RESEARCH ARTICLE

Early evidence of delayed oligodendrocyte maturation in themouse model of mucolipidosis type IVMolly Mepyans1,*, Livia Andrzejczuk2,*, Jahree Sosa2, Sierra Smith1, Shawn Herron1, Samantha DeRosa1,Susan A. Slaugenhaupt1, Albert Misko1, Yulia Grishchuk1,‡,§ and Kirill Kiselyov2,‡,§

ABSTRACTMucolipidosis type IV (MLIV) is a lysosomal disease caused bymutations in the MCOLN1 gene that encodes the endolysosomaltransient receptor potential channel mucolipin-1, or TRPML1. MLIVresults in developmental delay, motor and cognitive impairments, andvision loss. Brain abnormalities include thinning and malformation ofthe corpus callosum, white-matter abnormalities, accumulation ofundegraded intracellular ‘storage’ material and cerebellar atrophy inolder patients. Identification of the early events in theMLIV course is keyto understanding the disease and deploying therapies. The Mcoln1−/−

mouse model reproduces all major aspects of the human disease. Wehave previously reported hypomyelination in the MLIV mouse brain.Here, we investigated the onset of hypomyelination and comparedoligodendrocyte maturation between the cortex/forebrain andcerebellum. We found significant delays in expression of matureoligodendrocyte markersMag,Mbp andMobp in theMcoln1−/− cortex,manifesting as early as 10 days after birth and persisting later in life.Such delays were less pronounced in the cerebellum. Despite ourprevious finding of diminished accumulation of the ferritin-bound iron inthe Mcoln1−/− brain, we report no significant changes in expression ofthe cytosolic iron reporters, suggesting that iron-handling deficits inMLIV occur in the lysosomes and do not involve broad iron deficiency.These data demonstrate very early deficits of oligodendrocytematuration and critical regional differences in myelination between theforebrain and cerebellum in the mouse model of MLIV. Furthermore,they establish quantitative readouts of the MLIV impact on early braindevelopment, useful to gauge efficacy in pre-clinical trials.

KEY WORDS: Mucolipidosis type IV, Lysosome, TRPML1,Mucolipin-1, Myelination, Oligodendrocyte

INTRODUCTIONMucolipidosis type IV (MLIV) is an autosomal-recessive lysosomalstorage disease caused by mutations in the MCOLN1 gene, located inhumans in the 19p13.2 locus (Acierno et al., 2001). The disease wasfirst 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, which has a range of reportedfunctions including zinc and iron homeostasis, regulation of lysosomalacidification and calcium release driving membrane fusion/fissionevents, 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 ofmembrane traffic, autophagy andmitochondriahave been reported in variousMLIV tissues andmodels (Coblentz et al.,2014; Colletti et al., 2012; Jennings et al., 2006; Li et al., 2016; Miedelet 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 withpsychomotor delay and visual impairment, which stems from acombination 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 appreciatedduring the first decade of life, although axial hypotonia, appendicularhypertonia and upper motor neuron weakness prevent ambulation andlimit fine motor function in the majority of patients. Although a staticcourse has been classically reported, progressive psychomotor declinehas been documented. It begins in the second decade, eventuallyresulting in severe spastic quadriplegia (A.M., personal observations).In congruence with the clinical course, ancillary brain imaging hasdemonstrated stable white-matter abnormalities (corpus callosumhypoplasia/dysgenesis and white-matter lesions), with the emergenceof subcortical volume loss and cerebellar atrophy in older patients.Despite a 19-year-long effort in the MLIV natural history program(NCT00015782, NCT01067742), owing to the low number of patientsand poor availability of the human brain tissue for research (single casetissues are now available at the University of Maryland Brain andTissue Bank, Baltimore, MD, USA), the mechanism(s) ofMLIV braindisease is still not well understood. Pinpointing the early events inMLIV brain pathology is important to define the therapeutic windowfor intervention and set expectations for clinical trial outcomes.

Some important insights regarding brain pathology in MLIV havebeen obtained from the MLIVmouse model [Mcoln1 knockout (KO)mouse] developed by us (Grishchuk et al., 2014, 2016; Micsenyiet al., 2009; Venugopal et al., 2007). The mouse model shows all thehallmarks of MLIV, including motor deficits, retinal degenerationand reduced secretion of chloric acid by stomach parietal cells,leading to elevated plasma gastrin (Schiffmann et al., 1998). Thesehallmarks were also supported by the murineMLIV model generatedby the Muallem group (Chandra et al., 2011). Similar to MLIVpatients, we observed intracellular storage formations, thinning of thecorpus callosum in the Mcoln1−/− mouse brain, micro- andastrogliosis, and partial loss of Purkinje cells at the late stage of thedisease. Electronmicroscopy analysis shows reduced thickness of the

