First posted online on 25 June 2020 as …...2020/06/25 · Brain abnormalities include thinning...
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Early evidence of delayed oligodendrocyte maturation in the mouse model of mucolipidosis
type IV.
Molly Mepyans1,*, Livia Andrzejczuk2,*, Jahree Sosa2, Sierra Smith1, Shawn Herron1, Samantha
DeRosa1, Susan Slaugenhaupt1, Albert Misko1, Yulia Grishchuk1,** and Kirill Kiselyov2,**
1 Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital
Research Institute and Harvard Medical School, Boston, MA, 02114, USA
2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, 15260, USA
*, **: equal contribution
Address correspondence to Yulia Grishchuk at [email protected] or Kirill Kiselyov at
Keywords: mucolipidosis IV, lysosome, TRPML1, mucolipin-1, myelination, oligodendrocyte
Summary statement:
We show that loss of the lysosomal channel TRPML1, responsible for mucolipidosis IV, leads to
delayed maturation of oligodendrocytes in early post-natal development resulting in brain
hypomyelination.
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http://dmm.biologists.org/lookup/doi/10.1242/dmm.044230Access the most recent version at First posted online on 25 June 2020 as 10.1242/dmm.044230
Abstract.
Mucolipidosis type IV (MLIV) is a lysosomal disease caused by mutations in the MCOLN1 gene that
encodes the endolysosomal transient receptor potential channel mucolipin-1, or TRPML1. MLIV
results in developmental delay, motor and cognitive impairments, and vision loss. Brain abnormalities
include thinning and malformation of the corpus callosum, white matter abnormalities, accumulation
of undegraded intracellular “storage” material and cerebellar atrophy in older patients. Identification
of the early events in the MLIV course is key to understanding the disease and deploying therapies.
Mcoln1-/- mouse model reproduces all major aspects of the human disease. We have previously
reported hypomyelination in the MLIV mouse brain. Here we investigated the onset of
hypomyelination and compared oligodendrocyte maturation between the cortex/forebrain and
cerebellum. We found significant delays in expression of mature oligodendrocyte markers Mag, Mbp,
and Mobp in the Mcoln1-/- cortex manifesting as early as 10 days after birth and persisting later in life.
Such delays were less pronounced in the cerebellum. Despite our previous finding of diminished
accumulation of the ferritin-bound iron in the Mcoln1-/- brain, we report no significant changes in
expression of the cytosolic iron reporters, suggesting that iron-handling deficits in MLIV occur in the
lysosomes and do not involve broad iron deficiency. These data demonstrate very early deficits of
oligodendrocyte maturation and critical regional differences in myelination between the forebrain and
cerebellum in the mouse model of MLIV. Furthermore, they establish quantitative readouts of the
MLIV impact on early brain development, useful to gauge efficacy in pre-clinical trials.
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Introduction.
Mucolipidosis type IV (MLIV) is the autosomal-recessive lysosomal storage disease caused by
mutations in the MCOLN1 gene located in humans in the 19p13.2 locus (Acierno et al., 2001). The
disease was first described in 1974 (Berman et al., 1974) and the corresponding gene was reported
in 1999 (Bargal et al., 2000; Raas-Rothschild et al., 1999; Slaugenhaupt et al., 1999). MCOLN1
encodes the late endosomal/lysosomal ion channel TRPML1 whose range of reported functions
includes zinc and iron homeostasis, regulation of the lysosomal acidification and calcium release
driving membrane fusion/fission events as well as lysosomal biogenesis (Cheng et al., 2014; Dong et
al., 2008; Eichelsdoerfer et al., 2010; Kukic et al., 2013; Miao et al., 2015; Samie et al., 2013;
Soyombo et al., 2006; Venkatachalam et al., 2008; Wong et al., 2012; Xu and Ren, 2015; Xu et al.,
2006; Zhang et al., 2012). Abnormalities of membrane traffic, autophagy and mitochondria have been
reported in various MLIV tissues and models (Coblentz et al., 2014; Colletti et al., 2012; Jennings et
al., 2006; Li et al., 2016; Miedel et al., 2008; Venkatachalam et al., 2008; Vergarajauregui et al., 2008;
Wong et al., 2012; Xu et al., 2006; Zhang et al., 2016).
MLIV patients typically present in the first year of life with psychomotor delay and visual impairment,
which stems from a combination of corneal clouding, retinal dystrophy and optic atrophy (Abraham et
al., 1985; Altarescu et al., 2002; Riedel et al., 1985; Schiffmann et al., 2014). Developmental gains
can be appreciated during the first decade of life, though axial hypotonia, appendicular hypertonia
and upper motor neuron weakness preventing ambulation and limiting fine motor function in the
majority of patients. While a static course has been classically reported, progressive psychomotor
decline has been documented. It begins in the second decade, eventually resulting in severe spastic
quadriplegia (personal observations). In congruence with the clinical course, ancillary brain imaging
has demonstrated stable white matter abnormalities (corpus callosum hypoplasia/dysgenesis and
white matter lesions) with the emergence of subcortical volume loss and cerebellar atrophy in older
patients. Despite a 19 year-long effort in MLIV natural history program (NCT00015782,
NCT01067742), due to low number or patients and poor availability of the human brain tissue for
research* (*single case tissues are now available at the University of Maryland Brain and Tissue
Bank), the mechanism of MLIV brain disease is still not well understood. Pinpointing the early events
in MLIV brain pathology is important to define the therapeutic window for intervention and set
expectations for clinical trial outcomes.
