© 2015. Published by The Company of Biologists Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium

provided that the original work is properly attributed.

Impaired myelination and reduced ferric iron in mucolipidosis IV brain

Yulia Grishchuk1, Karina A. Peña2, Jessica Coblentz2, Victoria E. King1, Daniel M. Humphrey1,

Shirley L. Wang1, Kirill I. Kiselyov2# and Susan A. Slaugenhaupt1#

#-co-senior authors

1- Center for Human Genetic Research and Department of Neurology, Massachusetts

General Hospital and Harvard Medical School, 185 Cambridge Street, Boston, MA

02114;

2- Department of Biological Sciences, University of Pittsburgh, 519 Langley Hall, 4249

Fifth Avenue, Pittsburgh, PA 15260.

Corresponding author: Yulia Grishchuk; e-mail: ygrishchuk@mgh.harvard.edu.

Keywords: lysosome, transient receptor potential channel mucolipin-1, myelination,

oligodendrocytes

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.021154Access the most recent version at DMM Advance Online Articles. Posted 17 September 2015 as doi: 10.1242/dmm.021154http://dmm.biologists.org/lookup/doi/10.1242/dmm.021154Access the most recent version at

First posted online on 17 September 2015 as 10.1242/dmm.021154

Abstract.

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the

MCOLN1 gene, which encodes the lysosomal transient receptor potential ion channel mucolipin-

1 (TRPML1). MLIV causes impaired motor and cognitive development, progressive loss of

vision and gastric achlorhydria. How loss of TRPML1 leads to severe psychomotor retardation is

currently unknown and there is no therapy for MLIV. White matter abnormalities and a

hypoplastic corpus callosum are the major hallmarks of MLIV brain pathology. Here we report

that loss of TRPML1 in mice results in developmental aberrations of brain myelination due to

deficient maturation and loss of oligodendrocytes. Defective myelination is evident in Mcoln1-/-

mice at post-natal day 10, an active stage of post-natal myelination in the mouse brain.

Expression of mature oligodendrocyte markers is reduced in Mcoln1-/- mice at post-natal day 10

and remains lower throughout the course of disease. We observed reduced Perls’ staining in

Mcoln1-/- brain indicating lower levels of ferric iron. Total iron content in unperfused brain is not

significantly different between Mcoln1-/- and wild-type littermate mice, suggesting that the

observed maturation delay or loss of oligodendrocytes may be caused by impaired iron handling,

rather than global iron deficiency. Overall, these data emphasize a developmental rather than a

degenerative disease course in MLIV, and argue for a stronger focus on oligodendrocyte

maturation and survival in the search for answers to MLIV pathogenesis and treatment.

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Introduction.

MLIV was originally described in 1974 based on clinical manifestations that resembled

other mucolipidoses, but with a distinct biochemical profile (Berman ER, 1974). In 2000, the

gene mutated in MLIV was identified as MCOLN1, which encodes the protein mucolipin-1

(Bargal et al., 2000; Bassi et al., 2000; Sun et al., 2000). Based on homology with transient

receptor potential (TRP) ion channel proteins, mucolipin-1 is classified as a founding member of

the TRPML family and is also known as TRPML1. Due to its localization in the lysosomes and

the observed lysosomal storage disease phenotype of MLIV, TRPML1 is presumed to regulate

membrane traffic, lysosomal biogenesis, or lysosomal ion content (Colletti and Kiselyov, 2011;

Wang et al., 2014). The diagnostic ultrastructural hallmark of MLIV is the accumulation of

storage bodies filled with mixed lamellar and granular electron-dense content, called

“compound” bodies (Bargal and Bach, 1989; Berman ER, 1974). Biochemical composition of

this storage material includes gangliosides, phospholipids and acidic mucopolysaccharides (Bach

et al., 1975; Bach and Desnick, 1988; Bach et al., 1977; Bagdon et al., 1989; Bargal and Bach,

1988; Bargal and Bach, 1989; Slaugenhaupt et al., 1989).

MLIV is primarily found in the Ashkenazi Jewish population (approximately 75% of

patients), with a carrier frequency of ~1:100 (Altarescu et al., 2002; Bach, 2001). There are

approximately 100 diagnosed patients in the US. Worldwide numbers are largely unknown, but

patients with Arab (Mirabelli-Badenier et al., 2014), African-American (Noffke et al., 2001),

French Canadian (Berman ER, 1974) and Turkish descent (Tuysuz et al., 2009) have been

reported. Over two dozen mutations causing MLIV have been described, the vast majority of

which lead to mRNA instability and complete loss of protein (Wakabayashi et al., 2011). A small

fraction of MLIV-causing mutations, some of which may be associated with a milder phenotype,

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lead to aberrant localization and/or ion channel dysfunction (Chen et al., 2014; Kiselyov et al.,

2005). Establishing a genotype-phenotype correlation in MLIV is complicated due to the low

incidence of the disease.

MLIV is a developmental disorder that causes motor and cognitive deficiencies, which

become noticeable during the first year of life. On average, maximum neurologic development in

MLIV patients corresponds to the level of 15-18 months of age and remains stable throughout

the third decade of life (Altarescu et al., 2002). In most patients neurologic symptoms include

spasticity, hypotonia, an inability to walk independently, ptosis, myopathic faces, drooling,

difficulties in chewing and swallowing, and severely impaired fine-motor function (Altarescu et

al., 2002). Ophthalmic symptoms are progressive and result in blindness in the second decade of

life (Riedel et al., 1985; Smith et al., 2002; Wakabayashi et al., 2011).

Human brain pathology data in MLIV is limited to only two autopsy cases, in which

gliosis, abnormal white matter and numerous storage inclusions in all types of brain cells have

been reported (Folkerth RD, 1995; Tellez-Nagel I, 1976). Brain magnetic resonance imaging

studies revealed dysgenic corpus callosum, impaired myelination in the white matter and

decreased T2 signal intensities in the thalamus due to increased ferritin deposition (Frei KP,

1998; Schiffmann et al., 2014).

At present, there is no therapy for MLIV and one of the central factors hindering the

search for treatments is a limited understanding of MLIV brain pathology. Mouse models of

human disease provide an opportunity to gain a deeper insight into disease pathogenesis and

progression. The Mcoln1-/- knock-out mouse is an excellent model of the human disease, as all

hallmarks of MLIV are present in the mice with the exception of corneal clouding (Venugopal et

al., 2007). MLIV mice display brain and gastric phenotypes identical to those reported in human

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MLIV patients (Chandra et al., 2011; Grishchuk et al., 2014). Mcoln1-/- animals display no

immediate phenotype at birth. At two months of age, they start to display motor and cognitive

deficits that can be detected in the open field test (Grishchuk et al., 2014). Motor deficits in

MLIV mice progress to total hind limb paralysis and death by 8-9 months of age (Venugopal et

al., 2007). Post-mortem brain analysis shows lysosomal inclusions in multiple cell types

including neurons, astrocytes, oligodendrocytes, microglia and endothelial cells. Defective

myelination and gliosis, the hallmarks of human MLIV, are present in the young adult mice at

the onset of cognitive and motor deficits (Grishchuk et al., 2014). However, we observed no

neuronal loss in the Mcoln1-/- forebrain even at the late stage of disease (Grishchuk et al., 2014).

These findings prompted a look at glial cell involvement in MLIV. In the present study we use

the MLIV mouse model to analyze the dynamics of myelination and oligodendrocyte

development in the MLIV mouse and to investigate the role of iron dyshomeostasis in MLIV

pathogenesis. We show abnormal oligodendrocyte development and reduced ferric iron levels in

the white matter of the Mcoln1-/- mice. These findings highlight the role of TRPML1 in proper

brain myelination and argue for a stronger focus on oligodendrocyte maturation and survival in

the search for potential therapeutic approaches in MLIV.