Handling Editor: Steven J. ClapcoteReceived 27 January 2020; Accepted 16 June 2020

1Center for Genomic Medicine and Department of Neurology, MassachusettsGeneral Hospital Research Institute and Harvard Medical School, Boston,MA 02114, USA. 2Department of Biological Sciences, University of Pittsburgh,Pittsburgh, PA 15260, USA.*These authors contributed equally to this work‡These authors contributed equally to this work

§Authors for correspondence (ygrishchuk@partners.org; kiselyov@pitt.edu)

Y.G., 0000-0002-4684-4159; K.K., 0000-0001-6683-2895

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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myelin sheaths consistent with central nervous system (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 ashaving two distinct phases: the developmental phase, associated withearly-onset delayed oligodendrocyte maturation and gliosis (Grishchuket 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 MLIVpatients (Frei et al., 1998). Understanding the timing and mechanismsbehind these distinct events is important for designing and testingtherapies. Here, we investigate the role of TRPML1 in oligodendrocytematuration and myelin deposition in the developing brain. Our datashow early problems with oligodendrocyte maturation andmyelinationin higher-functioning brain regions such as the cerebral cortex. The roleof TRPML1 in early brain development is further supported by the factthat its expression gradually rises in postnatal brain and expression ishigher in the regions that display the most prominent deficits inmyelination in TRPML1-deficient (Mcoln1−/−) mice (cerebral cortex).Finally, these data establish quantitative readouts of the TRPML1impact on early brain development, which will be useful to gaugeefficacy readouts in future therapy trials.

RESULTSMcoln1/TRPML1expression increases with age in thepostnatal brainDespite 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; Zhanget al., 2016) and,more recently, aging and cancer (Jung et al., 2019; Xuet al., 2019), the time course of Mcoln1/TRPML1 expression duringthe development of living tissue has not been tracked. To answer howMcoln1/TRPML1 is regulated during brain development we analyzedits expression using quantitative reverse transcription PCR (qRT-PCR)in 1-, 10-, 21- and 60-day-old mice. Actin B (Actb) was used as ahousekeeping gene. Primer specificity was confirmed usingMcoln1−/− samples. Our data show a steady increase in Mcoln1expression levels in the forebrain from birth to 2months of age (Fig. 1).Mcoln1mRNA levels increased 4-fold (4.45±0.56, n=3 or 4,P=0.003)in the whole-cerebrum samples of wild-type (WT) animals betweenpostnatal day (P)1 and P10 (Fig. 1A). Focusing specifically on theisolated cortical tissues, we found a 6-fold increase inMcoln1mRNAlevels between P10 and P60 (6.31±1.10, n=3, statistically significantlinear trend with P=0.0028 using one-way ANOVA; Fig. 1C). Bycontrast, although Mcoln1 mRNA levels also increased in thecerebellar samples between P1 and P10 (Fig. 1B) and P10 and P60(Fig. 1C), the increases were small and did not attain statisticalsignificance or significant linear trend. We found a similar trend in theMcoln1 expression data generated by the Functional Annotation ofMammalian Genome (FANTOM5) project (Ha et al., 2019; Lizioet al., 2015; Noguchi et al., 2017; Vitezic et al., 2014) (Fig. S1A).Although the design of the study does not allow direct comparison oftranscriptional activation of Mcoln1 between visual cortex andcerebellum, the stable expression of Mcoln1 in cerebellum in thepre- and early-postnatal phase and increased expression in postnatalvisual cortex are consistent with the data we report here. Our Mcoln1expression data are the first on the developmental dynamics ofMcoln1transcription that allow comparison between different brain regions,cortex and cerebellum.Mcoln1-encoded endolysosomal cation channel TRPML1 is

involved in lysosomal function and signaling (Xu and Ren, 2015).

We compared its expression profilewith that of another endolysosomalcation channel, TPC1 (encoded by Tpcn1) (Yamaguchi et al., 2011).Interestingly, Tpcn1 mRNA did not increase with age in the mousecerebral cortex (Fig. 1D). This, to our knowledge, is the first analysis ofTpcn1 expression in the developing brain. Therefore, Mcoln1 mRNAupregulation during development is specific to this gene rather than amore general increase in expression of the lysosomal channels. Thedifference between Mcoln1 and Tpcn1 expression implies thatfunctions of the corresponding ion channels in the developing brainare distinct. The rapid pace of Mcoln1 mRNA increase in thedeveloping brain suggests its importance in development.Interestingly, there was a significant increase in the Tpcn1 mRNAlevels in theMcoln1−/− samples at the P60 time point (7.83±2.27-foldincrease, n=3, statistically significant linear trend with P=0.047 usingone-way ANOVA). RNA sequencing analysis on isolated brain celltypes shows enrichment of the Tpcn1 transcripts specifically inastrocytes, macrophage/microglia and endothelial cells (Zhang et al.,2014); therefore, the increase in its expression in Mcoln1−/− brain