Some important insights regarding brain pathology in MLIV have been obtained from the MLIV mouse
model (Mcoln1 knock-out mouse) developed by us (Grishchuk et al., 2014; Grishchuk et al., 2016;
Micsenyi et al., 2009; Venugopal et al., 2007). The mouse model shows all the hallmarks of MLIV
including motor deficits, retinal degeneration, and reduced secretion of chloric acid by stomach
parietal cells leading to the elevated plasma gastrin (Schiffmann et al., 1998). These hallmarks were
also supported by the murine MLIV model generated by the Muallem group (Chandra et al., 2011).
Similar to MLIV patients, we observed intracellular storage formations, thinning of the corpus callosum
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in the Mcoln1-/- mouse brain, micro- and astrogliosis and partial loss of Purkinje cells at the late stage
of the disease. Electron microscopy analysis shows the reduced thickness of the myelin sheaths
consistent with CNS hypomyelination (Grishchuk et al., 2014)
Based on the existing natural history data in MLIV patients and our observations in the Mcoln1-/-
mouse, MLIV can be thought of having two distinct phases: the developmental phase, associated with
an early-onset delayed oligodendrocyte maturation and gliosis (Grishchuk et al., 2014; Weinstock et
al., 2018); and the late degenerative phase associated with partial loss of Purkinje cells in the MLIV
mouse (Micsenyi et al., 2009) and mild cerebellar atrophy in some MLIV patients (Frei et al., 1998).
Understanding the timing and mechanisms behind these distinct events is important for designing and
testing therapies. Here we investigate the role of TRPML1 in oligodendrocyte maturation and myelin
deposition in the developing brain. Our data show early problems with oligodendrocyte maturation
and myelination in the higher-functioning brain regions such as cerebral cortex. The role of TRPML1
in the early brain development is further supported by the fact that its expression gradually rises in
post-natal brain and expression is higher in the regions that display the most prominent deficits in
myelination in TRPML1 deficient (Mcoln1-/-) mice (cerebral cortex). Finally, this data establishes
quantitative readouts of the TRPML1 impact on early brain development, which will be useful to gauge
efficacy readouts in the future therapy trials.
Materials and Methods
Animals.
Mcoln1 knock-out mice were maintained and genotyped as previously described (Venugopal et al.,
2007). The Mcoln1+/- breeders for this study were obtained by backcrossing onto a C57Bl6J
background for more than 10 generations. Mcoln1+/+ littermates were used as controls. Experiments
were performed according to the institutional and National Institutes of Health guidelines and
approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee.
Immunohistochemistry and image analysis.
To obtain brain tissue for histological examination 1-, 10-, 21- and 60-day-old Mcoln1-/- and control
mice were sacrificed using a carbon dioxide chamber. Immediately after, they were transcardially
perfused with ice-cold phosphate-buffered saline (PBS). Brains were post-fixed in 4%
paraformaldehyde in PBS for 48 hours, washed with PBS, cryoprotected in 30% sucrose in PBS for
24 hours, split into cerebrum and cerebellum, frozen in isopentane, and stored at -800C. Brains were
bisected along the midline and one hemisphere was examined histologically. Coronal sections of the
cerebrum and sagittal sections of the cerebellum were cut at 10 μm using cryostat. Sections were
mounted onto glass SuperFrost plus slides. These sections were stored at -20°C before any staining
procedures. For PLP and SMI312 staining, sections were microwaved in citrate buffer (10 mM citric
acid, 0.05% Tween 20, pH 6.0) at low power for 18 min for antigen retrieval, blocked in 0.5% Triton
X-100 and 5% normal goat serum (NGS) in PBS and incubated with primary antibodies diluted in 1%
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NGS overnight. The following primary antibodies were used: PLP rabbit polyclonal, 1:500, ab28486;
SMI312 (pan axonal neurofilament) mouse monoclonal 1:1000, ab24574; all from Abcam
(Cambridge, MA, USA). Sections were incubated with secondary antibodies in 1% NGS for 1.5 hours
at room temperature. The following secondary antibodies were used: donkey anti-mouse AlexaFluor
488 and donkey anti-rabbit AlexaFluor 555 (1:500; Invitrogen, Eugene, OR, USA). For APC CC1
staining no antigen retrieval was performed. After blocking sections were incubated with anti-APC
CC1 antibody (mouse monoclonal, 1:100, OP80, Calbiochem, Billerica, MA, USA), followed by goat
anti-mouse AlexaFluor 488 (1:500, Invitrogen, Eugene, OR, USA). Sections were counterstained with
NucBlue nuclear stain (Life Technologies, Eugene, OR, USA).
