two-way
ANOVA with Fisher’s LSD post-hoc. Mean ± SEM, n = 2–11 animals/sex/group, detailed n described in Additional file 4: Table S1. *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001
Fig. 4 Sex differences in force plate actimeter results. Cln8mnd males weighed significantly more than Cln8mnd females at 2, 4, 6, and 8 months of
age and started losing weight at 6 months of age (A). No consistent differences were observed in distance travelled (B), bouts of low mobility (C), or
area covered (D). Cln8mnd males exhibited greater frequency of focused stereotypy at 2, 4, 6, and 8 months of age (E). Comparisons of tremor scores
revealed that Cln8mnd females had higher tremor scores than their male counterparts at frequencies 15–20 Hz and 20–25 Hz (FI). Comparisons
of wild type males versus wild type females^, Cln8mnd males versus Cln8mnd females*, Cln8mnd males versus wild type males#, and Cln8mnd females
versus wild type females#. Two-way ANOVA with Fisher’s LSD post-hoc. Mean ± SEM, n = 1–11 animals/sex/group, detailed n described in Additional
file 4: Table S1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
(See figure on next page.)
Page 7 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
Fig. 4 (See legend on previous page.)
Page 8 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
We previously described a robust reduction of ASM
and SubC accumulation in Cln8mnd animals treated with
AAV9-CLN8 [6]. is reduction was most pronounced
through 8months of age, with slight increases in accu-
mulation seen in treated animals from 10 to 24months
of age, though this accumulation did not reach the same
burden as end-stage untreated Cln8mnd mice. When split
by sex, there were overall no differences to AAV9-CLN8
response to ASM prevention between the sexes (Fig.5A,
B). Treated Cln8mnd males had increased SubC at 8, 10,
and 24months of age, although the general response was
similar between the sexes (Fig.5C, D).
In terms of glial reactivity, we previously reported sig-
nificant attenuation of GFAP+ astrocytosis and CD68+
microgliosis in AAV9-CLN8 treated Cln8mnd animals
through 8 months of age [6]. From 10 to 24 months
of age, however, both astrocytosis and microgliosis
increased in AAV9-CLN8 treated animals, indicating a
heightened and sustained inflammatory response. When
analyzing the data by sex, there were no consistent differ-
ences in gliosis between the sexes of AAV9-CLN8 treated
animals (Fig.5E–H), indicating these heightened inflam-
matory responses are not sex-specific.
Lastly, we previously reported that AAV9-CLN8 treat-
ment largely prevented behavioral deficits in Cln8mnd ani-
mals, including preservation of motor abilities through
24months of age (as measured by an accelerating rotarod
and vertical pole climb), prevention of tremors through
12–18months of age, and retention of a full lifespan of
24months [6]. When examining these outcomes by sex,
there were generally no differences between AAV9-CLN8
treatment response in lifespan, rotarod performance,
performance in a vertical pole test, or tremor presence
(Fig.6AI). A consistent difference was detected in the
number of falls from the vertical pole test, where male
AAV9-treated animals showed a slight but significant
increase in falls when compared to female counterparts,
though this resolved over time and was less than the
Fig. 5 Sex-dependent histopathological differences in AAV9-treated Cln8mnd mice. No sex-dependent histopathological differences were observed
when comparing ASM accumulation in AAV9-treated Cln8mnd males and females in the VPM/VPL (A). AAV9-treated Cln8mnd females exhibit greater
ASM accumulation within the S1BF at 4 months of age (B). AAV9-treated Cln8mnd males evidenced enhanced SubC burden at 8 and 10 months of
age within the VPM/VPL (C) and at 24 months of age within the S1BF (D). Greater astrocytosis (GFAP+) was observed in AAV9-treated Cln8mnd males
at 10 months of age within the VPM/VPL (E), but not within the S1BF (F). No sex-dependent differences observed in microgliosis (CD68+) in the
VPM/VPL (G) or S1BF (H). Two-way ANOVA with Fisher’s LSD post-hoc. Mean ± SEM, n = 1–4 animals/sex/group, detailed n described in Additional
file 4: Table S1. *p < 0.05, **p < 0.01
Page 9 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
fall frequency of untreated Cln8mnd mice (Fig.6E; Addi-
tional file1: Fig. S1D). In the MWM, where we previ-
ously reported that AAV9-treated animals performed
poorly at the task beginning at 6months of age, sex-split
analysis interestingly showed that AAV9-treated Cln8mnd
females were significantly longer to complete the task
than their male counterparts at 6 and 8months of age,
(Fig.6J, K). Surprisingly, this difference was not reflected
in the reversal test and was not explained by swim speed
(Fig.6K–M). Taken together, there were scant differences
in male and female response to AAV9-CLN8 gene ther-
apy in pathology, behavior, and survival outcomes, and
while AAV9-treated Cln8mnd females experienced poorer
MWM performance than their male counterparts early
in disease course, it is unclear if this is due to an altered
response to treatment or simply due to the trajectory of
disease in a typical female animal.
is study demonstrates sex differences in the progres-
sion of CLN8 disease in the Cln8mnd murine model.