Fig. 1. Increasing Mcoln1 expression in the brain during development.(A,B) qPCRanalysis ofMcoln1mRNA in thewhole forebrain (A) and cerebellum(B) inMcoln1+/+ (WT) andMcoln1−/− (KO) mice, showing a significant increasewith age. Analysis of expression was performed using ΔΔCt method; Actb wasused as a housekeeping gene. Statistical analysis was performedusing two-wayANOVA followed by Tukey’s multiple comparison tests. n=3-5; ***P<0.0005 and**P<0.005. (C) Mcoln1 expression dynamics in the cortical and cerebellumsamples of P10-P60 mice. The symbols represent an average of three to fourexperiments. Three to four biological replicates; **P<0.005 calculated using one-way ANOVA. (D) Tpcn1 mRNA expression in the Mcoln1+/+ and Mcoln1−/−

cerebral cortex. n=3-5; *P<0.05 calculated using one-way ANOVA. ns, P>0.05;error bars represent s.e.m.

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could reflect astrocytosis and microgliosis, a known prominent featureof brain pathology in MLIV (Grishchuk et al., 2014; Micsenyi et al.,2009; Weinstock et al., 2018) (Fig. S1B).

Early myelination deficits due to loss of Mcoln1/TRPML1 arecaused by delayed oligodendrocyte maturationWe have previously shown hypomyelination in the mouse model ofMLIV (Grishchuk et al., 2015), suggesting oligodendrocyteinvolvement. The original data, however, did not clarify whether thehypomyelination is a result of a lower number of oligodendrocytes dueto loss of precursors or caused by their defective maturation. To gainmore insight, we performed an expression time course ofoligodendrocyte maturation markers in postnatal developmentstarting at birth to 2 months of age. To assess oligodendrocyte

maturation, we used three of the key myelin genes: myelin-associatedglycoprotein precursor (Mag), myelin basic protein (Mbp) and myelin-associated oligodendrocyte basic protein (Mobp) (Han et al., 2013;Huang et al., 2013; Pérez et al., 2013; Robinson et al., 2014). In theforebrain samples, the three markers showed linear increase betweenP1 and P21, allowing us to compare the dynamics of oligodendrocytematuration during brain development (Fig. 2). In both WT and KOsamples, Mobp mRNA showed a statistically significant linear trend(P=0.0021 and P=0.0438, respectively, calculated using one-wayANOVA, n=3 or 4; Fig. 2A). At P21, Mobp mRNA increase wassignificantly higher in the Mcoln1+/+ relative to Mcoln1−/− samples(P=0.037 using unpaired Student’s t-test, n=5-7). This trend persistedfor Mbp (Fig. 2B). Interestingly, while Mag mRNA levels showedlinear increase between P1 and P21, the rate of Mag mRNA increase

Fig. 2. Delayed oligodendrocytematuration in the Mcoln1−/−

cerebral cortex. (A-D) qPCR analysisof cerebrum (P1) and cortex (P10, P21and P60) of Mcoln1+/+ and Mcoln1−/−

littermate mice, showing expression ofthe mature oligodendrocyte markersMobp, Mbp and Mag (A-C) andoligodendrocyte precursor markerNg2 (D). The analysis was performedusing the ΔΔCt method; Actb was usedas a housekeeping gene. Slopes wereobtained using simple linearregression, all of which weresignificant except WT in D. **P<0.005and *P<0.05 using two-way ANOVAfollowed by Tukey’s multiplecomparison tests; n=3-5; ns, P>0.05;error bars represent s.e.m.

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was significantly lower than that ofMobp andMbpmRNA and did notdiffer significantly between Mcoln1+/+ relative toMcoln1−/− samples(Fig. 2C), suggesting that Mcoln1 loss actuates specific changes inoligodendrocyte maturation rather than the loss of entire cellpopulations. The relative abundance of Mbp and Mobp mRNAdecreased between P21 and P60 (Fig. 2A,B), whereas levels of MagmRNA continued to grow (Fig. 2C). These data are compatible withother published results (Ehmann et al., 2013). The observation that theexpression of all three maturation markers was lower in theMcoln1−/−

cortices compared with Mcoln1+/+ cortices demonstrates earlymyelination delays (Fig. 2).To answer whether the observed delays in the acquisition of the

mature oligodendrocyte gene expression in the cortex are the result ofdelayed oligodendrocyte maturation or caused by loss ofoligodendrocytes precursors, we analyzed the expression of theoligodendrocyte precursor marker neural/glial antigen 2 (Ng2; alsoknown as Cspg4) (Hill et al., 2014). Interestingly, we found that

expression of Ng2 increased in theMcoln1−/− cortices compared withMcoln1+/+ cortices (Fig. 2D). Increase inNg2 expression might reflectthe attempt to overcome the downstream myelination deficits duringthe active phase of brain myelination in postnatal development.Therefore, we conclude that brain hypomyelination in MLIV is causedby delayed oligodendrocytematuration and reducedmyelin productionrather than the death of the oligodendrocyte precursors.