Images were acquired on DM8i Leica Inverted Epifluorescence Microscope with Adaptive Focus
(Leica Microsystems, Buffalo Grove, IL, USA) with Hamamatsu Flash 4.0 camera and advanced
acquisition software package MetaMorph 4.2 (Molecular Devices, LLC, San Jose, CA, USA) using an
automated stitching function. The exposure time was kept constant for all sections (wild-type and
Mcoln1-/- sections within the same IHC experiment). Image analysis was performed using Fiji software
(NIH, Bethesda, Maryland, USA). Areas of interest were selected in each section. Area and mean
pixel intensity values were compared between genotypes using GraphPad Prism 7.04 software
(GraphPad, La Jolla, CA, USA) using Student’s T-test. Gaussian distribution was tested using the
Shapiro-Wilk normality test.
RNA extraction and q-RT-PCR
For q-RT-PCR experiments, total RNA was extracted from either whole cerebra or dissected cerebella
from P1 pups. From the brains of 10-, 21 day-old and 2 month-old mice, whole cerebella and cortical
samples were used. Cortical samples were dissected to include motor and somatosensory areas and
didn’t contain corpus callosum tissue. RNA was extracted using TRizol (Invitrogen, Carlsbad, CA),
according to manufacturer's protocol. All experimental groups have been designed to contain at least
4 animals per genotype and time-point, lower numbers in some conditions are due to low mRNA
yields that prevented to include them in q-RT-PCR experiments. For cDNA synthesis, MuLV Reverse
Transcriptase (Applied Biosystems, Foster City, CA, USA) was used with 2 μg of total RNA and
oligo(dT)18 (IDT, Coralville, IA, USA) as a primer. q-RT-PCR was carried out using 1:75 dilutions of
cDNA, 2X SYBR Green/ROX Master Mix (Fermentas, Glen Burnie, MD, USA), and 4 μM primer mix
per 10 μl reaction. For gene expression analysis, the following primers were used (IDT, Coralville, IA,
USA):
Actb, forward 5’-GCTCCGGCATGTGCAAAG-3’ and reverse 5’-CATCACACCCTGGTGCCTA-3’.
Fpn, forward 5’-ATCCTCTGCGGAATCATCCT-3’ and reverse 5’-CAGACAGTAAGGACCCATCCA-
3’;
Fth1, forward 5’-TGATCCCCACTTATGTGACTTCAT-3’ and reverse 5’-
ACGTGGTCACCCAGTTCTTT-3’;
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Heph, forward 5’-ACTAAAGAGATTGGAAAAGCAGTGA-3’ and reverse 5’-
TCATGCCCAGCATCTTCACA-3’;
Mag, forward 5’-CGGGATTGTCACTGAGAGC-3’ and reverse 5’-AGGTCCAGTTCTGGGGATTC-3’;
Mbp, forward 5’-GTGCCACATGTACAAGGACT-3’ and reverse 5’-TGGGTTTTCATCTTGGGTCC-
3’;
Mobp, forward 5’-CACCCTTCACCTTCCTCAAC-3’ and reverse 5’-TTCTGGTAAAAGCAACCGCT-
3’;
Ng2, forward 5’- CATTTCTTCCGAGTGGTGGC-3’ and reverse 5’-CACAGACTCTGGACAGACGG-
3’;
Tfrc, forward 5’- GAGGCGCTTCCTAGTACTCC-3’ and reverse 5’-TGGTTCCCCACCAAACAAGT-
3’.
To avoid amplification of genomic DNA and ensure amplification of cDNA, all primers were designed
to span exons and negative RT reactions (without reverse transcriptase) were performed as a control.
Samples were amplified in triplicates on the 7300 Real-Time System (Applied Biosystems, Foster
City, CA, USA) using the following program: 2 min at 50ºC, 10 min at 95ºC, and 40 cycles at 95ºC for
15 seconds followed by 1 min at 60ºC. In addition, a dissociation curve step was run to corroborate
that amplification with specific primers resulted in one product only. The ΔΔCt method was used to
calculate relative gene expression, where Ct corresponds to the cycle threshold. Ct values for a
representative set of genes are plotted in Supplementary Figure 1. ΔCt values were calculated as the
difference between Ct values from the target gene and the housekeeping gene Actb. Experiments
were performed as a double-blind using numerically coded samples; the sample identity was
disclosed at the final stage of data analysis. Data are presented as fold change.
Results
Mcoln1/TRPML1expression increases with age in the post-natal brain.