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Results.

Immunostaining of brain coronal sections with antibodies against mature myelinating

oligodendrocyte marker proteolipid protein (PLP) (Baumann and Pham-Dinh, 2001; Han et al.,

2013) showed reduced myelination in Mcoln1-/- mice (Fig. 1). We observed significant reduction

in the PLP immunoreactivity measured in the whole hemispheric coronal section or in the

cortical region in adult mice (Fig. 1 A, B). We also observed a significant reduction in the

surface area of the myelinated corpus callosum. Both findings were present in the early-

symptomatic (two months of age) and the late stage (seven months of age) Mcoln1-/- mice, but

were not progressive in the course of disease. Similar data were obtained on the brain sections

from two month-old Mcoln1-/- and wild-type littermate mice, immunostained with the antibodies

against myelin basic protein (MBP) (Huang et al., 2013) (Suppl. Fig. 1).This striking pattern of

reduced myelination and thinned corpus callosum is similar to the previously reported

FluoroMyelin Green staining performed in Mcoln1-/- mice at two and seven months of age

(Grishchuk et al., 2014). Reduced and non-progressive myelination in Mcoln1-/- mice was also

confirmed by Western blotting, which showed 4-fold decrease in the levels of PLP and MBP in

the cerebral cortex at the age of two and seven months (Fig.1 C, D). Interestingly, myelination

deficits (shown as reduced levels of MBP) were also detected outside of CNS, in sciatic nerves

of Mcoln1-/- mice (Suppl. Fig. 2).

To determine if reduced myelination in the white and gray matter of the Mcoln1-/- brain is

secondary to lower density of axons, we performed staining with pan-axonal marker SMI312

(Vasung et al., 2011). Strikingly, we observed no changes in surface area of the SMI312-stained

corpus callosum and in the SMI312 immunoreactivity in the whole hemi-brain sections, cortical

region, or corpus callosum between genotypes at two and seven months of age (Fig. 2 A, B).

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These data show that white matter abnormalities and reduced gray matter myelination are not

due to reduced density of axonal fibers in Mcoln1-/- mice.

To determine if the loss of TRPML1 alters the developmental stage of myelin deposition

we analyzed myelination markers at post-natal day 10, an active phase of brain myelination in

mice. PLP immunostaining of brain sections showed a disorganized pattern of myelinating fibers

in the corpus callosum and in the cortex of Mcoln1-/- pups (Fig. 3 A), while the overall intensity

was not significantly different between genotypes at this developmental stage. Quantitative

analysis of fiber morphology in the corpus callosum showed significant reduction in the number

of branches, number of junctions, number of triple and quadruple points in Mcoln1-/- pups (Table

1 and Suppl. Fig. 3), reflecting decelerated development of myelinated network due to loss of

TRPML1. Moreover, Western blot analysis of cortical homogenates showed reduced levels of

PLP and MBP in Mcoln1-/- mice (2.5 and 3-fold, correspondingly, Fig. 3 B). All together these

data indicate early developmental changes in brain myelination due to loss of TRPML1.

The observed reduction of myelination resulting from the loss of TRPML1 could reflect

1) the loss of differentiated oligodendrocytes or their abnormal differentiation; 2) deficient

myelin formation by Mcoln1-/- oligodendrocytes; or a combination of both. To determine if

reduced myelination in the Mcoln1-/- brain is caused by reduced numbers of oligodendrocytes,

we performed immunostaining with adenomatous polyposis coli (APC-CC1), a marker for post-

mitotic pre-myelinating oligodendrocytes, in 10 day-old and two month-old mice. Analysis of

CC1+ cell density revealed that the number of oligodendrocytes is significantly reduced in the

Mcoln1-/- corpus callosum at 10 days of age, but the CC1+ cell density improved by two months

of age (Fig. 4 A). Interestingly, in the cortical region the CC1+ cell density was the same at 10

days of age, but a loss of cortical oligodendrocytes was evident by two months. The observed

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difference between the corpus callosum and the cortex suggests that there are region-specific

oligodendrocyte responses in the Mcoln1-/- brain, which could be caused by differences in the

microenvironment in these brain structures or cell-intrinsic differences between white- and gray-

matter oligodendrocytes. While the mechanism is largely unknown, differences in

oligodendrocyte differentiation and survival, as well as differences in pathology in multiple

sclerosis lesions between white and gray matter have been recently reported (Hill et al., 2014;

Hill et al., 2013; Stadelmann et al., 2008).

We next performed q-RT-PCR gene-expression analysis using whole-brain RNA extracts

from 10 day-old, two and seven month-old mice. We observed reduced levels of mRNA

encoding mature oligodendrocyte markers myelin-associated glycoprotein (MAG) and myelin-

associated oligodendrocyte basic protein (MOBP) (Han et al., 2013) in Mcoln1-/- mice at 10 days

(Fig. 5A). Significant decline in MAG an MOBP mRNA levels persisted in Mcoln1-/- mice at

two and seven months of age (Fig. 5B), but wasn’t progressive in the course of disease as shown

by significant effect of genotype (MAG: Fgenotype = 32.96, p<0.0001; MOBP: Fgenotype = 23.67,

p=0.0004) and no significant effect of age (MAG: Fage = 0.12, p=0.73; MOBP: Fage = 0.19,

p=0.67) by two-way ANOVA analysis. These data further confirm that deficient myelination in

Mcoln1-/- brain occurs during development and persists throughout the course of disease.

To determine if impaired differentiation of oligodendrocyte precursors contributes to

decreased density of post-mitotic oligodendrocytes and hypomyelination in the Mcoln1-/- brain,

we analyzed expression of the oligodendrocyte precursor marker PDGFRα (Han et al., 2013;

Perez et al., 2013) in Mcoln1-/- and wild-type mice at 10 days, two and seven months of age. We

observed significant reduction in PDGFRα mRNA levels in 10 day-old Mcoln1-/- mice, and no

significant differences between genotypes in adult mice when myelination is largely complete

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(Fig. 5 A, B). Lower levels of PDGFRa transcripts at 10 days of age may indicate a reduced

number of oligodendrocyte precursors in Mcoln1-/- brain at this critical stage of myelination,

perhaps limiting their differentiation, maturation and myelin deposition. Restoration of the

PDGFRa mRNA levels in adult Mcoln1 knockout mice is not accompanied by restoration of the

mature oligodendrocyte/myelin markers, highlighting the importance of the time-dependent

regulation of this process and perhaps multiple roles of TRPML1 in the oligodendrocyte cell

lineage.

Since iron is an important factor for oligodendrocyte differentiation and maturation

(Todorich et al., 2009), and TRPML1 is directly involved in intracellular iron trafficking

(Coblentz et al., 2014; Dong et al., 2008b), we set out to test the iron status in the Mcoln1-/- brain.

We stained brain sections with Perls’ stain, which detects ferric iron bound to ferritin (Meguro et

al., 2005). As the most iron-rich cells in the brain, mature oligodendrocytes are frequently

visualized using this technique (Schonberg and McTigue, 2009). Mcoln1+/+ brains contained

clearly identifiable Perls’ stain, but such staining is significantly reduced in white-matter tracts in

Mcoln1-/- brains, indicating a loss of H-ferritin-bound iron (Fig. 6 A, B and C). Measurement of

total iron content (Fe2+ and Fe3+) in the unperfused brain by inductively coupled plasma mass

spectrometry (ICP-MS) showed no statistically significant differences in total iron levels

between Mcoln1-/- and wild-type mice, which rules out systemic iron deficiency in Mcoln1-/-

mice (Fig. 6 D). These data show that loss of Mcoln1 results in iron dyshomeostasis in the brain,

which likely plays a role in the observed impaired brain myelination in MLIV.