Next, we used immunohistochemistry to analyze developmentalmyelination of the corpus callosum in 10-day-old Mcoln1+/+ andMcoln1−/− mice using antibodies for the mature oligodendrocytemarker myelin proteolipid protein (PLP1) (Wang et al., 2006), one ofthe most abundant proteins in the myelin, and the neuronalneurofilament marker SMI312 (Vasung et al., 2011) (Fig. 3).Previously, we reported that thinning of the corpus callosum in 2-and 7-month-oldMcoln1−/−mice, while the thickness of the underlyingneuronal fibers in the corpus callosum remained intact, was caused bydefective myelination (Grishchuk et al., 2015). Similar to these

Fig. 3. Reduced myelination of the corpuscallosum in the MLIV mouse model is notaccompanied by significant reduction inneuronal fiber thickness. (A) Representativeimages of Mcoln1+/+ and Mcoln1−/− P10 brainsections stained with the mature oligodendrocyte/myelin marker PLP1 (red) and neuronal filamentmarker SMI312 (green). Corpus callosum andcortical areas are shown. Arrows showoligodendrocyte-deficient lesions in theMcoln1−/−

brain. Scale bar: 1 μm. (B) Analysis of the corpuscallosum area identified via either PLP1 orSMI312 staining, showing reduction of the PLP1-positive but not SMA312-positive area andsignificant reduction of the PLP1- to SMI312-positive area ratio in Mcoln1−/− mice, indicatingdeficient myelination but normal underlyingstructure in the forming corpus callosum in theMLIV mouse. n=4 (Mcoln1+/+) and n=6(Mcoln1−/−); ***P<0.001 using unpaired Student’st-test; n.s, P>0.05; error bars represent s.e.m.(C) Enlarged representative images of theMcoln1+/+ and Mcoln1−/− P10 brain sectionsimmunostained for PLP1, showing dysmorphicmyelinating fibers in the Mcoln1−/− brain. Scalebar: 200 nm.

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observations made in adult mice, we here detect no significant decreasein the SMI312-positive area of the corpus callosum in P10 pups,representing the normally forming tract of the callosal neuronal fibers,but observed a significant decrease in myelin deposition by matureoligodendrocytes, demonstrated by reduced PLP1-positive area aloneand decreased ratio of the PLP1-positive area to the correspondingSMI312-positive area (Fig. 3A,B). Furthermore, we observed theirregular distribution of PLP1-positive cells and forming myelinatedfibers in theMcoln1−/− cortex, the islands of tissue that are devoid of thePLP1 staining (Fig. 3C and arrows in A), further supporting defectivematuration of oligodendrocyte precursors in this model.

Delayed maturation of oligodendrocytes in the Mcoln1−/−

brain is region specificTo answer whether the impact of Mcoln1/TRPML1 loss onmyelination is different in the cerebellum, we performedquantitative PCR (qPCR) analysis on the cerebellar samples fromP1-P60 mice. We found that the overall differences in expression ofthe mature oligodendrocyte maturation markers in Mcoln1−/− andcontrol Mcoln1+/+ cerebella were not as pronounced as they were inthe cortical samples (Fig. 4A). This qPCR analysis was alsosupported by immunohistochemistry, using which we detected nodifference in the percentage of PLP1-positive area, nor in the countsof oligodendrocytes labeled by APC-CC1 (Fig. 4B,C), inMcoln1−/−

cerebella compared with Mcoln1+/+ cerebella. Therefore, the impactof Mcoln1/TRPML1 loss on oligodendrocyte maturation andmyelination is brain-region specific; this should be taken intoconsideration when discussing the impact of MLIV and its treatment.

Transcriptional analysis of the iron-regulated genes failedto reveal signs of iron depletion in the whole-corticalhomogenatesIron handling by the lysosomes has been shown to be impaired inMcoln1/TRPML1-deficient cells and brain tissues (Coblentz et al.,2014; Dong et al., 2008; Grishchuk et al., 2015). Despite the existingevidence for iron involvement, the mechanism by which ironmishandling 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 theendolysosomes (Todorich et al., 2009, 2011; Valerio, 2007).TRPML1 may be involved in this by transferring ferrous iron fromthe lysosomal lumen to the cytoplasm. It is, therefore, possible thatMcoln1/TRPML1 loss traps ferrous iron in the lysosomes, deprivingthe cytoplasm of iron and causing lysosomal oxidative stress.Importantly, cytoplasmic iron is key to oligodendrocyte maturationand myelin production as it is a co-factor in ATP production, which ishighly important due to extremely high energy requirements ofoligodendrocytes during myelination, and in the activity of keyenzymes of lipid catabolism and biosynthesis required for myelinproduction (Todorich et al., 2009). Iron retention in the lysosome dueto loss of Mcoln1/TRPML1 might therefore directly affectoligodendrocyte maturation by depriving the reactions of iron.