Specifically, female Cln8mnd mice performed worse on
the MWM assessment, perished earlier, and showed
increased astrocyte and microglia reactivity over their
Cln8mnd male counterparts at several time points. Our
reported results of ASM and SubC accumulation com-
parisons between Cln8mnd male and female mice dem-
onstrated contrasting data in that storage accumulation
was more pronounced at different time stages of patho-
genesis. Generally, Cln8mnd male mice had greater ASM
accumulation within the VPM/VPL and S1BF whereas
Cln8mnd female mice had greater SubC burden within
both areas and the striatum. Accumulation is thought
to occur due to any disruption in the basic processes of
autophagy, lysosomal function, or oxidative damage;
however, other mechanisms of accumulation may exist
[38]. e primary storage material of ASM within CLN8-
Batten disease is SubC, although, other disease subtypes
may have a differing primary constituent like sphin-
golipid activator proteins in CLN1 and CLN10-Batten
disease [38, 39]. Other ASM accumulation components
include neutral lipids, phospholipids, dolichol pyrophos-
phate linked oligosaccharides, lipid linked oligosaccha-
rides, dolichol esters, and metal ions [38, 40, 41]. Based
on our data suggesting Cln8mnd female mice having a
greater SubC component of ASM, it is thus presumed
their male comparisons are accumulating other molecu-
lar components from an unknown mechanism.
Interestingly, there was a marked increase in glial activ-
ity between 2 and 4months of age, indicating this may
Fig. 6 Analysis of sex-dependent differences in life span and motor-behavioral outcomes of AAV9-treated Cln8mnd mice. AAV9-treated Cln8mnd mice
live similar lifespans regardless of sex (A). AAV9-treated Cln8mnd mice perform similarly in an accelerated rotarod test (B). Transient sex-dependent
differences detected at 8 and 18 months of age in the pole climb measurement “time to descend pole” (C), while no differences were detected in
“time to turn down pole” (D), and differences detected at 10 and 12 months of age in “number of falls from the pole” (E). No differences detected
in tremor presence between the sexes of AAV9-treated Cln8mnd mice (F-I) Cln8mnd females treated with AAV9-CLN8 were significantly slower at
the Morris water maze (MWM) at 6 and 8 months of age (J), which was not accounted for by swim speed (K). No differences were observed in
completion time of the reverse MWM (L) but AAV9-treated Cln8mnd females had a higher swim velocity (M). Survival curve: log-rank (Mantel–Cox);
n = 15–16 animals/sex. Two-way ANOVA with Fisher’s LSD post-hoc. Mean ± SEM, n = 5–11 animals/sex/group, detailed n described in Additional
file 4: Table S1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Page 10 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
be the critical time point in which pathological change
from both these processes occurs. It is possible that
this increase in gliosis may be a contributing factor to
the poorer MWM performance and decreased lifespan
seen within Cln8mnd females. Previous investigations of
neural injuries in mice offer support for an association
between enhanced gliotic activity and poorer motor-
behavioral outcomes in assessments like the MWM
[4244]. However, it is worth noting that prior investiga-
tion of sex differences of a CLN6 disease mouse model
revealed Cln6nclf males experience greater microgliosis
than Cln6nclf females at 6months of age within the S1BF
despite Cln6nclf females perishing earlier and exhibiting
poorer motor-behavioral outcomes [31]. ese differ-
ences in pathological variations, such as increases in male
ASM versus female SubC and increases in female gliosis
in one NCL model versus male gliosis in another, high-
light the complexity of interpreting pathological changes
and their relation to disease progression and treatment
outcomes, and specifically suggest that a more holistic
approach may be required for this purpose. Unfortu-
nately, these sex-dependent murine model differences
cannot be correlated with clinical outcomes in humans
with CLN8 disease since there have been no such detailed
human investigations, likely due to small patient popula-
tions and the difficulty in comparing human subjects due
to environmental differences and genetic heterogeneity
of CLN8 mutations [45].
Greater pathological visual deficits and/or dysfunc-
tion are another possible explanation for worse MWM
performance by Cln8mnd females. Cln8mnd females previ-
ously demonstrated harsher retinal histopathologic pro-
files and retina cell apoptosis compared to Cln8mnd male
comparisons [32], and we hypothesize these differences
and increased activated glia may contribute to Cln8mnd
females’ greater visual aberrations and poorer perfor-
mance [46, 47]. Prior investigations have highlighted glial
dysfunction in NCL murine models coinciding with sub-
sequent neuronal damage of the visual cortex and retina,
resulting in deterioration of visual perception and retinal
function [4850]. Moreover, attenuation of inflammatory
microglia via therapeutic agents in Batten disease animal
models improved visual acuity, reduced retinal thinning,
and improved retinal ganglion cell survival [49, 5153].
