© 2020. Published by The Company of Biologists Ltd.

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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 ygrishchuk@partners.org or Kirill Kiselyov at

kiselyov@pitt.edu.

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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