Iron dyshomeostasis is known to lead to oxidative stress in the brain (Abeliovich and

Flint Beal, 2006; Chen et al., 2013; Hare et al., 2013; Schrag et al., 2011). Therefore, we

evaluated the levels of the marker HMOX1 mRNA in the brain of Mcoln1-/- and wild-type mice.

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Importantly, HMOX mRNA was significantly elevated in Mcoln1-/- brain at all ages evaluated,

indicating prolonged oxidative stress. (Fig. 6 E).

Overall, our data suggest that myelination deficits in Mcoln1-/- mice are caused by defects

in the formation of the myelin sheath, rather than by axonal loss. Our demonstration that markers

of oligodendrocyte precursors, pre-myelinating and mature myelinating oligodendrocytes are all

disrupted in Mcoln1-/- mouse brain suggests that loss of TRPML1 affects all stages of

oligodendrocyte development. Further, our results show impaired iron handling in the brain due

to loss of TRPML1. Since iron is an important trophic factor for oligodendrocyte differentiation

and maturation, improper iron handling due to loss of TRPML1 might account for the observed

dysmyelination in mucolipidosis IV. Our results, although descriptive in nature, provide the first

evidence of impaired iron handling and early myelination defects in MLIV, highlight the

developmental nature of mucolipidosis IV and emphasize the rational for a stronger focus on

oligodendroglia and early-stage myelination as potential targets for the development of

therapeutics.

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Discussion.

In the present study we show that hypomyelination in the MLIV mouse brain is

associated with decreased expression of both precursor and mature oligodendrocyte markers, as

well as a decreased number of post-mitotic oligodendrocytes. Our observation of delayed myelin

deposition during early post-natal brain development highlights the developmental character of

brain pathology in MLIV. Interestingly, our data also indicate that the fate of developing

oligodendrocytes is affected differently in the white (corpus callosum) versus grey (neocortex)

matter of the brain as indicated by changes in CC+ cell counts (Fig. 4) or PLP and MBP staining

intensities in these brain regions. This suggests that tissue microenvironment and factors released

by neighboring cells can contribute to the ability of Mcoln1-/- oligodendrocytes to develop,

survive and/or produce myelin. Despite these region-specific changes within the brain, our data

demonstrate that TRPML1 is an important regulator of myelination in both, central nervous

system and in peripheral nerves. Given the absence of overt neuronal loss in MLIV (Grishchuk et

al., 2014), combined with the preservation of axonal fibers (Fig. 2) in the Mcoln1-/- brain,

oligodendrocyte development and survival should be high-priority targets for the development of

therapeutics.

The main function of oligodendrocytes is myelin synthesis and the formation of myelin

sheaths around axons to facilitate action potential propagation. Pediatric disorders of myelin such

as periventricular white matter injury (PWMI) and hereditary leukodystrophies result in cerebral

palsy-like and cognitive dysfunction similar to those documented in MLIV (Pouwels et al.,

2014). Proliferation of oligodendrocyte precursors, differentiation and maturation relies on

several trophic factors including FGF, PDGF, IGF-1, thyroid hormone and iron (Morath and

Mayer-Proschel, 2001; Rouault, 2013; Todorich et al., 2009). Oligodendrocytes are the most

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iron-rich cells in the brain. As the most metabolically active cells in the brain, oligodendrocytes

require massive amounts of ATP, and iron is involved in oxidative phosphorylation. Iron is a

cofactor for a number of enzymes directly or indirectly involved in myelin synthesis, including

lipid saturase and desaturase (Nakamura and Nara, 2004; Rao et al., 1983; Todorich et al., 2009).

Further, iron deficiency causes a delay in oligodendrocyte maturation and hypomyelination in

both humans and rodents (Hare et al., 2013; Todorich et al., 2009). Injection of ferritin-bound

iron into white matter leads to an increase in the levels of oligodendrocyte progenitor and mature

oligodendrocyte markers (Schonberg et al., 2012; Schonberg and McTigue, 2009). An increase

in intracellular ferritin levels is also evident during the oligodendrocyte maturation process (Li et

al., 2013).

TRPML1 is clearly required for oligodendrocyte development and function, and this is

perhaps what underlies the observed brain pathology in MLIV. The precise role of TRPML1 in

oligodendrocyte development is currently unknown; however several studies highlight

possibilities and suggest a link to the TRPML1 role in iron handling. How might mucolipin-1

regulate iron handling? One possibility is through its function in endo-lysosomal trafficking.

Mucolipin-1 localizes to late endosomes and lysosomes (Kiselyov et al., 2005; Miedel et al.,

2006; Vergarajauregui and Puertollano, 2006) and is known to regulate endo-lysosomal fusion

and membrane trafficking (Dong et al., 2010; LaPlante et al., 2004; Pryor et al., 2006; Samie et

al., 2013; Thompson et al., 2007; Vergarajauregui et al., 2008). In most tissues, iron is absorbed

by endocytosis of Fe3+-transferrin or Fe3+-ferritin (Valerio, 2007). In the endo-lysosomal

compartment Fe3+ is liberated from the protein complex, converted to Fe2+ and absorbed into the

cytoplasm via endo/lysosomal membranes. In the cytoplasm, Fe2+ is oxidized to Fe3+ and binds

to cytoplasmic ferritin. From the cytoplasm, iron enters mitochondria for energy production,

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binds to transferrin for trafficking or exporting, serves as a cofactor for enzymatic reactions such

as myelin production, or binds to cytoplasmic ferritin and other scavengers for storage. Hence,

based on the role of mucolipin-1 in endocytic membrane trafficking, it may regulate ferritin-Fe3+

delivery to the lysosomes.

The second possibility is based on the TRPML1 permeability to divalent heavy metal

ions, including Fe2+ (Dong et al., 2008a), so it could facilitate the direct transport of Fe2+ from

lysosome to cytoplasm. The common transferrin-dependent iron entry pathway relies on the

divalent transporter DMT1 (Siah et al., 2006) for iron release from endo-lysosomes into cytosol.

Interestingly, oligodendrocytes strictly depend on extracellular ferritin as a source of iron (Hulet

et al., 2000; Todorich et al., 2009; Todorich et al., 2011; Todorich et al., 2008), and the role of

DMT1 in iron handling specifically in oligodendrocytes has been disputed (Burdo et al., 2001;

Song et al., 2007). Thus, due to its Fe2+ permeability, mucolipin-1 may supplant DMT1 in

oligodendrocytes totally, or during some stages of development, thus making oligodendrocytes

especially vulnerable to the loss of mucolipin-1. Our demonstration of reduced levels of ferritin-

bound ferric iron in Mcoln1-/- brain by Perls’ stain experimentally supports this idea. Iron bound

to ferritin, specifically to H-ferritin is prevalent in oligodendrocytes and can be visualized using

Perls’ stain. Reduced Perls’ staining in white-matter tracts of Mcoln1-/- mice indicates loss of

biologically available, H-ferritin-bound iron. Since other brain cell types, including neurons, rely

on transferrin endocytosis and DMT1 for iron delivery (Hare et al., 2013; Moos and Morgan,

2004; Paz Soldan and Pirko, 2012; Perez et al., 2013; Todorich et al., 2011; Valerio, 2007), it is

possible that mucolipin-1 loss might not affect neuronal iron handling or total iron content.