Despite the emerging role of TRPML1 in a range of cellular functions including cationic lysosomal
homeostasis, cell repair (Colletti et al., 2012; Li et al., 2016; Miedel et al., 2008; Venkatachalam et al.,
2008; Vergarajauregui et al., 2008; Wong et al., 2012; Xu et al., 2006; Zhang et al., 2016), and more
recently, aging and cancer (Jung et al., 2019; Xu et al., 2019), time course of Mcoln1/TRPML1
expression during the development of living tissue has not been tracked. To answer how
Mcoln1/TRPML1 is regulated during brain development we analyzed its expression using qRT-PCR
at the 1-, 10-, 21- and 60-day-old mice. Actin B (ActB) was used as a housekeeping gene. Primer
specificity was confirmed using Mcoln1-/- samples. Our data shows a steady increase in Mcoln1
expression levels in the forebrain from birth to two months of age (Fig. 1). Mcoln1 mRNA levels
increased four-fold (4.45±0.56, n=3 or 4, p=0.003) in the whole cerebrum samples of wild type animals
between P1 and P10 (Fig. 1A). Focusing specifically on the isolated cortical tissues we found a 6-fold
increase in Mcoln1 mRNA levels between P10 and P60 (6.31±1.10, n=3, statistically significant linear
trend with p=0.0028 using one-way ANOVA, Fig. 1C). By contrast, while Mcoln1 mRNA levels also
increased in the cerebellar samples between P1 and P10 (Fig. 1B) and P10 and P60 (Fig. 1C); the
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increases were small and did not attain statistical significance or significant linear trend. We found
similar trend in Mcoln1 expression data generated by FANTOM5 (Functional ANnoTation Of
Mammalian Genome) project (Ha et al., 2019; Lizio et al., 2015; Noguchi et al., 2017; Vitezic et al.,
2014) (Supplementary Figure 2A). While the design of the study doesn’t allow direct comparison of
transcriptional activation of Mcoln1 between visual cortex and cerebellum, the stable expression of
Mcoln1 in cerebellum in the pre- and early-post-natal phase and increased expression in post-natal
visual cortex is consistent with the data we report here. Our Mcoln1 expression data this is the first
report of the developmental dynamics of Mcoln1 transcription that allows comparison between
different brain regions, cortex and cerebellum.
Mcoln1-encoded endolysosomal cation channel TRPML1 is involved in the lysosomal function and
signaling (Xu and Ren, 2015). We compared its expression profile with that of another endolysosomal
cation channel, TPC1 (Tpcn1) (Yamaguchi et al., 2011). Interestingly, Tpcn1 mRNA did not increase
with age in the mouse cerebral cortex (Fig. 1D). This, to our knowledge, is the first analysis of Tpcn1
expression in the developing brain. Therefore, Mcoln1 mRNA upregulation during development is
specific to this gene rather than a more general increase in expression of the lysosomal channels.
The difference between Mcoln1 and Tpcn1 expression implies that functions of the corresponding ion
channels in the developing brain are distinct. The rapid pace of Mcoln1 mRNA increase in the
developing brain suggests its importance in development. Interestingly, there was a significant
increase in the Tpcn1 mRNA levels in the Mcoln1-/- samples at the P60 time-point (7.83±2.27-fold
increase, n=3, statistically significant linear trend with p=0.047 using one-way ANOVA). Since RNA
seq analysis on isolated brain cell-types shows enrichment of the Tpcn1 transcripts specifically in
astrocytes, macrophage/microglia and endothelial cells (Zhang et al., 2014), rise of its expression in
the Mcoln1-/- brain may reflect astrocytosis and microgliosis, a known prominent feature of brain
pathology in MLIV (Grishchuk et al., 2014; Micsenyi et al., 2009; Weinstock et al., 2018)
(Supplementary Figure 2B)
Early myelination deficits due to loss of Mcoln1/TRPML1 are caused by delayed
oligodendrocyte maturation.
We have previously shown hypomyelination in the mouse model of MLIV (Grishchuk et al., 2015),
suggesting oligodendrocyte involvement. The original data, however, did not clarify whether the
hypomyelination is a result of lower number of oligodendrocytes due to loss of precursors or it is
caused by their defective maturation. To gain more insight, we performed an expression time course
of oligodendrocyte maturation markers in post-natal development starting at birth to two months of
age. To assess oligodendrocyte maturation we used three of the key myelin genes: myelin-associated
glycoprotein precursor (Mag), myelin basic protein (Mbp) and myelin-associated oligodendrocyte
basic protein (Mobp) (Han et al., 2013; Huang et al., 2013; Perez et al., 2013; Robinson et al., 2014).
In the forebrain samples, the three markers showed linear increase between P1 and P21, allowing us
to compare the dynamics of oligodendrocyte maturation during brain development (Fig. 2). In both
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WT and KO samples, Mobp mRNA showed statistically significant linear trend (p=0.0021 and
p=0.0438 respectively, calculated using one-way ANOVA, n=3 or 4, Fig 2A). At P21 Mobp mRNA
increase was significantly higher in the Mcoln1+/+ relative to Mcoln1-/- samples (p=0.037 using t-test,
n=5 to 7). This trend persisted for Mbp (Fig 2B). Interestingly, while Mag mRNA levels showed linear
increase between P1 and P21, the rate of Mag mRNA increase was significantly lower than that of
Mobp and Mbp mRNA and did not differ significantly between Mcoln1+/+ relative to Mcoln1-/- samples
(Fig 2C), suggesting that Mcoln1 loss actuates specific changes in oligodendrocyte maturation rather
than the loss of entire cell populations. The relative abundance of Mbp and Mobp mRNA decreased
between P21 and P60 (Fig. 2A, B), while levels of Mag mRNA continued to grow (Fig. 2C). This data
is compatible with other published results (Ehmann et al., 2013). The observation that the expression
of all three maturation markers was lower in the Mcoln1-/- cortices compared with Mcoln1+/+ littermates
demonstrates early myelination delays (Fig. 2).
