Sonic Hedgehog Signaling Drives Mitochondrial ......1.00E + 07 1.50E + 07 2.00E + 07 2.50E + 07...

Signal Transduction

Sonic Hedgehog Signaling Drives MitochondrialFragmentation by Suppressing Mitofusins inCerebellar Granule Neuron Precursors andMedulloblastomaAnshu Malhotra1, Abhinav Dey1, Niyathi Prasad1, and Anna Marie Kenney1,2

Abstract

Sonic hedgehog (Shh) signaling is closely coupled with bio-energetics of medulloblastoma, the most common malignantpediatric brain tumor. Shh-associated medulloblastoma arisesfrom cerebellar granule neuron precursors (CGNP), a neuralprogenitor whose developmental expansion requires signalingby Shh, a ligand secreted by the neighboring Purkinje neurons.Previous observations show that Shh signaling inhibits fatty acidoxidation although driving increased fatty acid synthesis. Prolif-erating CGNPs and mouse Shh medulloblastomas feature highlevels of glycolytic enzymes in vivo and in vitro. Because both ofthese metabolic processes are closely linked to mitochondrialbioenergetics, the role of Shh signaling in mitochondrial biogen-esis was investigated. This report uncovers a surprising decrease inmitochondrial membrane potential (MMP) and overall ATPproduction in CGNPs exposed to Shh, consistent with increasedglycolysis resulting in high intracellular acidity, leading to mito-

chondrial fragmentation. Ultrastructural examination of mito-chondria revealed a spherical shape in Shh-treated cells, in con-trast to the elongated appearance in vehicle-treated postmitoticcells. Expression of mitofusin 1 and 2 was reduced in these cells,although their ectopic expression restored the MMP to the non-proliferating state and themorphology to a fused, interconnectedstate. Mouse Shh medulloblastoma cells featured drasticallyimpaired mitochondrial morphology, restoration of which byectopic mitofusin expression was also associated with a decreasein the expression of Cyclin D2 protein, a marker for proliferation.

Implications: This report exposes a novel role for Shh in regu-latingmitochondrial dynamics and rescue of themetabolic profileof tumor cells to that of nontransformed, nonproliferating cellsand represents a potential avenue for development of medullo-blastoma therapeutics. Mol Cancer Res; 14(1); 114–24. �2015 AACR.

IntroductionMedulloblastoma is the most common malignant pediatric

brain tumor, typically arising in children before the age of 10,although infrequently late adolescent or adult patients do presentwith this tumor. Medulloblastomas arise in the cerebellum, abrain region which regulates posture and coordination as well assome aspects of cognition, and whose development takes placelargely postnatally. The current treatment regimen for thesetumors includes cranio-spinal radiation, surgical resection, andchemotherapy, whose cumulative effects on the young brainleaves survivors with long-term cognitive and behavioral defects(1) that severely impact quality of life. Thus, there is a need forunderstanding tumor-specific processes and characteristics thatcould in the future be targeted by more specific therapeuticapproaches, leaving intact the patients' developing brains.

Concomitant studies by several groups have divided medullo-blastoma into four genetically and histologically defined sub-groups (2–9). The unique sample clusters identified in this studyinclude:WNT, Sonic hedgehog (SHH), groupC, and groupD. Thetumors bearing the SHH signature are thought to arise fromneuronal progenitors known as cerebellar granule neuron pre-cursors (CGNP; ref. 10). Activation of the Shh pathway is requiredfor CGNPs to undergo rapid population expansion in the externalgranule layer (EGL) of the cerebellum during postnatal develop-ment. The Shh ligand, secreted by the Purkinje neurons under-lying the EGL, binds to Patched (Ptc), which is a 12-pass trans-membrane domain receptor. Ptc inhibits smoothened (Smo), a 7-pass transmembrane protein. Binding of Shh to Ptc relieves theinhibition of Smo, leading to activation of the Shh pathway andtranslocation of theGli family transcription factors to the nucleus.This causes activation of target genes that drive proliferation andinhibit differentiation of CGNPs (11–13).

CGNPs can be cultured in vitro by harvesting the cells from thecerebellum of postnatal day 4/5 mice and can be maintained in aproliferative state for up to 72 hours by adding Shh protein to themedium. We and others have used CGNP cultures to modelmitogenic Shh signaling in vitro, to study mechanisms drivingcell-cycle progression, interactions between Shh and other sig-naling pathways, and proliferation-associated intracellular pro-cesses (14). Importantly, robust mouse models for the SHHsubclass of medulloblastomas have been generated, such as bydeletion of Ptc or transgenic expression of activated Smoothened(SmoA1; refs. 15–17).

1Department of Pediatric Oncology, Emory University, Atlanta, Geor-gia. 2Winship Cancer Institute, Atlanta, Georgia.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

CorrespondingAuthor:AnnaMarie Kenney, Department of Pediatric Oncology,Emory University, 1760 Haygood Drive NE, Atlanta, GA 30322. Phone: 404-727-1536; Fax: 404-727-4455; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-15-0278

�2015 American Association for Cancer Research.