Supporting this hypothesis, our ICP-MS data show no changes in the total amount of iron in the

unperfused brain tissue between Mcoln1-/- and control mice. Therefore, the

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maturation/myelination deficits in MLIV cannot be explained by a general brain iron deficiency.

Rather, they are directly related to oligodendrocyte malfunction.

Given that TRPML1 is permeable to Fe2+ but not Fe3+, it is possible that loss of TRPML1

leads to lysosomal buildup of Fe2+ which is known to catalyze Fenton-like reactions resulting in

the formation of reactive oxygen species (ROS). Previously, oxidative stress has been shown in

both in vitro cellular models (Coblentz et al., 2014) and in a Drosophila model (Venkatachalam

et al., 2008), and in this study we show for the first time evidence of oxidative stress in vivo in

the MLIV mouse brain.

Regardless of the precise role of the ion channel TRPML1 in regulating iron in the brain

our studies suggest that it is a new target for pharmacological intervention for conditions linked

to brain iron handling and myelination. Our data also indicate that restoration of myelin is an

attractive therapeutic strategy for MLIV, and further studies will reveal whether mechanisms

leading to impaired myelination in MLIV rely solely on oligodendrocyte intrinsic (cell-

autonomous) mechanisms.

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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 US National Institute 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 10 day-, two and seven month-old Mcoln1-/-

and control mice were transcardially perfused under isoflurane anesthesia with ice-cold

phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were

postfixed in 4% paraformaldehyde in PBS for 24 hours, washed with PBS, cryoprotected in 30%

sucrose in PBS overnight, frozen in isopentane and stored at -800C. Brains were bisected along

the midline and one hemisphere was examined histologically. 40μm coronal sections were cut

using a Microm freezing microtome and collected into 96 well plates containing TBSAF (TBS,

30% ethylene glycol, 15% sucrose, 0.05% sodium azide). These sections were stored at 4°C

prior to any staining procedures. For experiments, the corresponding serial sections were used

for each brain. For PLP, MBP and SMI312 staining sections were microwaved in citrate buffer

(10 mM citric acid, 0.05% Tween 20, pH 6.0) at low power for 10 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% NGS overnight. The following primary antibodies were used:

PLP rabbit polyclonal, 1:500, ab28486; MBP (SMI99), mouse monoclonal, 1:1000, ab24568;

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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: goat 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 633 (1:500, Invotrogen, Eugene,

OR, USA). Sections were counterstained with NucBlue nuclear stain (Life Technologies,

Eugene, OR, USA) and mounted on microscopic slides.

For PLP, MBP and SMI312 staining analysis images were acquired on Nikon 80i upright

epifluorescence microscope with Hamamatsu Orca CCD camera (Nikon, Tokyo, Japan) with x5

objective and NIS-Elements 4.2 software (Nikon, Tokyo, Japan) using an automated stitching

function. The exposure time was set to avoid saturation and was the same 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 (whole hemi-section,

cortical region or corpus callosum) were selected in each section. Area and mean pixel intensity

values were compared between genotypes using GraphPad Prizm 5 software (GraphPad, La

Jolla, CA, USA) using Student’s T-test.

Quantitative morphological analysis of corpus callosum was done using AnalyzeSkeleton plugin

of ImageJ (Arganda-Carreras et al., 2010). 200 x 200 px regions of interests (ROI) of corpus

callosum were selected on images of PLP-stained sections of 10 day-old brains. Selections were

converted to 8 bit images, made binary and skeletonized using Skeletonize 2D/3D option and

analyzed by AnalyzeSkeleton plugin. An investigator was blind to genotypes. Total counts of

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number of branches, junctions, end-point voxels, junction voxels, slab voxels, triple and

quadruple points, average and maximal branch lengths per region of interest per mouse were

averaged across animals in genotype groups (n=5 (WT); n=4 (Mcoln1-/-)) and compared in

GraphPad Prizm 5 software (GraphPad, La Jolla, CA, USA) using Student’s T-test.

For APC CC1 analysis we acquired images of two non-overlapping fields of view in

somatosensory/motor cortex and corpus callosum using x20 objective (HCX PL APO CS

20.0x0.70 DRY UV) and confocal laser scanning microscope Leica TCS SP5 (Leica

Microsystems Inc., Wetzlar, Germany). Number of CC1+ cells was measured in FIJI in

corresponding areas of interest (cortex or corpus callosum). CC1 cell density expressed as

number of cells in mm2 of the tissue. Statistical analysis of data (two-way ANOVA with

Bonferroni correction to assess the effect of genotype and age) was performed using GraphPad

Prizm 5 (GraphPad, La Jolla, CA, USA).

Perls’ staining

For Perls’ staining sections were incubated in Potassium Ferrocyanide with HCl (10% Potassium

Ferrocyanide: 10% HCl (2:1)) for 60 minutes with rocking. Sections were washed with water,

incubated with inactivated DAB (no H2O2 added) and activated DAB, for 30 minutes each time.

After washing in water, sections were mounted on microscopic slides, dried and coverslipped.

Bright-field images were acquired using Leica DMI6000 microscope using DFC425 camera and

HCX PL S-APO 5.0x0.15 objective (Leica Microsystems Inc., Wetzlar, Germany). Inverted

mean pixel intensities were measured in FIJI (NIH, Bethesda, Maryland, USA). For background

normalization, inverted mean pixel intensity measured outside of the brain tissue was subtracted

from inverted mean pixel intensity values for every image. Students T-test was used for

comparisons between genotypes.

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Western blot analysis

For immunoblotting fresh-frozen pieces of sensory-motor cortex were homogenized in the lysis

buffer (20 mmol/L HEPES, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, 2.5 mmol/L EGTA,

0.1 mmol/L dithiothreitol, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1% Triton X-100 (reagents from

Sigma, LaJolla, CA, USA), and a protease inhibitor cocktail (Roche, 11873580001, Mannheim,

Germany)). Homogenates were centrifuged at 5000 rpm for 10 min at 4oC, supernatants

collected and protein concentration was determined using Pierce BSA Protein Assay kit

(ThermoFisher Scientific, Waltham, MA, USA). Sciatic nerves were homogenized in

radioimmunoprecipitation assay buffer (RIPA, Boston BioProducts, Ashland, MA, USA)

supplemented with 0.1 mmol/L EDTA and a protease inhibitor cocktail (Roche, 11873580001,

Mannheim, Germany) according to (Vargas et al., 2010). Proteins (20 μg for cortex and sciatic

nerve homogenates from 2 and 7 month-old mice; 40μg for 10 day-old cortex homogenates)

were separated by SDS-PAGE on a 4-12% Bis-Tris gels 3-(N-morpholino)propansulfonic acid

(MOPS) buffer (Invitrogen, Waltham, MA, USA) and transferred onto nitrocellulose membrane

(ThermoFisher Scientific, Waltham, MA, USA). The following primary antibodies were used:

anti-proteolipid protein (ab28486, 1/1000, rabbit polyclonal, Abcam, Cambridge, MA, USA),

anti-myelin basic protein (NE1019, mouse monoclonal, 1/1000, EMD Millipore, Billerica, MA,

USA), anti-β-actin (A5441, 1/3000, mouse monoclonal, Sigma, LaJolla, CA, USA). After

incubation with primary antibody, the following secondary antibodies were applied: polyclonal

goat anti-mouse or goat anti-rabbit IgG conjugated with IRDye 680 or IRDye 800 both from LI-

COR (Lincoln, NE, USA). Protein bands were visualized using the Odyssey Infrared Imaging

System and analyzed by Odyssey v3.0 software (LI-COR, Lincoln, NE, USA). In densitometric

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analysis OD values were normalized by b-actin. All data were expressed as mean ± SEM. Data

were analyzed statistically by Student’s t-test.