To answer whether Mcoln1/TRPML1 loss is associated with thedepletion of the cytoplasmic iron, we analyzed the expression of theiron-regulated genes, such as transferrin receptor (Tfrc), hephaestin(Heph), ferritin heavy chain 1 (Fth1) and ferroportin (Fpn; alsoknown as Slc40a1), by qPCR. All these genes are regulated by

Fig. 4. Cerebellar myelination is preserved in MLIV mice. (A) qPCR analysis of P1, P10, P21 and P60 cerebella from Mcoln1+/+ (WT) and Mcoln1−/− (KO)littermate mice, showing expression of the mature oligodendrocyte markersMobp,Mbp andMag. The analysis was performed using the ΔΔCt method; Actb wasused as a housekeeping gene. ns, P>0.05 using two-way ANOVA followed by Tukey’s multiple comparison test; n=3-5; error bars represent s.e.m.(B) Representative images of the Mcoln1+/+ and Mcoln1−/− P10 brain sections stained with the mature oligodendrocyte/myelin marker PLP1 (red),oligodendrocyte marker APC-CC1 (green) and nuclear counterstain Nuc-Blue (blue). Scale bars: 500 nm (left), 100 nm (right). (C) Statistical analysis of the APC-CC1-positive cell counts per section (left) and PLP1-positive area (right) in the cerebellar sections show no significant changes betweenMcoln1+/+ andMcoln1−/−

mice. n.s, P>0.05 using unpaired Student’s t-test; n=4 (Mcoln1+/+) and n=5 (Mcoln1−/−).

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cytoplastic iron through Hif1α, Hif2α and other transcription factorsinvolved in metal handling and oxidative stress (Acikyol et al.,2013; Li et al., 2013). qPCR showed no distinct difference betweenthe WT and Mcoln1−/− samples (Fig. 5). We, therefore, concludethat whole-tissue transcriptomic analysis reports no brain-widecytoplasmic iron depletion. Owing to the relatively low abundanceof oligodendroglial mRNA in the whole-tissue samples, however,we cannot rule out whether dysregulation of these genes inMcoln1−/− animals takes place only in oligodendrocytes and may bemasked by the signal from more-abundant cell types.

DISCUSSIONIn the present study, we show that loss of the lysosomal channelTRPML1 is associated with delayed oligodendrocyte maturation

leading to brain hypomyelination, which is a characteristic of both theMLIVmousemodel and the human disease. A dramatically significantaspect ofMLIV uncovered by these studies is the extremely early onsetof the oligodendrocyte maturation delay, manifesting already by P10.Importantly, these early myelination deficits are not reflecting orcausing underlying structural abnormalities of neuronal fibers or theirloss. This is a key piece of information for designingMLIV treatments.

We have previously reported reduced myelination in Mcoln1−/−

mice via expression analysis of oligodendrocyte markers MAG,MBP, MOBP and platelet-derived growth factor receptor (PDGFR;also known as PDGFRB) in whole-brain homogenates at 10 days,2 months and 7 months of age (Grishchuk et al., 2015). We alsoshowed lower protein levels of the mature oligodendrocyte markerPLP1 in the Mcoln1−/− brain and reduced thickness of the

Fig. 5. Cytoplasmic iron markers in the MLIV mouseforebrain. (A-D) qPCR analysis of mRNA for cytoplasmiciron markers Tfrc (A),Heph (B), Fth1 (C) and Fpn (D) in thecerebrum (P1) and cortex (P10-P60) samples of WT(Mcoln1+/+) and Mcoln1−/− (KO) mice. The analysis wasperformed using the ΔΔCt method; Actb was used as ahousekeeping gene. *P<0.05 using two-way ANOVAfollowed by Tukey multiple comparison test; n=3-5; ns,P>0.05; error bars represent s.e.m.