To answer whether the observed delays in the acquisition of the mature oligodendrocyte gene
expression in the cortex is the result of the delayed oligodendrocyte maturation or caused by loss of
oligodendrocytes precursors, we analyzed the expression of the oligodendrocyte precursor marker
Ng2 (Neural/glial antigen 2 (Hill et al., 2014)). Interestingly, we found that expression of Ng2 increased
in the Mcoln1-/- cortex as compared to Mcoln1+/+ littermates (Fig. 2D). Increase of Ng2 expression
may reflect the attempt to overcome the downstream myelination deficits during the active phase of
brain myelination in post-natal development. Therefore, we conclude that brain hypomyelination in
MLIV is caused by delayed oligodendrocyte maturation and reduced myelin production rather than
the death of the oligodendrocyte precursors.
Next, we used immunohistochemistry to analyze developmental myelination of the corpus callosum
in the 10-day-old Mcoln1+/+ and Mcoln1-/- mice using antibodies for the mature oligodendrocyte marker
PLP1 (myelin proteolipid protein) (Wang et al., 2006), one of the most abundant proteins in the myelin,
and the neuronal neurofilament marker SMI312 (Vasung et al., 2011) (Fig. 3). Previously we reported
that thinning of the corpus callosum in 2- and 7-months old Mcoln1-/- mice was caused by defective
myelination while the thickness of the underlying neuronal fibers in the corpus callosum remained
intact (Grishchuk et al., 2015). Similar to these observations made in adult mice, here we detect no
significant decrease in the SMI312-positive area of the corpus callosum in P10 pups, representing
normally forming tract of the callosal neuronal fibers, but observed significant decrease in myelin
deposition by mature oligodendrocytes, presented by reduced PLP-positive area alone and
decreased ratio of the PLP-area to the corresponding SMI312-area (Fig. 3A, B). Furthermore, we
observed the irregular distribution of PLP1-positive cells and forming myelinated fibers in the Mcoln1-
/- cortex: the islands of tissue that are devoid of the PLP staining (Fig. 3C and arrows in Fig. 3A),
further supporting defective maturation of oligodendrocyte precursors in this model.
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Delayed maturation of oligodendrocytes in the Mcoln1-/- brain is region-specific.
To answer whether the impact of Mcoln1/TRPML1 loss on myelination is different in the cerebellum,
we performed q-PCR analysis on the cerebellar samples from P1-P60 mice. We found that the overall
differences in expression of the mature oligodendrocyte maturation markers in Mcoln1-/- and control,
Mcoln1+/+ cerebella were not as pronounced as they were in the cortical samples (Fig. 4A). This q-
PCR analysis was also supported by immunohistochemistry, where we detected neither difference in
the percentage of the PLP-positive area, nor in the counts of oligodendrocytes labeled by APC-CC1
(Fig. 4 B, C) in Mcoln1-/- cerebella as compared to the Mcoln1+/+ littermates. Therefore, the impact of
Mcoln1/TRPML1 loss on ODC maturation and myelination is brain-region specific; this should be
taken into consideration when discussing the impact of MLIV and its treatment.
Transcriptional analysis of the iron-regulated genes failed to reveal signs of iron-depletion in
the whole cortical homogenates.
Iron handling by the lysosomes has been shown to be impaired in Mcoln1/TRPML1-deficient cells
and brain tissues (Coblentz et al., 2014; Dong et al., 2008; Grishchuk et al., 2015). Despite the existing
evidence for iron involvement, the mechanism through which iron mishandling impacts
Mcoln1/TRPML1-deficient cells is unclear. Iron uptake in many cell types is facilitated through
receptor-mediated endocytosis and therefore its late events are handled by the endolysosomes
(Todorich et al., 2009; Todorich et al., 2011; Valerio, 2007). TRPML1 may be involved in this by
transferring ferrous iron from the lysosomal lumen to the cytoplasm. It is, therefore, possible that
Mcoln1/TRPML1 loss traps ferrous iron in the lysosomes resulting in depriving the cytoplasm of iron
and causing lysosomal oxidative stress. Importantly, cytoplasmic iron is key to oligodendrocyte
maturation and myelin production as it is a cofactor in ATP production, which is highly important due
to extremely high energy requirements of oligodendrocytes during myelination, and in the activity of
key enzymes of lipid catabolism and biosynthesis required for myelin production (Todorich et al.,
2009). Iron retention in the lysosome due to loss of Mcoln1/TRPML1 might therefore directly affect
oligodendrocyte maturation by depriving iron to these reactions.
To answer whether Mcoln1/TRPML1 loss is associated with the depletion of the cytoplasmic iron, we
analyzed the expression of the iron-regulated genes such as transferrin receptor (Tfrc), hephaestin
(Heph), ferritin heavy chain 1 (Fth1) and ferroportin (Fpn) by q-PCR. All these genes are regulated by
cytoplastic iron through Hif1α and Hif2α and other transcription factors involved in metal handling and
oxidative stress (Acikyol et al., 2013; Li et al., 2013). q-PCR showed no distinct difference between
the wild-type and Mcoln1-/- samples (Fig. 5). We, therefore, conclude that whole-tissue transcriptomic
analysis reports no brain-wide cytoplasmic iron depletion. Due to the relatively low abundance of
oligodendroglial mRNA in the whole-tissue samples, however, we cannot rule out whether
dysregulation of these genes in Mcoln1-/- animals takes place only in oligodendrocytes and may be
masked by the signal from more abundant cell types.