RNA extraction and q-RT-PCR

For q-RT-PCR experiments, total RNA was extracted from brain tissue from 10 day-, two- and

seven-month-old Mcoln1-/- and control mice using TRizol (Invitrogen, Carlsbag, CA), according

to manufacturer’s protocol. 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,

Cralville, IA, USA) as 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, Cralville,

IA, USA): Pdgfrα, forward 5’-TGGAAGCTTGGGGCTTACTTT-3’ and reverse 5’-

TAGCTCCTGAGACCTTCTCCT-3’; Plp, forward 5’-GTTCCAGAGGCCAACATCAA-3’ and

reverse 5’-CCACAAACTTGTCGGGATGT-3’; Mbp, forward 5’-

GTGCCACATGTACAAGGACT-3’ and reverse 5’-TGGGTTTTCATCTTGGGTCC-3’; Mag,

forward 5’-CGGGATTGTCACTGAGAGC-3’ and reverse 5’-AGGTCCAGTTCTGGGGATTC-

3’; Mobp, forward 5’-CACCCTTCACCTTCCTCAAC-3’ and reverse 5’-

TTCTGGTAAAAGCAACCGCT-3’; Hmox1, forward 5’-CACAGATGGCGTCACTTCCGTC-

3’ and reverse 5’-GTGAGGACCCACTGGGAGGAG-3’; Actb, forward 5’-

GCTCCGGCATGTGCAAAG-3’ and reverse 5’-CATCACACCCTGGTGCCTA-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 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

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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

(Suppl. Fig. 4). The ΔΔCt method was used to calculate relative gene expression, where Ct

corresponds to the cycle threshold. ΔCt values were calculated as the difference between Ct

values from the target gene and the housekeeping gene Actb (Suppl. Table 1). 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 represented as fold change.

ICP-MS

ICP-MS analysis of mouse cortical tissues was performed at the Department of Geology and

Planetary Science, University of Pittsburgh. Frozen brain samples were coded and analyzed by in

a blind manner. Mouse cortical tissues were weighed, dissolved in pure nitric acid, dried,

reconstituted in 2% nitric acid and analyzed using PerkinElmer NexION 350 ICM-MS

Spectrometer (PerkinElmer Inc., Waltham, MA, USA). Iron concentration was normalized to the

total wet tissue weight. In addition to iron, sodium and other metals were analyzed. As an

additional analysis tool, iron concentration was normalized to sodium content.

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Acknowledgements:

This work was supported by grant from ML4 foundation to S.A.S. and Y.G. and NIH grants

HD058577 and ES01678 to K.K. Authors thank Drs. James Connor and Amanda Snyder for help

with the Perls’ staining protocol. We thank Dr. Daniel Bain (University of Pittsburgh) for help

with ICP-MS.

Competing interests:

Authors report no competing interests.

Author contribution:

Y.G., and K.K. designed the study, Y.G., K.A.P., J.C., V.E.K., D.M.H., S.L.W. performed

experiments and data analysis, Y.G., K.K. and S.A.S wrote the manuscript.

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References:

Abeliovich, A. and Flint Beal, M. (2006). Parkinsonism genes: culprits and clues. J

Neurochem 99, 1062-72.

Altarescu, G., Sun, M., Moore, D. F., Smith, J. A., Wiggs, E. A., Solomon, B. I.,

Patronas, N. J., Frei, K. P., Gupta, S., Kaneski, C. R. et al. (2002). The neurogenetics of

mucolipidosis type IV. Neurology 59, 306-13.

Arganda-Carreras, I., Fernandez-Gonzalez, R., Munoz-Barrutia, A. and Ortiz-De-

Solorzano, C. (2010). 3D reconstruction of histological sections: Application to mammary gland

tissue. Microsc Res Tech 73, 1019-29.

Bach, G. (2001). Mucolipidosis type IV. Mol Genet Metab 73, 197-203.

Bach, G., Cohen, M. M. and Kohn, G. (1975). Abnormal ganglioside accumulation in

cultured fibroblasts from patients with mucolipidosis IV. Biochem Biophys Res Commun 66,

1483-90.

Bach, G. and Desnick, R. J. (1988). Lysosomal accumulation of phospholipids in

mucolipidosis IV cultured fibroblasts. Enzyme 40, 40-4.

Bach, G., Ziegler, M., Kohn, G. and Cohen, M. M. (1977). Mucopolysaccharide

accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV. American

Journal of Human Genetics 29, 610-8.

Bagdon, M. M., Lubs, M. L., Phillips, J. A., 3rd, Murray, J. C., Slaugenhaupt, S. A.,

Chakravarti, A., Adler, W. H. and Bach, G. (1989). Lysosomal accumulation of phospholipids

in mucolipidosis IV cultured fibroblasts. American Journal of Human Genetics 44, 48-50.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Bargal, R., Avidan, N., Ben-Asher, E., Olender, Z., Zeigler, M., Frumkin, A., Raas-

Rothschild, A., Glusman, G., Lancet, D. and Bach, G. (2000). Identification of the gene

causing mucolipidosis type IV. Nat Genet 26, 118-23.

Bargal, R. and Bach, G. (1988). Phospholipids accumulation in mucolipidosis IV

cultured fibroblasts. J Inherit Metab Dis 11, 144-50.

Bargal, R. and Bach, G. (1989). Phosphatidylcholine storage in mucolipidosis IV. Clin

Chim Acta 181, 167-74.

Bassi, M. T., Manzoni, M., Monti, E., Pizzo, M. T., Ballabio, A. and Borsani, G.

(2000). Cloning of the gene encoding a novel integral membrane protein, mucolipidin-and

identification of the two major founder mutations causing mucolipidosis type IV. Am J Hum

Genet 67, 1110-20.

Baumann, N. and Pham-Dinh, D. (2001). Biology of oligodendrocyte and myelin in the

mammalian central nervous system. Physiol Rev 81, 871-927.

Berman ER, L. N., Shapira E, Merin S, Levij IS. (1974). Congenital corneal clouding

with abnormal systemic storage bodies: a new variant of mucolipidosis. J Pediatr. 84, 519-26

Burdo, J. R., Menzies, S. L., Simpson, I. A., Garrick, L. M., Garrick, M. D., Dolan,

K. G., Haile, D. J., Beard, J. L. and Connor, J. R. (2001). Distribution of divalent metal

transporter 1 and metal transport protein 1 in the normal and Belgrade rat. J Neurosci Res 66,

1198-207.

Chandra, M., Zhou, H., Li, Q., Muallem, S., Hofmann, S. L. and Soyombo, A. A.

(2011). A role for the Ca2+ channel TRPML1 in gastric acid secretion, based on analysis of

knockout mice. Gastroenterology 140, 857-67.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Chen, C. C., Keller, M., Hess, M., Schiffmann, R., Urban, N., Wolfgardt, A.,

Schaefer, M., Bracher, F., Biel, M., Wahl-Schott, C. et al. (2014). A small molecule restores

function to TRPML1 mutant isoforms responsible for mucolipidosis type IV. Nat Commun 5,

4681.

Chen, J., Marks, E., Lai, B., Zhang, Z., Duce, J. A., Lam, L. Q., Volitakis, I., Bush,

A. I., Hersch, S. and Fox, J. H. (2013). Iron accumulates in Huntington's disease neurons:

protection by deferoxamine. PLoS One 8, e77023.