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PLP1-immunostained corpus callosum. Importantly, theneurofilament marker SMI312 remained unchanged in the earlysymptomatic Mcoln1−/− mice (2 months of age), showing normallyformed neuronal fibers in the corpus callosum. In the present study,we tracked and compared oligodendrocyte maturation markers in theforebrain/cortex and cerebellum in Mcoln1−/− mice at 1, 10, 21 and60 days after birth. We found dramatic differences betweengenotypes and brain regions. More specifically, while we reportsignificant myelination deficits in the anterior regions (cortex, corpuscallosum) of the Mcoln1−/− brain, myelination in the posterior brain(cerebellum) was normal. This regional and temporal pattern ofmyelination deficits in the Mcoln1−/− mouse, along with the timecourse of Mcoln1/TRPML1 expression in postnatal braindevelopment, provides new insights into the role of the TRPML1channel in the myelination process. It is well recognized thatmyelination progresses from caudal to rostral pathways and in theorder of increasing complexity of brain function (Gerber et al., 2010;Purger et al., 2016), starting from the areas responsible for basichomeostatic functions (pons, cerebellum) and proceeding to the areascontrolling more complex tasks (frontal cortex). In the mouse,deposition of myelin in the cerebellum starts in the deepest regionsearlier than P6 and is largely accomplished by P23 (Purger et al.,2016). In the corpus callosum, the first myelinating cells are noticedon P9. In TRPML1-deficient brains, the regions that normally acquiremyelination later in development are affected the most. Interestingly,we show that, in parallel with brain myelination, expression ofTRPML1 steadily increases in the postnatal brain and is moreprominent 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−/− KO mice,

specific abnormalities in cerebral white matter have been observedin MLIV patients. In a cross-sectional cohort analysis of 15 MLIVpatients ranging in age from 16 months to 22 years (Frei et al.,1998), hypoplasia of the corpus callosum and elevated T2 signal inthe subcortical white matter were reported in all patients, whilecortical atrophy was only noted in two of the oldest (15 and22 years). A subsequent prospective study of brain magneticresonance imaging (MRI) changes in five MLIV patients, rangingfrom 7 to 18 years of age, found increasing cortical gray matter anddecreasing subcortical white-matter volumes across a 2- to 3-yearfollow-up period (Schiffmann et al., 2014). Although additionallongitudinal data in individual patients are still needed to supportconclusions, the available data in humans illustrate an earlydevelopmental defect followed by a late degenerative processspecifically affecting white-matter tracts. The possibility that axonaldegeneration could accompany white matter loss in MLIV patientswas 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, canbe seen in the setting of neuronal mitochondrial dysfunction withoutneuronal degeneration. Taken together with our current findings, wepropose that the neurological deficits observed in the Mcoln1 KOmice and MLIV patients mainly arise from abnormalities in CNSwhite matter.Our findings in Mcoln1−/− mice have important implications for

understanding the disease pathogenesis in MLIV patients, for whomdata on the earliest stages of myelination are not available. In regardsto myelination, P10 in mice is roughly equivalent to the full-termhuman neonate (Hagberg et al., 2002; Semple et al., 2013). Whilecorpus callosum hypoplasia/dysgenesis and subcortical white-matterabnormalities have consistently been reported in older individuals(Frei et al., 1998), brain MRI data for MLIV patients younger than

16 months of age have not been reported, largely due to the firstsymptoms presenting around 6 months of age and diagnosis rarelyoccurring before the first year. The delayed manifestation of motoricdysfunction in MLIV patients might reflect selective myelinationdeficit in higher-cortical regions. This is also supported by the factthat in the first months of life (prior to MLIV symptom onset) motorfunction is mostly attributable to primitive brain regions, where,based on our mouse data, myelination is not affected by the loss ofMcoln1/TRPML1.

Our understanding of leukodystrophies, genetically determineddisorders that selectively affect white matter in the CNS, has rapidlyevolved with the recent application of brain imaging techniques pairedwith next-generation sequencing (van der Knaap and Bugiani, 2017).Surprisingly, mutations in myelin- or oligodendrocyte-specificproteins are only responsible for a portion of clinically discernibleleukodystrophies, while genes important for housekeeping processesin multiple cell linages are largely responsible for the remainder. As aresult, the new extended classification of the leukodystrophies nowincludes more than 50 genetic syndromes with mutations in myelin- oroligodendrocyte-specific genes (hypomyelinating and demyelinatingleukodystrophies; leukodystrophies with myelin vacuolization), andother white-matter components including axons (leuko-axonopathies),glia (astrocytopathies and microgliopathies) and blood vessels(leukovasculopathies). We believe that the prominent and earlyinvolvement of developmental hypomyelination strongly advocatesfor MLIV to be included to this list.