Sex comparisons of microglia contribution to pathology
and response to therapy in vision related systems may
better elucidate this process.
An increasing body of evidence indicates that aber-
rant glial cell function contributes to the disruption
of CNS homeostasis and resulting neurodegeneration
in Batten disease [54, 55]. Broadly, activation of astro-
cytes and microglia predicts subsequent neuron
degeneration within the local area in various Batten
disease models, and in the Cln8mnd mouse model spe-
cifically, enhanced gliosis coincides with further dis-
ease progression [10, 56]. More recently, investigation
of invitro glial cultures derived from CLN1 and CLN3
murine models demonstrates the negative influence of
glia on neuron survival through differing phenotypic
functional states [5759]. Ppt1/ microglia cultures
were shown to exist in a basally activated state with
increased secretion of cytokines and chemokines that
induce neuron death, and similarly, cultured Cln3Δex7/8
microglia behave in a reactionary state where stimuli
elicit a caspase-1 mediated pro-inflammatory response
that includes cytokine/chemokine production, gluta-
mate release, and hemichannel activity that induces cell
death [57, 59]. Furthermore, depletion of microglia via
pharmacologic targeting can improve CLN1 disease in
mice, and interestingly, Berve etal. observed surprising
sex and anatomical region biases: greater preservation
of Ppt1/ female microglia was observed as they were
less responsive to pharmacologic treatment, especially
within the S1BF, and females experienced subsequently
poorer treatment outcomes compared to their male
counterparts [51].
Nonetheless, the question remains why Cln8mnd
females exhibit enhanced microglial activation within
the S1BF and VPM/VPL nuclei of the thalamus. Within
murine brains, sexual dimorphism has been noted in
microglia function, morphology, and colonization of
brain structures–stemming from variance in sex-specific
gene expression, circulating sex steroidal hormones and
response to hormones, and epigenetic interactions [60
63]. Female-derived mouse microglia tend to be more
reactive and inflammatory than male-derived micro-
glia, characterized by higher inflammatory cytokines,
inflammatory-related receptor expression, and differ-
ential expression of estrogen receptor subtypes [61].
Comparison of microglial number within the amygdala,
hippocampus, and parietal cortex revealed that male
mice had more microglia in the initial post-natal period,
coinciding with their testosterone surge, until the tran-
sition into adolescence when females exhibited greater
microglia with an activated phenotype in the same
regions [60]. e sex differences in microglial coloniza-
tion may be influenced by disparate levels of sex hor-
mones and chemokines, as evidenced by a 200-fold influx
of CCL20 and 50 fold increase of CCL4 during the tes-
tosterone surge in early male mouse development [60, 64,
65]. erefore, sex-dependent chemokine expression in
Cln8mnd mice is a possible explanation for the relatively
increased microgliosis observed in Cln8mnd females at
later life stages, and should be investigated further.
Sexual dimorphism in genetic architecture and X-chro-
mosome gene regulation may promote the chronic
Page 11 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
inflammatory process in Batten disease, and thus may
partially explain the exacerbated phenotype observed
within females [6669]. e X-chromosome is the locus
of numerous genes related to immune function and regu-
lation and through mechanisms like mosaic X-chromo-
some inactivation and “gene escape” from the inactivated
X-chromosome, may lead to differential and bi-allelic
expression of proinflammatory genes respectively [67,
68, 70, 71]. An estimated 3–7% and 15–23% of genes on
the inactivated X-chromosome escape in female mice
and humans respectively [70, 72, 73]. For example, clus-
ter of differentiation (CD) 40 and 99 ligand are expressed
on the X-chromosome and increased CD receptor-CD
ligand engagement activates proinflammatory cascades
involving T and B cells, monocyte derivatives like mac-
rophages and microglia, and cytokine upregulation which
is implicated in a multitude of neurologic disease [74, 75].
To our knowledge, no such studies have investigated the
degree to which X-chromosome inactivation escape may
influence the poorer histopathologic and motor-behav-
ioral outcomes observed within female sex in Batten dis-
ease. Elucidation of the likely mechanism(s) by which this
process occurs may provide insight for potential thera-
peutic targets to alleviate disease burden.