Coblentz, J., St Croix, C. and Kiselyov, K. (2014). Loss of TRPML1 promotes

production of reactive oxygen species: is oxidative damage a factor in mucolipidosis type IV?

Biochem J 457, 361-8.

Colletti, G. A. and Kiselyov, K. (2011). Trpml1. Adv Exp Med Biol 704, 209-19.

Dong, X., Cheng, X., E., M., M., D., F., W., Kurz, T. and Xu, H. (2008a). The type IV

mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel. Nature

455, 992-996.

Dong, X. P., Cheng, X., Mills, E., Delling, M., Wang, F., Kurz, T. and Xu, H.

(2008b). The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron

release channel. Nature 455, 992-6.

Dong, X. P., Shen, D., Wang, X., Dawson, T., Li, X., Zhang, Q., Cheng, X., Zhang,

Y., Weisman, L. S., Delling, M. et al. (2010). PI(3,5)P(2) controls membrane trafficking by

direct activation of mucolipin Ca(2+) release channels in the endolysosome. Nat Commun 1, 38.

Folkerth RD, A. J., Lomakina I, Skutelsky E, Raghavan SS, Kolodny EH. (1995).

Mucolipidosis IV: morphology and histochemistry of an autopsy case. J Neuropathol Exp Neurol

54, 154-64.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Frei KP, P. N., Crutchfield KE, Altarescu G, Schiffmann R (1998) Mucolipidosis

type IV: characteristic MRI findings. Neurology. 51: 565-569. (1998). Mucolipidosis type IV:

characteristic MRI findings. Neurology 51, 565-569.

Grishchuk, Y., Sri, S., Rudinskiy, N., Ma, W., Stember, K. G., Cottle, M. W., Sapp,

E., Difiglia, M., Muzikansky, A., Betensky, R. A. et al. (2014). Behavioral deficits, early

gliosis, dysmyelination and synaptic dysfunction in a mouse model of mucolipidosis IV. Acta

Neuropathol Commun 2, 133.

Han, H., Myllykoski, M., Ruskamo, S., Wang, C. and Kursula, P. (2013). Myelin-

specific proteins: a structurally diverse group of membrane-interacting molecules. Biofactors 39,

233-41.

Hare, D., Ayton, S., Bush, A. and Lei, P. (2013). A delicate balance: Iron metabolism

and diseases of the brain. Front Aging Neurosci 5, 34.

Hill, R. A., Patel, K. D., Goncalves, C. M., Grutzendler, J. and Nishiyama, A. (2014).

Modulation of oligodendrocyte generation during a critical temporal window after NG2 cell

division. Nat Neurosci 17, 1518-27.

Hill, R. A., Patel, K. D., Medved, J., Reiss, A. M. and Nishiyama, A. (2013). NG2

cells in white matter but not gray matter proliferate in response to PDGF. J Neurosci 33, 14558-

66.

Huang, H., Zhao, X. F., Zheng, K. and Qiu, M. (2013). Regulation of the timing of

oligodendrocyte differentiation: mechanisms and perspectives. Neurosci Bull 29, 155-64.

Hulet, S. W., Heyliger, S. O., Powers, S. and Connor, J. R. (2000). Oligodendrocyte

progenitor cells internalize ferritin via clathrin-dependent receptor mediated endocytosis. J

Neurosci Res 61, 52-60.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Kiselyov, K., Chen, J., Rbaibi, Y., Oberdick, D., Tjon-Kon-Sang, S., Shcheynikov,

N., Muallem, S. and Soyombo, A. (2005). TRP-ML1 is a lysosomal monovalent cation channel

that undergoes proteolytic cleavage. J Biol Chem 280, 43218-23.

LaPlante, J. M., Ye, C. P., Quinn, S. J., Goldin, E., Brown, E. M., Slaugenhaupt, S.

A. and Vassilev, P. M. (2004). Functional links between mucolipin-1 and Ca2+-dependent

membrane trafficking in mucolipidosis IV. Biochem Biophys Res Commun 322, 1384-91.

Li, Y., Guan, Q., Chen, Y., Han, H., Liu, W. and Nie, Z. (2013). Transferrin receptor

and ferritin-H are developmentally regulated in oligodendrocyte lineage cells. Neural Regen Res

8, 6-12.

Meguro, R., Asano, Y., Odagiri, S., Li, C., Iwatsuki, H. and Shoumura, K. (2005).

The presence of ferric and ferrous iron in the nonheme iron store of resident macrophages in

different tissues and organs: histochemical demonstrations by the perfusion-Perls and -Turnbull

methods in the rat. Arch Histol Cytol 68, 171-83.

Miedel, M. T., Weixel, K. M., Bruns, J. R., Traub, L. M. and Weisz, O. A. (2006).

Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1. J

Biol Chem 281, 12751-9.

Mirabelli-Badenier, M., Severino, M., Tappino, B., Tortora, D., Camia, F.,

Zanaboni, C., Brera, F., Priolo, E., Rossi, A., Biancheri, R. et al. (2014). A novel

homozygous MCOLN1 double mutant allele leading to TRP channel domain ablation underlies

Mucolipidosis IV in an Italian Child. Metab Brain Dis.

Moos, T. and Morgan, E. H. (2004). The significance of the mutated divalent metal

transporter (DMT1) on iron transport into the Belgrade rat brain. J Neurochem 88, 233-45.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Morath, D. J. and Mayer-Proschel, M. (2001). Iron modulates the differentiation of a

distinct population of glial precursor cells into oligodendrocytes. Dev Biol 237, 232-43.

Nakamura, M. T. and Nara, T. Y. (2004). Structure, function, and dietary regulation of

delta6, delta5, and delta9 desaturases. Annu Rev Nutr 24, 345-76.

Noffke, A. S., Feder, R. S., Greenwald, M. J., O'Grady, R. B. and Roth, S. I. (2001).

Mucolipidosis IV in an African American patient with new findings on electron microscopy.

Cornea 20, 536-9.

Paz Soldan, M. M. and Pirko, I. (2012). Biogenesis and significance of central nervous

system myelin. Semin Neurol 32, 9-14.

Perez, M. J., Fernandez, N. and Pasquini, J. M. (2013). Oligodendrocyte

differentiation and signaling after transferrin internalization: a mechanism of action. Exp Neurol

248, 262-74.

Pouwels, P. J., Vanderver, A., Bernard, G., Wolf, N. I., Dreha-Kulczewksi, S. F.,

Deoni, S. C., Bertini, E., Kohlschutter, A., Richardson, W., Ffrench-Constant, C. et al.

(2014). Hypomyelinating leukodystrophies: translational research progress and prospects. Ann

Neurol 76, 5-19.

Pryor, P. R., Reimann, F., Gribble, F. M. and Luzio, J. P. (2006). Mucolipin-1 is a

lysosomal membrane protein required for intracellular lactosylceramide traffic. Traffic 7, 1388-

98.

Rao, G. A., Crane, R. T. and Larkin, E. C. (1983). Reduction of hepatic stearoyl-CoA

desaturase activity in rats fed iron-deficient diets. Lipids 18, 573-5.

Riedel, K. G., Zwaan, J., Kenyon, K. R., Kolodny, E. H., Hanninen, L. and Albert,

D. M. (1985). Ocular abnormalities in mucolipidosis IV. Am J Ophthalmol 99, 125-36.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Rouault, T. A. (2013). Iron metabolism in the CNS: implications for neurodegenerative

diseases. Nat Rev Neurosci 14, 551-64.