At the cellular level, TRPML1 has been shown to be involved invarious cell functions including membrane fission and fusion, as wellas 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; Soyomboet al., 2006; Venkatachalam et al., 2008; Wong et al., 2012; Xu andRen, 2015; Xu et al., 2006; Zhang et al., 2012). The latter effects wereor can be attributed to TRPML1 iron conductance, or its role in thetraffic of iron or iron-bound proteins in the endocytic pathway.Impairment of TRPML1-dependent iron uptake is an attractivehypothesis for the oligodendrocyte maturation deficits in the MLIVbrain, as cytoplasmic iron is a key co-factor of the myelin synthesispathway (Todorich et al., 2009). Indeed, iron deficiency andabnormal cellular iron absorption are associated withhypomyelination (Morath and Mayer-Pröschel, 2001; Todorichet al., 2009, 2011). Iron handling deficits have been reported by usin the brain of theMcoln1−/− mouse model (Grishchuk et al., 2015).At that time, we were unable to provide a definitive answer regardingthe impact of iron mishandling in the Mcoln1−/− brain. Here, weextended our studies using a battery of iron-reporter genes but foundno evidence of the cytoplasmic iron deficit in theMcoln1−/− corticaltissue homogenates. We, therefore, conclude that iron mishandlingcaused by Mcoln1/TRPML1 loss is either (1) limited to a specificsubset of cells (such as mature oligodendrocytes) and being maskedin the whole-tissue preparations, (2) taking place at certaindevelopmental stages, and/or (3) only manifesting as a lysosomaliron buildup without significantly affecting the cytoplasmic ironuptake and therefore unable to be detected via analysis of expressionof the iron-related genes. To this end, isolated Mcoln1−/−

oligodendrocyte cultures and co-cultures with neurons and glia willbe instrumental in delineating the mechanism by directly measuringcytosolic iron reporters, lysosomal iron content, oxidative stress andtheir impact on myelination, and will be the focus of our future study.

In conclusion, we present evidence of the role of the TRPML1lysosomal channel in developmental myelination. Genetic ablation ofTRPML1 leads to deficient oligodendrocyte maturation, resulting in

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hypomyelination of the cortex and corpus callosum already noticeableat P10. However, mechanisms of how the loss of TRPML1 leads tosuch deficits remain unknown. Brain myelination in development is acomplex and highly regulated process that depends on both cell-intrinsic (oligodendroglial) and -extrinsic (neuronal, astrocytic andmicroglial) signaling and cues. Indeed, reduced myelination in theearly-postnatal Mcoln1−/− cortex is accompanied by the observationsof robust activation of astrocytes, resulting in elevated cytokine/chemokine release and neuroinflammation (Weinstock et al., 2018).Therefore, better understanding of the role of TRPML1 inoligodendrocyte maturation and myelin deposition will require newgenetic in vivo (using the Cre-LoxP system for cell-type-specificmanipulation of Mcoln1 expression) and in vitro oligodendrocyteand other brain cell co-culture models and this will be the scope ofour future investigations. Elucidating the involvement of otherbrain cell types, neurons and glia, affected by the loss of TRPML1in the development of the oligodendrocyte lineage cells is important,given the complex picture of brain pathology and dysfunction inthis disease.

MATERIALS AND METHODSAnimalsMcoln1 KO mice were maintained and genotyped as previously described(Venugopal et al., 2007). The Mcoln1+/− breeders for this study wereobtained by backcrossing onto a C57Bl6J background for more than tengenerations.Mcoln1+/+ littermates were used as controls. Experiments wereperformed according to the institutional and National Institutes of Healthguidelines and approved by the Massachusetts General HospitalInstitutional Animal Care and Use Committee.

Immunohistochemistry and image analysisTo obtain brain tissue for histological examination 1-, 10-, 21- and 60-day-oldMcoln1−/− 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 inPBS for 48 h, washed with PBS, cryoprotected in 30% sucrose in PBS for24 h, split into cerebrum and cerebellum, frozen in isopentane and stored at−80°C. Brains were bisected along the midline and one hemisphere wasexamined histologically. Coronal sections of the cerebrum and sagittalsections of the cerebellum were cut at 10 μm using a cryostat. Sections weremounted onto glass SuperFrost plus slides. These sections were stored at−20°C before any staining procedures. For PLP1 and SMI312 staining,sections were microwaved in citrate buffer (10 mM citric acid, 0.05% Tween20, 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 withprimary antibodies diluted in 1% NGS overnight. The following primaryantibodies were used: PLP1 rabbit polyclonal (1:500; ab28486) and SMI312(pan axonal neurofilament) mouse monoclonal (1:1000; ab24574), both fromAbcam (Cambridge, MA, USA). Sections were incubated with secondaryantibodies in 1% NGS for 1.5 h at room temperature. The followingsecondary antibodies were used: donkey anti-mouse AlexaFluor 488 anddonkey 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-mouseAlexaFluor 488 (1:500; Invitrogen). Sectionswere counterstainedwithNucBlue nuclear stain (Life Technologies, Eugene, OR, USA).

Images were acquired on DM8i Leica Inverted EpifluorescenceMicroscope with Adaptive Focus (Leica Microsystems, Buffalo Grove,IL, USA) with Hamamatsu Flash 4.0 camera and advanced acquisitionsoftware package MetaMorph 4.2 (Molecular Devices, San Jose, CA, USA)using an automated stitching function. The exposure time was kept constantfor all sections (WT and Mcoln1−/− sections within the sameimmunohistochemistry experiment). Image analysis was performed usingFiji software (National Institutes of Health, Bethesda, MD, USA). Areas ofinterest 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 unpaired Student’s t-test. Gaussiandistribution was tested using the Shapiro–Wilk normality test.