We also reported that AAV9 gene therapy was well
received and generally efficacious to the same degree in
Cln8mnd mice regardless of sex, with one exception where
AAV9-treated female mice performed worse on MWM
assessments than their male counterparts, which as dis-
cussed may be due to the relatively worse retinal damage
experienced by Cln8mnd females [32]. ere have been
few publications on sexual dimorphism in AAV-medi-
ated gene therapy, though reports have indicated dif-
ferences in tissue transduction depending on serotype,
route of administration, tissue type, and the presence of
single or double-stranded genomes, with the most com-
monly affected tissues being the liver, skeletal muscle,
and gonads [7678]. Specifically, one detailed report
described how male-specific increases in liver transduc-
tion were the result of androgen-dependent pathways,
and that modulating these pathways led to improved
transduction in the livers of female mice [79]. While there
is limited data on sex-dependent differences of AAV-gene
therapy in the CNS, one recent study demonstrated sex-
specific responses to intracerebroventricularly delivered
AAV9 in a mouse model of Dravet syndrome, a debilitat-
ing seizure disorder caused by mutations in the α subunit
of NaV1.1 channels (SCN1A) [80]. e authors specu-
lated that these sex-specific differences occurred due to
basal differences in voltage-gated sodium channel pres-
ence in male and female mice, indicating that any sex-
specific differences in response to gene therapies, or lack
of differences, may be due to whether there are sexually
dimorphic functions already present for the protein
product in question.
Taken together, the results from this investigation pro-
vide further evidence of sex-dependent differences in
lifespan, histopathology, and motor-behavioral outcomes
within the Cln8mnd mouse model of Batten disease, and
gives insight into sex-dependent responses to CNS-deliv-
ered AAV9 gene therapy. Although sex discrepancies
have been observed in human subjects with CLN3-Bat-
ten disease, sparse information exists for other forms
of NCLs. As such, based on the surmounting body of
evidence demonstrating the importance of sex as a bio-
logic modifier, prospective and retrospective analysis of
sex differences in other forms of Batten disease should
be conducted to yield a better understanding of disease
pathogenesis and treatment response.
Materials andmethods
e majority of the data presented in this manuscript was
previously published as a mixed-sex cohort in Johnson
etal. [6] where the authors showed AAV9-gene therapy
of CLN8 prevented CLN8 Batten disease characteristics
within Cln8mnd mice. e present manuscript primar-
ily examines that previously collected data as a sex-split
cohort, and adds additional analyses not previously
Ethics statement/animals
Wild type and homozygous Cln8mnd mice on C57BL/6J
backgrounds were used for all studies and were housed
under identical conditions in an AAALAC accredited
facility in accordance with IACUC approval (Protocol #:
178-02-24D Sanford Research, Sioux Falls, SD). Animals
were bred from standing colonies at Sanford Research.
Cln8mnd animals exhibit a single nucleotide insertion
(267–268C, codon 90) predicting a premature termina-
tion codon. Wild type animals lacked this mutation.
Cln8mnd mice were treated with scAAV9.pT-MecP2.
CLN8 via intracerebral ventricular injection (ICV) on
postnatal day 1 as previously described at a dose of
5.0 × 1010 vg/animal [6].
Mice were CO2 euthanized, cardiac perfused with phos-
phate-buffered saline, and the left hemisphere of the
brain fixed in 4% paraformaldehyde. e brain was sec-
tioned on a vibratome into 50μm slices and immuno-
histochemistry was performed on free-floating sections
as previously described using anti-ATP synthase subunit
Page 12 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
C (Abcam, ab181243), anti-GFAP (Dako, Z0334), and
anti-CD68 (AbD Serotec, MCA1957) antibodies [6]. Sec-
ondary antibodies included anti-rat and anti-rabbit bioti-
nylated (Vector Labs, BA-9400). Sections were imaged
and analyzed using an Aperio Digital Pathology Slide
Scanner (VERSA) and associated software. Regions of
interest were extracted in triplicate and subdivided into
4 quadrants for analysis. Immunolabeling was quantified
using ImageJ.
ASM data was collected by methods previously
described [6] with right hemisphere placed on a 1 mm
sagittal brain block. Tissue blocks from 0 to 3mm right
of the midline were flash-frozen, brain sections sliced
on a cryostat at 16μm, and placed on slides. Slides were
briefly post-fixed in 10% NBF and series dehydrated, with
nuclei labeled using DAPI and coverslips applied using
antifade mounting media (Dako Faramount, Agilent
Technologies). Sections were imaged using a Nikon fluo-
rescent microscope and quanitified using ImageJ.
Cortical thickness measurements were obtained in the
motor and somatosensory cortex of coronal tissue sec-
tions labeled with nuclear dye. Measurements were taken
as previously described [31], as triplicates of the cortical
plate encompassing layers 1–6 of the cerebral cortex.
Neurobehavior testing
Animals participated in an accelerating Rotarod proto-
col as previously described to assess motor coordination
(Columbus Instruments, Columbus, OH, USA) [6]. e
machine was set to accelerate 0.3rpm every two seconds,
with a starting speed of 0.3rpm and a maximum speed
of 36rpm. Briefly, mice were trained for nine trials in the
morning (3 sets of three consecutive trials followed by a
30min rest), given a four-hour rest period, and tested in
nine trials in the afternoon (3 sets of three consecutive
trials followed by a 30min rest). e latency to fall from
the rod (time in seconds) was averaged from each of the
nine afternoon testing sessions to produce one value per
Pole climb
e pole climb descent test was performed as previously
described [6]. Mice were placed downward on a metal
pole for 5 trials and given 60s to descend the pole each
trial. Mice were then placed upward on a metal pole for
4 trials and given 60s to turn downward on the pole for
each trial. Lastly, the number of falls made by each mouse
during the 9 trials was recorded.