Samie, M., Wang, X., Zhang, X., Goschka, A., Li, X., Cheng, X., Gregg, E., Azar,

M., Zhuo, Y., Garrity, A. G. et al. (2013). A TRP channel in the lysosome regulates large

particle phagocytosis via focal exocytosis. Dev Cell 26, 511-24.

Schiffmann, R., Mayfield, J., Swift, C. and Nestrasil, I. (2014). Quantitative

neuroimaging in mucolipidosis type IV. Mol Genet Metab 111, 147-51.

Schonberg, D. L., Goldstein, E. Z., Sahinkaya, F. R., Wei, P., Popovich, P. G. and

McTigue, D. M. (2012). Ferritin stimulates oligodendrocyte genesis in the adult spinal cord and

can be transferred from macrophages to NG2 cells in vivo. J Neurosci 32, 5374-84.

Schonberg, D. L. and McTigue, D. M. (2009). Iron is essential for oligodendrocyte

genesis following intraspinal macrophage activation. Exp Neurol 218, 64-74.

Schrag, M., Mueller, C., Oyoyo, U., Smith, M. A. and Kirsch, W. M. (2011). Iron,

zinc and copper in the Alzheimer's disease brain: a quantitative meta-analysis. Some insight on

the influence of citation bias on scientific opinion. Prog Neurobiol 94, 296-306.

Siah, C. W., Ombiga, J., Adams, L. A., Trinder, D. and Olynyk, J. K. (2006). Normal

iron metabolism and the pathophysiology of iron overload disorders. Clin Biochem Rev 27, 5-16.

Slaugenhaupt, S. A., Lewis, J. G., Chakravarti, A., Antonarakis, S. E. and Bargal,

R. (1989). Phosphatidylcholine storage in mucolipidosis IV. Genomics 4, 579-91.

Smith, J. A., Chan, C. C., Goldin, E. and Schiffmann, R. (2002). Noninvasive

diagnosis and ophthalmic features of mucolipidosis type IV. Ophthalmology 109, 588-94.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Song, N., Jiang, H., Wang, J. and Xie, J. X. (2007). Divalent metal transporter 1 up-

regulation is involved in the 6-hydroxydopamine-induced ferrous iron influx. J Neurosci Res 85,

3118-26.

Stadelmann, C., Albert, M., Wegner, C. and Bruck, W. (2008). Cortical pathology in

multiple sclerosis. Curr Opin Neurol 21, 229-34.

Sun, M., Goldin, E., Stahl, S., Falardeau, J. L., Kennedy, J. C., Acierno, J. S., Jr.,

Bove, C., Kaneski, C. R., Nagle, J., Bromley, M. C. et al. (2000). Mucolipidosis type IV is

caused by mutations in a gene encoding a novel transient receptor potential channel. Hum Mol

Genet 9, 2471-8.

Tellez-Nagel I, R. I., Iwamoto T, Johnson AB, Norton WT, Nitowsky H. (1976).

Mucolipidosis IV. Clinical, ultrastructural, histochemical, and chemical studies of a case,

including a brain biopsy. Arch Neurol Dec 33, 828-35

Thompson, E. G., Schaheen, L., Dang, H. and Fares, H. (2007). Lysosomal trafficking

functions of mucolipin-1 in murine macrophages. BMC Cell Biol 8, 54.

Todorich, B., Pasquini, J. M., Garcia, C. I., Paez, P. M. and Connor, J. R. (2009).

Oligodendrocytes and myelination: the role of iron. Glia 57, 467-78.

Todorich, B., Zhang, X. and Connor, J. R. (2011). H-ferritin is the major source of iron

for oligodendrocytes. Glia 59, 927-35.

Todorich, B., Zhang, X., Slagle-Webb, B., Seaman, W. E. and Connor, J. R. (2008).

Tim-2 is the receptor for H-ferritin on oligodendrocytes. J Neurochem 107, 1495-505.

Tuysuz, B., Goldin, E., Metin, B., Korkmaz, B. and Yalcinkaya, C. (2009).

Mucolipidosis type IV in a Turkish boy associated with a novel MCOLN1 mutation. Brain Dev

31, 702-5.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Acce

pted

man

uscr

ipt

Valerio, L. G. (2007). Mammalian iron metabolism. Toxicol Mech Methods 17, 497-517.

Vargas, M. E., Watanabe, J., Singh, S. J., Robinson, W. H. and Barres, B. A. (2010).

Endogenous antibodies promote rapid myelin clearance and effective axon regeneration after

nerve injury. Proc Natl Acad Sci U S A 107, 11993-8.

Vasung, L., Jovanov-Milosevic, N., Pletikos, M., Mori, S., Judas, M. and Kostovic, I.

(2011). Prominent periventricular fiber system related to ganglionic eminence and striatum in the

human fetal cerebrum. Brain Struct Funct 215, 237-53.

Venkatachalam, K., Long, A. A., Elsaesser, R., Nikolaeva, D., Broadie, K. and

Montell, C. (2008). Motor deficit in a Drosophila model of mucolipidosis type IV due to

defective clearance of apoptotic cells. Cell 135, 838-51.

Venugopal, B., Browning, M. F., Curcio-Morelli, C., Varro, A., Michaud, N.,

Nanthakumar, N., Walkley, S. U., Pickel, J. and Slaugenhaupt, S. A. (2007). Neurologic,

gastric, and opthalmologic pathologies in a murine model of mucolipidosis type IV. Am J Hum

Genet 81, 1070-83.

Vergarajauregui, S., Connelly, P. S., Daniels, M. P. and Puertollano, R. (2008).

Autophagic dysfunction in mucolipidosis type IV patients. Hum Mol Genet 17, 2723-37.

Vergarajauregui, S. and Puertollano, R. (2006). Two di-leucine motifs regulate

trafficking of mucolipin-1 to lysosomes. Traffic 7, 337-53.

Wakabayashi, K., Gustafson, A. M., Sidransky, E. and Goldin, E. (2011).

Mucolipidosis type IV: an update. Mol Genet Metab 104, 206-13.

Wang, W., Zhang, X., Gao, Q. and Xu, H. (2014). TRPML1: an ion channel in the

lysosome. Handb Exp Pharmacol 222, 631-45.

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Figures

Figure 1. Reduced myelination in Mcoln1-/- mouse brain. (A, B) PLP immunostaining on

coronal brain hemi-sections from two (A) and seven month-old (B) Mcoln1-/- mice and control

littermates shows reduced mean pixel intensity in both the hemi-section and cortical region, and

reduced surface area of corpus callosum in Mcoln1-/- sections. Right panels represent magnified

images of the regions of interest shown in white selection areas on the images in the left panel.

Scale bar, left panel = 500 µm; right panel = 100 µm. N= 5 per genotype (2 mo), n=3 per

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genotype (7 mo). (C, D) Western blot images and quantification show significant reduction in

the level of PLP and MBP in cerebral cortex homogenates from Mcoln1-/- mice at the age of two

(C) and seven (D) months. Samples from three individual mice per genotype group are loaded in

duplicates. All graphs represent average values ± S.E.M. Student’s T-Test: *** p<0.0001,

**p<0.01, *p<0.05, n.s p>0.05.

Figure 2. Preserved axonal projections in Mcoln1-/- mouse brain. Immunostaining with pan-

axonal marker SMI312 on coronal brain hemi-sections from two (A) and seven (B) Mcoln1-/-

mice and control littermates shows no significant difference in SMI312 mean pixel intensity or

surface area of corpora callosa between control and Mcoln1-/- sections, except for slight increase

in SMI312 mean pixel intensity in the corpus callosum of 7 month-old Mcoln1-/- mice. All

graphs represent average values ± S.E.M. Student’s T-Test: *** p<0.0001, **p<0.01, *p<0.05,

n.s p>0.05.