RNA extraction and qRT-PCRFor qRT-PCR experiments, total RNA was extracted from whole cerebra ordissected cerebella from P1 pups. Whole-cerebella and cortical samples fromthe brains of 10-day-, 21-day- and 2-month-old mice were used. Corticalsamples were dissected to include motor and somatosensory areas and did notcontain corpus callosum tissue. RNAwas extracted using TRizol (Invitrogen,Carlsbad, CA, USA), according to the manufacturer’s protocol. Allexperimental groups were designed to contain at least four animals pergenotype and time point; lower numbers in some conditions were due to lowmRNA yields that prevented them being included in qRT-PCR experiments.For complementary DNA (cDNA) synthesis, MuLV Reverse Transcriptase(Applied Biosystems, Foster City, CA, USA) was used with 2 μg total RNAand oligo(dT)18 (IDT, Coralville, IA, USA) as a primer. qRT-PCR wascarried out using 1:75 dilutions of cDNA, 2× SYBRGreen/ROXMaster Mix(Fermentas, Glen Burnie,MD, USA) and 4 μMprimermix per 10 μl reaction.For gene expression analysis, the following primers were used (IDT,Coralville, IA, USA): Actb, forward 5′-GCTCCGGCATGTGCAAAG-3′ andreverse 5′-CATCACACCCTGGTGCCTA-3′; Fpn, forward 5′-ATCCTCT-GCGGAATCATCCT-3′ and reverse 5′-CAGACAGTAAGGACCCATCCA-3′; Fth1, forward 5′-TGATCCCCACTTATGTGACTTCAT-3′ and reverse5′-ACGTGGTCACCCAGTTCTTT-3′; Heph, forward 5′-ACTAAAGAG-ATTGGAAAAGCAGTGA-3′ and reverse 5′-TCATGCCCAGCATCTTC-ACA-3′;Mag, forward 5′-CGGGATTGTCACTGAGAGC-3′ and reverse 5′-AGGTCCAGTTCTGGGGATTC-3′; Mbp, forward 5′-GTGCCACATGTA-CAAGGACT-3′ and reverse 5′-TGGGTTTTCATCTTGGGTCC-3′; Mobp,forward 5′-CACCCTTCACCTTCCTCAAC-3′ and reverse 5′-TTCTGGT-AAAAGCAACCGCT-3′; Ng2, forward 5′-CATTTCTTCCGAGTGGTGG-C-3′ and reverse 5′-CACAGACTCTGGACAGACGG-3′; Tfrc, forward 5′-GAGGCGCTTCCTAGTACTCC-3′ and reverse 5′-TGGTTCCCCACCAA-ACAAGT-3′.

To avoid amplification of genomic DNA and ensure amplification ofcDNA, all primers were designed to span exons and negative reversetranscription reactions (without reverse transcriptase) were performed as acontrol. Samples were amplified in triplicate on a 7300 Real-Time System(Applied Biosystems, Foster City, CA, USA) using the following program:2 min at 50°C, 10 min at 95°C, and 40 cycles at 95°C for 15 s followed by1 min at 60°C. In addition, a dissociation curve step was run to corroboratethat amplification with specific primers resulting in one product only. TheΔΔCt method was used to calculate relative gene expression, where Ctcorresponds to the cycle threshold. Ct values for a representative set of genesare plotted in Fig. S2. ΔCt values were calculated as the difference between Ctvalues from the target gene and the housekeeping gene Actb. Experimentswere performed double blind using numerically coded samples; the sampleidentity was disclosed at the final stage of data analysis. Data are presented asfold change.

AcknowledgementsWe thank Dr Franca Cambi for fruitful discussions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.A.S., Y.G., K.K.; Methodology: S.H., Y.G., K.K.; Formalanalysis: L.A., Y.G., K.K.; Investigation: M.M., L.A., J.S., S.S., S.H., S.D., Y.G., K.K.;Data curation: Y.G., K.K.; Writing - original draft: A.M., Y.G., K.K.; Writing - review &editing: A.M., Y.G., K.K.; Supervision: S.A.S., Y.G., K.K.; Funding acquisition:S.A.S., Y.G., K.K.

FundingThis work was supported by the National Institutes of Health (R01NS096755).

Supplementary informationSupplementary information available online athttps://dmm.biologists.org/lookup/doi/10.1242/dmm.044230.supplemental

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RESEARCH ARTICLE Disease Models & Mechanisms (2020) 13, dmm044230. doi:10.1242/dmm.044230

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