Water maze
Mice were tested in a 4 foot diameter Morris Water
Maze apparatus as previously described [6]. Briefly,
the apparatus was filled with water to ~ 26 inches, the
goal platform submerged by 0.5 cm at 315°, and the
tub aligned with four distinct visual cues at 0, 90, 180,
and 270° to aid in spatial memory. After mice were
trained in a clear pool with a flagged platform, mice
were trained to find a hidden platform in opaque water
over four trials in the morning (60 s consecutive tri-
als). Mice were then given a three-hour rest period
and tested over four trials in the afternoon (60s con-
secutive trials). Mice were tested for four consecutive
days, each day starting at a different visual cue. Mice
were recorded using Any-maze video tracking software
(Stoelting Co., Wood Dale, IL, USA), and test duration
and swim speed were averaged from the sixteen after-
noon trials performed by each mouse.
Clasping, ledge, andgait tests
Tests were performed as previously described [6]. For
hind limb clasping measurements, animals were scored
on the extent to which their limbs clasped into their
abdomen when held by the base of their tail (score 0–3).
For ledge lowering measurements, animals were scored
on their ability to climb down from the edge of their
home cage (score 0–3). For gait measurements, animals
were scored on their overall ease of walking, including
whether their abdomen dragged on the ground and if
their limbs were splayed out while walking (score 0–3).
e scores were examined as individual tests and col-
lectively as a score from 0 to 9. e same blinded experi-
menter determined all scores.
Force plate
A force plate actimeter was used to measure locomotion
and tremors as previously described [6]. Animals were
recorded in a sound-proof chamber for 20min and data
was processed using FPA Analysis Software (BASi, West
Lafayette, IN) (Additional file3: Fig. S3).
Statistical analysis
Statistical analyses were performed using GraphPad
Prism (v9.0.2 or equivalent) and details are noted in
the figure legends. In general, a two-way ANOVA was
employed with Fisher’s LSD, and outliers were removed
with the ROUT method, Q = 0.1%. If appropriate, an
unpaired t-test was used. For the survival curve analy-
sis, the log-rank (Mantel–Cox) test was used. *p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001. Detaile d sample n’s
are described in Additional file4 TableS1.
Page 13 of 15
Holmesetal. Orphanet Journal of Rare Diseases (2022) 17:411
e data utilized within this study was previously
published by Johnson etal. as a combined sex dataset
only [6], and the current study expands on this data by
doing an in-depth sex split analysis.
NCL(s): Neuronal ceroid lipofuscinoses; vLINCL: Variant late-infantile neuronal
ceroid lipofuscinoses; JNCL: Juvenile neuronal ceroid lipofuscinoses; CLN (1,
2, 3, 6, 8, 10): Ceroid lipofuscinosis neuronal genes, designated as CLN1, CLN2,
CLN3, CLN6, CLN8, CLN10, etc.; Cln8mnd: Mouse model of CLN8 disease; Cln6nclf:
Mouse model of CLN6 disease; Cln3Δex7/8: Mouse model of CLN3 disease; ER:
Endoplasmic reticulum; CNS: Central nervous system; VPM/VPL: Ventral pos-
teromedial/ventral posterolateral nuclei of the thalamus; S1BF: Somatosensory
cortex, barrel field; CA3: Cornu ammonis 3, region of the hippocampus; ASM:
Autofluorescent storage material; SubC: Mitochondrial atp synthase subunit c;
GFAP: Glial fibrillary acidic protein; CD68, CD40, CD99: Cluster of differentiation
protein 68; 40; 99; CCL4, CCL20: Chemokine ligand 4; 20; SCN1A: Sodium chan-
nel protein type 1 subunit alpha; CO2: Carbon dioxide; NBF: Neutral buffered
formalin; PBS: Phosphate buffered saline; DAPI: 4,6-Diamidino-2-phenylindole;
AAV9: Adeno associated virus, serotype 9; scAAV9.pT-MecP2.CLN8 (AAV9-
CLN8): Self-complimentary adeno associated virus serotype 9, targeting
CLN8 with a truncated methyl-CpG-binding protein promoter. Designated
as AAV9-CLN8; ICV: Intracerebroventricular; Vg: Viral genomes; MWM: Morris
water maze; RPM: Rotations per minute; FPA: Force plate actimeter; ANOVA:
Analysis of variance; LSD: Least significant difference; ROUT: Robust regression
and outlier removal; SEM: Standard error of the mean; Hz: Hertz; AAALAC:
Association for Assessment and Accreditation of Laboratory Animal Care;
IACUC : Institutional Animal Care and Use Committee; NIH: National Institutes
of Health.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13023- 022- 02564-7.