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Figure 3. Myelin disruption in 10 day-old Mcoln1-/- mice. (A). PLP immunostaining on

coronal brain hemi-sections shows morphologically altered, “patchy” myelination in Mcoln1-/-

mice compared to wild-type littermates, while no significant changes in the overall mean pixel

intensity or surface area was observed. Right panel is magnified images of the regions of interest

shown in white selection areas on the images in the left panel. Scale bar, left panel = 500 µm;

right panel = 100 µm. (B). Western blot images and quantification showing significant reduction

in the levels of PLP and MBP in cerebral cortex of Mcoln1-/- pups. Samples from three individual

mice per genotype group are loaded in duplicates. All graphs represent average values ± S.E.M,

n=3 per genotype. Student’s T-Test: *** p<0.0001, **p<0.01, *p<0.05, n.s p>0.05.

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Figure 4. Decreased density of oligodendrocytes in Mcoln1-/- brain. A. APC CC1

immunostaining in the corpus callosum (outlined by white lines) of 10 day- and 2 month-old

Mcoln1-/- mice and their wild-type littermates. Scale bars =50 µm. Two-way ANOVA analysis of

CC1+ cell (post-mitotic oligodendrocytes) density shows significant effect of age (F=47.11;

p<0.0001) and genotype (F=12.34; p=0.0034). CC1+ cell density significantly decreased in

Mcoln1-/- corpora callosa at 10 days of age (Bonferroni post-test, p<0.01). N (P10) = 4 per

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genotype; n (2 mo) = 5 per genotype. B. APC CC1 immunostaining in cerebral cortex of 10 day-

and 2 month-old Mcoln1-/- mice and their wild-type littermates. Scale bars =50 µm. Two-way

ANOVA analysis of CC1+ cell density shows significant interaction between age and genotype

(F=14.74; p=0.0024). CC1+ cell density significantly decreased in Mcoln1-/- cortex at 2 months

of age (Bonferroni post-test, p<0.01); n (P10) = 3 per genotype; n (2 mo) = 5 per genotype.

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Figure 5. Decreased markers of oligodendrocyte precursor and mature oligodendrocyte in

Mcoln1-/- mouse brain. Q-RT-PCR analysis of Pdgfrα, Mag and Mobp expression in 10 day-old

(A), two and seven month-old (B) Mcoln1-/- and wild-type brains (n=4 per genotype for each

time-point). RNA of each sample was extracted from whole brain. Q-RT-PCR reactions

performed blind to investigator using numerically-coded samples. Each reaction was performed

in duplicate. The data are analyzed using comparative dCt method and presented as percentage

of WT values. T-test was used for statistical analysis of P10 data; two-way ANOVA test with

Bonferroni correction was used to assess the effect of genotype and age on mRNA levels for 2

and 7 months-old data. * : p<0.05; **: p<0.01; n.s: p>0.05.

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Figure 6. Decreased levels of ferric iron and oxidative stress in the Mcoln1-/- brain. A.

Coronal brain sections stained for Fe3+ content by modified Perls’ protocol. Note an increase of

iron deposits with age in the white matter of control mice, and reduced staining in the Mcoln1-/-

brain sections. Scale bar =1 mm. B. Higher magnification images of Perls’ stained WT and

Mcoln1-/- brain sections from white quadrants selections shown in A demonstrate lower staining

density in Mcoln1-/- white matter. Scale bar =50 µm C. Quantification of Perls’ staining shows

significantly reduced staining in Mcoln1-/- brains at both two and seven months of age (Students’

T-test: p (2mo) = 0.025; p (7mo) = 0.02); n=5 (WT), n= 4 (KO) at two months, and n=4 (WT),

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n=5 (KO) per genotype at seven months. D. ICP-MS analysis of brain cortex homogenates of 7

month-old Mcoln1+/+ and Mcoln1-/- mice done in a blind experiment (n=4 per genotype). The

differences are not significant (p=0.52 and p=0.30). E. Oxidative stress in MLIV brain. Q-RT-

PCR analysis of Hmox1 expression in whole-brain homogenates of Mcoln1+/+ and Mcoln1-/-

mice (n=4 per genotype). Actb was used as a housekeeping gene. The experiments were

performed blind to investogator. The data presented as percentage of wild-type mRNA levels ±

SEM. T-test was used for comparison between genotypes for data obtained in 10 day-old

samples *: p<0.05. two-way ANOVA test of data sets at 2 and 7 months showed significant

increase of Hmox1 expression with age (Fgenotype x age = 7.2; p=0.014); Bonferroni post-test: ***:

p<0.0001; n.s: p>0.05.

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Table 1. Morphological analysis of corpus callosum in 10 day-old Mcoln1-/- mice.

Counts (± SEM) WT Mcoln1-/- p (T-test)

n=5 n=4

# branches 162.8 (±15.4) 108 (±11.1) 0.029

# junctions 76.8 (±8.9) 39 (±7.4) 0.016

# end-point voxels 86.4 (±9.6) 109 (±15.9) 0.242

# junction voxels 176.2 (±21.7) 87.75 (±18.3) 0.019

# slab voxels 835.6 (±43.9) 541.75 (±49.9) 0.003

Average branch length 138 (±28.5) 206.1 (±40.95) 0.201

# triple points 59.2 (±6.2) 32.25 (±6.02) 0.018

# quadruple points 14 (±2.1) 5 (±0.82) 0.009

Max branch length 199.48 (±40) 295.7 (±58.65) 0.199

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Translational impact:

Clinical Issue: Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by

mutations in MCOLN1, which encodes the lysosomal ion channel TRPML1 (mucolipin-1).

Delayed motor and cognitive development in MLIV patients becomes noticeable during the first

year of life. Neurologic development in MLIV patients corresponds on average to 15 months of

age and remains stable in the second and third decades of life. White matter abnormalities and a

hypoplastic corpus callosum are the major hallmarks of brain pathology in MLIV. At present,

there is no specific treatment for this disease due to poor understanding of its pathogenesis.

Results: We show that loss of TRPML1 in the mouse model of MLIV (Mcoln1 knock-out

mouse) results in reduced myelination in both the white matter tracts and the gray matter of the

brain, similar to what is observed in MLIV patients. Our data indicates that oligodendrocyte

differentiation, maturation, and region-dependent loss of oligodendrocytes contribute to

dysmyelination in Mcoln1-/- mice. Iron is a key factor for oligodendrocyte development and

myelination, and iron deficiency in children is often associated with hypomyelination and

neurological disabilities. We observed reduced Perls’ staining in Mcoln1-/- brain indicating lower

levels of ferric iron, while the total iron content in the brain was not changed. These findings

indicate that the dysmyelination in MLIV may be caused by impaired iron handling in the brain.

Mechanistically the role of TRPML1 in brain iron traffic could be explained either by its

function as a lysosomal iron channel releasing ferrous iron from the lysosome to cytoplasm, or

by its involvement in the endo-lysosomal membrane trafficking pathways important for iron

delivery and distribution within the brain.

Implications and future directions: Iron is vital to brain development, yet mechanisms of its

regulation are poorly understood and our data suggests that TRPML1 may be an important

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player. Our future work will be focused on determining the molecular mechanism underlying

dysmyelination in MLIV and will provide an experimental basis for new therapeutic strategies

for this incurable devastating disease, which might include iron supplementation and cell

transplantation. Beyond this rare disease, a better understanding of the role of TRPML1 in brain

iron regulation provides a new target for therapies aimed at overcoming the consequences of

childhood brain iron deficiency, the most common micronutrient deficiency world-wide.

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