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Discussion:
In the present study, we show that loss of the lysosomal channel TRPML1 is associated with delayed
oligodendrocyte maturation leading to brain hypomyelination, which is a characteristic of both,
mucolipidosis IV mouse model and the human disease. A dramatically significant aspect of MLIV
uncovered by these studies is the extremely early onset of the oligodendrocyte maturation delay,
manifesting already by post-natal day 10. Importantly, these early myelination deficits are not
reflecting or causing underlying structural abnormalities of neuronal fibers or their loss. This is a key
piece of information for designing MLIV treatments.
We have previously reported reduced myelination in Mcoln1-/- mice via expression analysis of
oligodendrocyte markers myelin-associated glycoprotein precursor (MAG), myelin basic protein
(MBP), myelin-associated oligodendrocyte basic protein (MOBP) and platelet-derived growth factor
receptor (PDGFR) in the whole-brain homogenates at 10 days, 2 and 7 months of age (Grishchuk et
al., 2015). We also showed lower protein levels of mature oligodendrocyte marker PLP in the Mcoln1-
/- brain and reduced thickness of the PLP-immunostained corpus callosum. Importantly, the
neurofilament marker SMI312 remained unchanged in the early symptomatic Mcoln1-/- mice (2 months
of age) showing normally formed neuronal fibers in the corpus callosum. In the present study, we
tracked and compared oligodendrocyte maturation markers in the forebrain/cortex and cerebellum in
Mcoln1-/- mice at 1, 10, 21 and 60 days after birth. We found dramatic differences between genotypes
and brain regions. More specifically, while we report significant myelination deficits in the anterior
regions (cortex, corpus callosum) of the Mcoln1-/- brain, myelination in the posterior brain (cerebellum)
was normal. This regional and temporal pattern of myelination deficits in the Mcoln1-/- mouse along
with the time-course of Mcoln1/TRPML1 expression in post-natal brain development provides new
insights into the role of TRPML1 channel in the myelination process. It is well recognized that
myelination progresses from caudal to rostral pathways and in the order of increasing complexity of
brain function (Gerber et al., 2010; Purger et al., 2016), starting from the areas responsible for basic
homeostatic functions (pons, cerebellum) and proceeding to the areas controlling more complex tasks
(frontal cortex). In the mouse, deposition of myelin in the cerebellum starts in the deepest regions
earlier than P6 and is largely accomplished by P23 (Purger et al., 2016). In the corpus callosum, the
first myelinating cells are noticed on postnatal day 9. In TRPML1 deficient brains, the regions that
normally acquire myelination later in development are affected the most. Interestingly, we show that
in parallel with brain myelination, expression of TRPML1 steadily increases in the postnatal brain and
is more prominent in the cortex at the final stage of the myelination process, indicating its need for
myelination of the higher-hierarchical regions.
In corroboration with our findings in Mcoln1-/- knockout mice, specific abnormalities in cerebral white
matter have been observed in MLIV patients. In a cross-sectional cohort analysis of 15 MLIV patients
ranging in age from 16 months to 22 years (Frei KP, 1998), hypoplasia of the corpus callosum and
elevated T2 signal in the subcortical white matter were reported in all patients, while cortical atrophy
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was only noted in two of the oldest (15 and 22 years). A subsequent prospective study of brain MRI
changes in five MLIV patients, ranging from 7 to 18 years of age, found increasing cortical gray matter
and decreasing subcortical white matter volumes across a 2-3 year follow up period (Schiffmann et
al., 2014). Though additional longitudinal data in individual patients is still needed to support
conclusions, the available data in humans illustrates an early developmental defect followed by a late
degenerative process specifically affecting white matter tracts. The possibility that axonal
degeneration could accompany white matter loss in MLIV patients was raised by the finding of
decreased N-acetylaspartate (NAA)/creatine or choline ratios in multiple cortical regions (Bonavita et
al., 2003; Cambron et al., 2012). Decreased NAA levels, however, can be seen in the setting of
neuronal mitochondrial dysfunction without neuronal degeneration. Taken together with our current
findings, we propose that the neurological deficits observed in the Mcoln1 knockout mice and MLIV
patients mainly arise from abnormalities in CNS white matter.
Our findings in Mcoln1-/- mice have important implications for understanding the disease pathogenesis
in MLIV patients, where data on the earliest stages of myelination are not available. In regards to
myelination, post-natal day 10 in mice is roughly equivalent to the full-term human neonate (Hagberg
et al., 2002; Semple et al., 2013). While corpus callosum hypoplasia/dysgenesis and subcortical white
matter abnormalities have been consistently reported in older subjects (Frei et al., 1998), brain MRI
data for MLIV younger than 16 months of age have not been reported, largely due to the first
symptoms presenting around 6 months of age and diagnosis rarely occurring before first year. The
delayed manifestation of motoric dysfunction in MLIV patients may reflect selective myelination deficit
in higher cortical regions. This is also supported by the fact that in the first months of life (prior MLIV
symptoms onset) motor function is mostly attributable to primitive brain regions, where, based on our
mouse data, myelination is not affected by the loss of Mcoln1/TRPML1.