Additional le1. Figure S1: Female Cln8mnd mice show enhanced subu-
nit c accumulation and glial activation in the striatum and hippocampus.
Cln8mnd females show enhanced SubC accumulation at 8 months within
the striatum, while no sex differences are detected in the CA3 region of
the hippocampus (A). Female Cln8mnd mice have greater GFAP+ astro-
cyte expression within the striatum at 8 months of age, but not in the CA3
(B). Female Cln8mnd mice exhibit enhanced microglial activation (CD68+)
at 8 months of age within the striatum and CA3 of the hippocampus (C).
Two-way ANOVA with Fisher’s LSD post-hoc. Mean ± SEM, n=2-3 animals/
sex/group, detailed n described in Additional file 4: Table S1. *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001. Scale Bars: 150 µm.
Additional le2. Figure S2: Cln8mnd mice show no thinning of the
cerebral cortex at 2 and 6 months of age.
Additional le3. Figure S3: Comparison of wild type and Cln8mnd mice
on rotarod and pole climb assessments. Cln8mnd animals perform poorly
in an accelerating rotarod test by 6 months of age, with Cln8mnd animals
performing similarly regardless of sex (A). Cln8mnd animals perform
poorly in pole climb assessment by 8 months of age regardless of sex
(B-D). Comparisons of wild type males vs. wild type females^, Cln8mnd
males vs. Cln8mnd females*, Cln8mnd males vs. wild type males#, and
Cln8mnd females vs. wild type females#. Two-way ANOVA with Fisher’s
LSD post-hoc. Mean ± SEM, n=1-11 animals/sex/group, detailed n
described in Additional file 4: Table S1. *p<0.05, **p<0.01, ***p<0.001,
Additional le4. TableS1: Detailed animal n for each experiment
(n=number of animals; Male/Female).
ADH was supported via scholarship from the University of South Dakota
Sanford School of Medicine Scholarship Pathways Program, and would like to
thank the Program for additional mentorship.
Author contributions
Conceptualization: JMW; methodology: KAW, MAP, TBJ, JMW; validation: ADH,
KAW, MAP, TBJ; formal analysis: ADH, MAP, KAW; investigation: ADH, KAW, MAP,
TBJ, JMW; resources: SL, KM; writing—original draft: ADH, KAW; writing—
review and editing: ADH, KAW, MAP, TBJ, SL, KM, JMW; visualization: ADH, MAP,
KAW; supervision: JMW; project administration: KAW, MAP, TBJ, JMW; funding
acquisition: JMW. All authors read and approved the final manuscript.
This work was supported by funding to JMW from the Cure Batten CLN8
Velona Foundation, Amicus Therapeutics, and the Sanford Research Imag-
ing Core within the Sanford Research Center for Pediatric Research (NIH
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Ethics approval and consent to participate
All animals were in an AAALAC accredited facility in accordance with IACUC
approval (Protocol #: 178-02-24D Sanford Research, Sioux Falls, SD).
Consent for publication
Not applicable.
Competing interests
JMW and TBJ are an employees of Amicus Therapeutics, Inc. and hold equity
in the company in the form of stock-based compensation. The other authors
declare no competing interests.
Author details
1 Pediatrics and Rare Diseases Group, Sanford Research, 2301 E 60Th St N, Sioux
Falls, SD, USA. 2 Department of Pediatrics, Sanford School of Medicine, Univer-
sity of South Dakota, Sioux Falls, SD, USA. 3 The Research Institute at Nation-
wide Children’s Hospital, Columbus, OH, USA. 4 Department of Pediatrics, The
Ohio State University, Columbus, OH, USA.
Received: 22 May 2022 Accepted: 23 October 2022
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Immune-inflammatory conditions in the central nervous system (CNS) rely on molecular and cellular interactions which are homeostatically maintained to protect neural tissue from harm. The CD40-CD40L interaction upregulates key proinflammatory molecules, a function best understood in the context of infection, during which B-cells are activated via CD40 signaling to produce antibodies. However, the role of CD40 in neurological disease of non-infectious etiology is unclear. We review the role of CD40-CD40L in traumatic brain injury, Alzheimer's Disease, Parkinson's Disease, stroke, epilepsy, nerve injury, multiple sclerosis, ALS, myasthenia gravis and brain tumors. We also highlight therapeutic advancements targeting the CD40 system to either attenuate the neuroinflammatory response or leverage the downstream effects of CD40 signaling for direct tumor cell lysis.
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The neuronal ceroid lipofuscinoses (NCLs) are a group of childhood-onset neurodegenerative lysosomal storage disorders mainly affecting the brain and the retina. In the NCLs, disease-causing mutations in 13 different ceroid lipofuscinoses genes (CLN) have been identified. The clinical symptoms include seizures, progressive neurological decline, deterioration of motor and language skills, and dementia resulting in premature death. In addition, the deterioration and loss of vision caused by progressive retinal degeneration is another major hallmark of NCLs. To date, there is no curative therapy for the treatment of retinal degeneration and vision loss in patients with NCL. In this review, the key findings of different experimental approaches in NCL animal models aimed at attenuating progressive retinal degeneration and the decline in retinal function are discussed. Different approaches, including experimental enzyme replacement therapy, gene therapy, cell-based therapy, and immunomodulation therapy were evaluated and showed encouraging therapeutic benefits. Recent experimental ocular gene therapies in NCL animal models with soluble lysosomal enzyme deficiencies and transmembrane protein deficiencies have shown the strong potential of gene-based approaches to treat retinal dystrophies in NCLs. In CLN3 and CLN6 mouse models, an adeno-associated virus (AAV) vector-mediated delivery of CLN3 and CLN6 to bipolar cells has been shown to attenuate the retinal dysfunction. Therapeutic benefits of ocular enzyme replacement therapies were evaluated in CLN2 and CLN10 animal models. Since brain-targeted gene or enzyme replacement therapies will most likely not attenuate retinal neurodegeneration, there is an unmet need for treatment options additionally targeting the retina in patients with NCL. The long-term benefits of these therapeutic interventions aimed at attenuating retinal degeneration and vision loss in patients with NCL remain to be investigated in future clinical studies.
Full-text available
While significant efforts have been made in developing pre-clinical treatments for the neuronal ceroid lipofuscinoses (NCLs), many challenges still remain to bring children with NCLs a cure. Devising effective therapeutic strategies for the NCLs will require a better understanding of pathophysiology, but little is known about the mechanisms by which loss of lysosomal proteins causes such devastating neurodegeneration. Research into glial cells including astrocytes, microglia, and oligodendrocytes have revealed many of their critical functions in brain homeostasis and potential contributions to neurodegenerative diseases. Genetically modified mouse models have served as a useful platform to define the disease progression in the central nervous system across NCL subtypes, revealing a wide range of glial responses to disease. The emerging evidence of glial dysfunction questions the traditional “neuron-centric” view of NCLs, and would suggest that directly targeting glia in addition to neurons could lead to better therapeutic outcomes. This review summarizes the most up-to-date understanding of glial pathologies and their contribution to the pathogenesis of NCLs, and highlights some of the associated challenges that require further research.
Full-text available
Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells.
Full-text available
The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders that affect children and adults. They share some similar clinical features and the accumulation of autofluorescent storage material. Since the discovery of the first causative genes, more than 530 mutations have been identified across 13 genes in cases diagnosed with NCL. These genes encode a variety of proteins whose functions have not been fully defined; most are lysosomal enzymes, or transmembrane proteins of the lysosome or other organelles. Many mutations in these genes are associated with a typical NCL disease phenotype. However, increasing numbers of variant disease phenotypes are being described, affecting age of onset, severity or progression, and including some distinct clinical phenotypes. This data is collated by the NCL Mutation Database which allows analysis from many perspectives. This article will summarise and interpret current knowledge and understanding of their genetic basis and phenotypic heterogeneity.
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Fetal growth restriction (FGR) is a common complication of pregnancy, resulting in a fetus that fails to reach its genetically determined growth potential. Whilst the fetal cardiovascular response to acute hypoxia is well established, the fetal defence to chronic hypoxia is not well understood due to experiment constraints. Growth restriction results primarily from reduced oxygen and nutrient supply to the developing fetus, resulting in chronic hypoxia. The fetus adapts to chronic hypoxia by redistributing cardiac output via brain sparing in an attempt to preserve function in the developing brain. This review highlights the impact of brain sparing on the developing fetal cardiovascular and cerebrovascular systems, as well as emerging long-term effects in offspring that were growth restricted at birth. Here, we explore the pathogenesis associated with brain sparing within the cerebrovascular system. An increased understanding of the mechanistic pathways will be critical to preventing neuropathological outcomes, including motor dysfunction such as cerebral palsy, or behaviour dysfunctions including autism and attention-deficit/hyperactivity disorder (ADHD).
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CLN8 is a ubiquitously expressed membrane-spanning protein that localizes primarily in the ER, with partial localization in the ER-Golgi intermediate compartment. Mutations in CLN8 cause late-infantile neuronal ceroid lipofuscinosis (LINCL). We describe a female pediatric patient with LINCL. She exhibited a typical phenotype associated with LINCL, except she did not present spontaneous myoclonus, her symptoms occurrence was slower and developed focal sensory visual seizures. In addition, whole-exome sequencing identified a novel homozygous variant in CLN8, c.531G>T, resulting in p.Trp177Cys. Ultrastructural examination featured abundant lipofuscin deposits within mucosal cells, macrophages, and monocytes. We report a novel CLN8 mutation as a cause for NCL8 in a girl with developmental delay and epilepsy, cerebellar syndrome, visual loss, and progressive cognitive and motor regression. This case, together with an analysis of the available literature, emphasizes the existence of a continuous spectrum of CLN8-associated phenotypes rather than a sharp distinction between them.