Our understanding of leukodystrophies, genetically determined disorders that selectively affect white
matter in the central nervous system, has rapidly evolved with the recent application of brain imaging
techniques paired with next generation sequencing (van der Knaap and Bugiani, 2017). Surprisingly,
mutations in myelin or oligodendrocyte specific proteins are only responsible for a portion of clinically
discernable leukodystrophies while genes important for house keeping processes in multiple cell
linages are largely responsible for the remainder. As a result, the new extended classification of the
leukodystrophies that now includes more than 50 genetic syndromes with mutations in myelin- or
oligodendrocyte-specific genes (hypomyelinating and demyelinating leukodystrophies;
leukodystrophies with myelin vacuolization), and other white matter components including axons
(leuko-axonopathies), glia (astrocytopathies and microgliopathies) and blood vessels
(leukovasculopathies). We believe that the prominent and early involvement of developmental
hypomyelination strongly advocates for MLIV to be included to this list.
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On the cellular level, TRPML1 has been shown to be involved in various cell functions including
membrane fission and fusion, as well as the redistribution of ions between the lysosomes and the
cytoplasm (Cheng et al., 2014; Dong et al., 2008; Eichelsdoerfer et al., 2010; Kukic et al., 2013; Miao
et al., 2015; Samie et al., 2013; Soyombo et al., 2006; Venkatachalam et al., 2008; Wong et al., 2012;
Xu and Ren, 2015; Xu et al., 2006; Zhang et al., 2012). The latter effects were or can be attributed to
TRPML1 iron conductance, or its role in the traffic of iron or iron-bound proteins in the endocytic
pathway. Impairment of the TRPML1-dependent iron uptake is an attractive hypothesis for the
oligodendrocyte maturation deficits in the MLIV brain, as cytoplasmic iron is a key co-factor of the
myelin synthesis pathway (Todorich et al., 2009). Indeed, iron deficiency and abnormal cellular iron
absorption are associated with hypomyelination (Morath and Mayer-Proschel, 2001; Todorich et al.,
2009; Todorich et al., 2011). Iron handling deficits have been reported by us in the brain of the Mcoln1-
/- mouse model (Grishchuk et al., 2015). At that time, we were unable to provide a definitive answer
regarding the impact of iron mishandling in the Mcoln1-/- brain. Here we extended our studies using a
battery of iron-reporter genes but found no evidence of the cytoplasmic iron deficit in the Mcoln1-/-
cortical tissue homogenates. We, therefore, conclude that iron mishandling caused by
Mcoln1/TRPML1 loss is either 1) limited to a specific subset of cells (such as mature
oligodendrocytes) and is being masked in the whole tissue preparations, 2) taking place on certain
developmental stages, and/or 3) it only manifests as a lysosomal iron buildup without significantly
affecting the cytoplasmic iron uptake and therefore cannot be detected via analysis of expression of
the Fe-related genes. To this end, isolated Mcoln1-/- oligodendrocytes cultures and co-cultures with
neurons and glia will be instrumental to delineate the mechanism by directly measuring cytosolic Fe-
reporters, lysosomal iron content, oxidative stress and their impact on myelination and will be in the
focus of our future study.
In conclusion, we present evidence of the role of the TRPML1 lysosomal channel in developmental
myelination. Genetic ablation of TRPML1 leads to deficient oligodendrocyte maturation resulting in
hypomyelination of the cortex and corpus callosum noticeable already at post-natal day 10. However,
mechanisms of how the loss of TRPML1 leads to such deficits remain unknown. Brain myelination in
development is a complex and highly regulated process, that depends on both cell-intrinsic
(oligodendroglial) and extrinsic (neuronal, astrocytic and microglial) signaling and cues. Indeed,
reduced myelination in the early post-natal Mcoln1-/- cortex is accompanied by the observations of
robust activation of astrocytes, resulting in the elevated cytokine/chemokine release and
neuroinflammation (Weinstock et al., 2018). Therefore, better understanding the role of TRPML1 in
oligodendrocyte maturation and myelin deposition will require new genetic in-vivo (using the Cre-LoxP
system for cell-type-specific manipulation of Mcoln1 expression) and in-vitro ODC and other brain cell
co-culture models and this will be a scope of our future investigations. Elucidating the involvement of
other brain cell types, neurons and glia, affected by the loss of TRPML1, in the development of the
oligodendrocyte lineage cells is important given the complex picture of brain pathology and
dysfunction in this disease.
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Acknowledgments:
The authors are thankful to Dr. Franca Cambi for a fruitful discussion.
Competing interests:
The authors report no competing interests.
Funding:
These studies were supported by NIH R01NS096755
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Figures
Fig 1. Increasing Mcoln1 expression in the brain during development. A. and B. qPCR analysis
of the Mcoln1 mRNA in the whole forebrain (A) and cerebellum (B) in Mcoln1+/+ (WT) and Mcoln1-/-
(KO) mice showing a significant increase with age. The analysis of expression was performed using
Ct method; Actb was used as a housekeeping gene. Statistical analysis was performed using two-
way ANOVA followed by Tukey’s multiple comparison tests. *** indicates p<0.0005 and ** indicates
p<0.005; errors bars are SEM. C. Mcoln1 expression dynamics in the cortical and cerebellum samples
of P10-P60 mice. The symbols represent average of 3-4 experiments. ** represents statistically
significant linear trend with p<0.005 calculated using one-way ANOVA. ns is “not significant”. C. Tpcn1
mRNA expression in the Mcoln1+/+ and Mcoln1-/- cerebral cortex. * indicates statistically significant
linear trend with p<0.05 calculated using one-way ANOVA.