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Background information: The endo-lysosomal system (ELS) comprises a set of membranous organelles responsible for transporting intracellular and extracellular components within cells. Defects in lysosomal proteins usually affect a large variety of processes and underlie many diseases, most of them with a strong neuronal impact. Mutations in the endoplasmic reticulum-resident CLN8 protein cause CLN8 disease. This condition is one of the 14 known Neuronal Ceroid Lipofuscinoses (NCL), a group of inherited diseases characterized by accumulation of lipofuscin-like pigments within lysosomes. Besides mediating the transport of soluble lysosomal proteins, recent research suggested a role for CLN8 in the transport of vesicles and lipids, and autophagy. However, the consequences of CLN8 deficiency on ELS structure and activity, as well as the potential impact on neuronal development, remain poorly characterized. Therefore, we performed CLN8 knockdown in neuronal and non-neuronal cell models to analyze structural, dynamic, and functional changes in the ELS and to assess the impact of CLN8 deficiency on axodendritic development. Results: CLN8 knockdown increased the size of the Golgi apparatus, the number of mobile vesicles, and the speed of endo-lysosomes. Using the fluorescent fusion protein mApple-LAMP1-pHluorin, we detected significant lysosomal alkalization in CLN8-deficient cells. In turn, experiments in primary rat hippocampal neurons showed that CLN8 deficiency decreased the complexity and size of the somatodendritic compartment. Conclusions: Our results suggest the participation of CLN8 in vesicular distribution, lysosomal pH, and normal development of the dendritic tree. We speculate that the defects triggered by CLN8 deficiency on ELS structure and dynamics underlie morphological alterations in neurons, which ultimately lead to the characteristic neurodegeneration observed in this NCL. Significance: This is, to our knowledge, the first characterization of the effects of CLN8 dysfunction on the structure and dynamics of the ELS. Moreover, our findings suggest a novel role for CLN8 in somatodendritic development, which may account at least in part for the neuropathological manifestations associated with CLN8 disease. This article is protected by copyright. All rights reserved.
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Sex is a significant variable in the prevalence and incidence of neurological disorders. Sex differences exist in neurodegenerative disorders (NDs), where sex dimorphisms play important roles in the development and progression of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the last few years, some sex specific biomarkers for the identification of NDs have been described and recent studies have suggested that microRNA (miRNA) could be included among these, as influenced by the hormonal and genetic background. Failing to consider the possible differences between males and females in miRNA evaluation could introduce a sex bias in studies by not considering some of these sex-related biomarkers. In this review, we recapitulate what is known about the sex-specific differences in peripheral miRNA levels in neurodegenerative diseases. Several studies have reported sex-linked disparities, and from the literature analysis miR-206 particularly has been shown to have a sex-specific involvement. Hopefully, in the near future, patient stratification will provide important additional clues in diagnosis, prognosis, and tailoring of the best therapeutic approaches for each patient. Sex-specific biomarkers, such as miRNAs, could represent a useful tool for characterizing subgroups of patients.
Full-text available
Neuropathic pain (NeP) in humans is often a lifelong condition with no effective therapy available. The higher incidence of female gender in NeP onset is worldwide reported, and although the cause is generally attributed to sex hormones the actual mechanisms and the players involved are still unclear. Glial and immune cells take part in NeP development, and orchestrate the neuroimmune and inflammatory response, releasing pro-inflammatory factors with chemoattractant properties that activate resident immune cells and recruit immune cells from circulation. The neuro-immune crosstalk is a key contributor to pain hypersensitivity following peripheral nervous system injury. Our previous works showed that in spite female mice had an earlier than males analgesic response following nerve lesion, the recovery from NeP was never complete, suggesting that this difference could occur in the very early stages after injury. To further investigate gender differences in immune and neuroimmune responses to NeP, we studied the main immune cells and mediators elicited both in plasma and sciatic nerves by peripheral nerve lesion. After injury, we found a different pattern of distribution of immune cell populations showing either a higher infiltration of T cells in nerves from females or a higher infiltration of macrophages in nerves from males. Moreover, in comparison to male mice, the levels of cytokines and chemokines were differently up-and down-regulated in blood and nerve lysates from female mice. Our study provides some novel insights for the understanding of gender-associated differences in the generation and perseveration of NeP as well as for the isolation of specific neurodegenerative mechanisms underlying NeP. The identification of gender-associated inflammatory profiles in neuropathy is of key importance for the development of differential biomarkers and gender-specific personalized medicine.