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Fig 2. Delayed oligodendrocyte maturation in the Mcoln1-/- cerebral cortex. qPCR analysis of
cerebrum (P1) and cortex (P10, P21 and P60) of Mcoln1+/+ and Mcoln1-/- littermate mice shows
expression of the mature ODC markers Mobp, Mbp and Mag (A-C) and oligodendrocyte precursor
marker Ng2 (D). The analysis was performed using the Ct method; Actb was used as a
housekeeping gene. Slopes were obtained using simple linear regression, all of which were significant
except WT in panel D. ** and * indicate p<0.005 and p<0.05 using two-way ANOVA followed by
Tukey’s multiple comparison tests ; n=3-5; ns means p>0.05; error bars represent SEM.
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Fig 3. Reduced myelination of the corpus callosum in the MLIV mouse model is not
accompanied by a significant reduction of its neuronal fiber thickness. A. Representative
images of the Mcoln1+/+ and Mcoln1-/- P10 brain sections stained with the mature ODC/myelin marker
PLP (red) and neuronal filament marker SMI312 (green). Corpus callosum and cortical areas are
shown. Arrows show ODC-deficient lesions in the Mcoln1-/- brain. Scale bar is equal to 1 micron. B.
Analysis of the corpus-callosum area identified via either PLP or SMI312 staining showing reduction
of the PLP-positive but not SMA312-positive area and significant reduction of the PLP to SMI312
positive area ratio in Mcoln1-/- mice indicating deficient myelination but normal underlying structure in
the forming corpus callosum in the MLIV mouse (n=4 (Mcoln1+/+) and n=6 (Mcoln1-/-); n.s means
P>0.05 using T-test; *** P<0.001). C. Enlarged representative images of the Mcoln1+/+ and Mcoln1-/-
P10 brain sections immunostained for PLP showing dysmorphic myelinating fibers in the Mcoln1-/-.
Scale bar is equal to 200 nm.
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Fig 4. Cerebellar myelination is preserved in the MLIV mice. A. qPCR analysis of P1, P10, P21
and P60 cerebella from Mcoln1+/+ (WT) and Mcoln1-/- (KO) littermate mice shows expression of the
mature ODC markers Mobp, Mbp and Mag. The analysis was performed using the Ct method; Actb
was used as a housekeeping gene. ns is p>0.05 using two-way ANOVA followed by Tukey’s multiple
comparison tests; n=3-5; ns means p>0.05; error bars represent SEM. B. Representative images of
the Mcoln1+/+ and Mcoln1-/- P10 brain sections stained with the mature ODC/myelin marker PLP (red),
ODC marker APC-CCI (green) and nuclear counterstain Nuc-Blue (blue). Scale bar for the lower
magnification panels is equal to 500 nm; for higher magnification panels is 100 nm. C. Statistical
analysis of the APC-CC1-positive cell counts per section (left panel) and PLP-positive area (rigdht
panel) in the cerebellar sections show no significant changes between Mcoln1+/+ and Mcoln1-/- mice
(T-test, n=4 (Mcoln1+/+) and n=5 (Mcoln1-/-); n.s P>0.05).
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Fig 5. Cytoplasmic iron markers in the MLIV mouse forebrain. qPCR analysis of mRNA for
cytoplasmic iron markers Tfrc, Heph, Fth1 and Fpn in the cerebrum (P1) and cortex (P10-P60)
samples of WT (Mcoln1+/+) and Mcoln1-/- (KO) mice. The analysis was performed using the Ct
method; Actb was used as a housekeeping gene. Actb was used as a housekeeping gene. * indicates
p<0.05 using two-way ANOVA followed by Tukey multiple comparison test; n=3-5; ns means p>0.05;
error bars represent SEM.
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Supplementary Figure 1.
Fig S1. Sample Ct values obtained using qPCR with Mobp and Actb primers. Vertical axis: individual Ct values.
Disease Models & Mechanisms: doi:10.1242/dmm.044230: Supplementary information
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Supplementary Figure 2
Fig S2. A: Mcoln1 expression in the mouse cerebellum or visual cortex from FANTOM (Functional ANnoTation Of Mammalian Genome) data set. Expression levels are shown as the average of (relative log expression) RLE normalized TPM counts; n = 3-4 for each time-point. Visual cortex values obtained from (Vitezic et al., 2014) and cerebellum data from (Ha et al., 2019). All data have been extracted using the FANTOM5 Table Extraction Tool. B: Cell-type specific expression of either Mcoln1 or Tpcn1 in the mouse brain from (Zhang et al., 2014);
Mcoln1, cerebellum Mcoln1, visual cortex
Age, days Age, days
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Disease Models & Mechanisms: doi:10.1242/dmm.044230: Supplementary information
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