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Extensive studies have been undertaken to understand thebiology of medulloblastoma, but little is known about theirmetabolic profile. Tumor metabolism was included as animportant trait when the hallmarks of cancer were revisited in2011 (18). Previously, we reported that Shh mitogenic/onco-genic signaling is tightly coupled to the reprogramming ofmitochondrial bioenergetics: Shh inhibits fatty acid oxidation(or b-oxidation) although driving increased fatty acid synthesis(FAS; ref. 19). A critical building block for FAS is acetyl CoA,the final product of glycolysis. We have also shown thatproliferating CGNPs and mouse Shh medulloblastomas featurehigh levels of glycolytic enzymes in vivo and in vitro (20)consistent with the "Warburg effect" (21). Indeed, the glycolyticenzyme Hexokinase 2 is required for cerebellar neurogenesisand its deletion abrogates Shh-associated mouse medulloblas-toma development and aggressiveness (22), underscoringthe importance of this metabolic pathway in rapid Shh-drivenCGNP proliferation both in context of normal developmentand tumorigenesis. Lactate secretion from elevated glycolysisis the main cause of acidic extracellular pH in most tumors(23, 24). Additionally, a striking feature of such glycolytic cellsis their fragmented mitochondrial profile (25). Mitochondriaare dynamic organelles that undergo fusion or fission reac-tions, collectively termed "biogenesis," based on their bioe-nergetic activity. Glycolytic cells present small, rounded mito-chondria with expanded matrices, whereas mitochondria ofrespirative cells, characterized by an abundance of substrate,appear interconnected, with enlarged intercristae compartment(26). Mitochondrial biogenesis is important in maintainingtheir physiology, morphology, and respiratory capacity (27).Thus, mitochondria are not only central to cellular energetics,but functional mitochondria are also required for a trans-formed cell (28).

Mitochondrial cristae carry out oxidative phosphorylation(Oxphos), which gives rise to an electro motive force across thetwo mitochondrial membranes and is known as the mitochon-drial membrane potential (MMP). MMP therefore, is a key indi-cator of cell health (29). Importantly, cancer cells do not shutdown energy metabolism completely, rather, they alter the mito-chondrial pathways (30). Even with elevated glycolytic levels,mitochondrial oxphos is still functional albeit, in a deregulatedmanner (21). Dysfunctional oxphos indicates a lower MMP, butmost cancer cells exhibit elevatedMMP, which can be understoodas a phenomenon to resist apoptosis (31) and favor high prolif-erative rates characteristic of cancer cells. Mitochondria are wellknown to signal pathways regulating apoptosis (32), and cellsundergoing apoptosis characteristically show a decrease in MMP(32). Thus, despite being deregulated, mitochondria of cancercells display a complex balance of metabolic and apoptoticsignals, which promotes proliferation.

Our present study highlights the effects of Shh pathwayactivity on mitochondria in proliferating CGNPs and medul-loblastoma cells (MBC). We report that Shh causes markedreduction in mitochondrial mass, accompanied by reducedmembrane potential, which is not associated with cell death,and reduces mitochondrial fusion by downregulating the mito-fusins 1 and 2. Overexpression of the mitofusins in CGNPs andMBCs not only restores the mitochondrial morphology but alsorestores their nonproliferative phenotype. These novel findingssuggest that in future studies it will be important to ask whethermanipulation of mitochondrial biogenesis can impact function

sufficiently to complement current medulloblastoma treatmentregimens.

Materials and MethodsAnimal studies

Harvest of neural precursors from neonatal mice and prepara-tion of cerebella and tumor tissue from wild-type and mutantmice for cell culture or histologic analysis were carried out incompliance with the Emory University Institutional Animal Careand Use Committee guidelines. NeuroD2-SmoA1 mice wereobtained from Jackson Research Laboratories.

Culture of CGNPs and MBCsCGNP cultures were generated as described previously (33).

Cells were plated on poly-DL-ornithine (Sigma) precoated platesor precoated glass coverslips. Where indicated, Shh was used at aconcentration of 3 mg/mL. Cyclopamine (R&D Systems) was usedat 1 mg/mL.

MBCs were harvested from SmoA1 mouse medulloblastomas.Briefly, tumors were disassociated and cells were incubated inPapain/DNAse solution for 30minutes, and then passed througha cell strainer. Cells were subsequently separated on a density stepgradient of 35%and60%Percoll solution (Sigma). PurifiedMBCswere enriched by preplating on uncoated tissue culture dishes toremove adherent fibroblasts and glial cells. Nonadherent cellswere plated on tissue culture dishes or glass coverslips precoatedwith poly D-lysine (Sigma) and Matrigel (BD Biosciences),infected with adenoviruses and cultured for 48 hours beforeWestern blotting.

Adenovirus production and infectionReplication incompetent adenoviruses were obtained from

Vector Biolabs. A GFP-only adenovirus was used as control. Forviral expansion, 60% to 80% confluent HEK293T cells wereinfected with the respective adenovirus and cultured for 48 to72 hours. The cells were harvested, pelleted by centrifugation, andsubjected to four cycles of freeze-thaw in freezingmixture. Viruseswere pelleted by ultracentrifugation at 24,000 rpm, for 3 hours at4�C. For infection, CGNPs or MBCs were cultured for 3 hoursbefore addition of viruses at 100 MOI. The cells were allowed togrow for 48 hours after infection.

Protein preparation and immunoblottingCells were washed once in PBS and protein extracts were

prepared as described in ref. (33). Protein estimation was per-formed using the Bradford method in the Bio-Rad protein assay.SDS-PAGE was performed according to standard procedures.Thirty micrograms of the total cell lysate was loaded on 10%polyacrylamide gels and transferred to activated Immobilonpolyvinylidene difluoride (Millipore) membrane, which wereblocked in 5% milk. Antibodies used for Western blotting wereMfn1, Mfn2 (Abcam), Drp1, CyclinD2 (Santa Cruz Biotechnol-ogy Inc.), Beta-actin, and GFP (Cell Signaling). Donkey anti-mouse horseradish peroxidase-linked secondary was from Jack-sonResearch Laboratories, and goat anti-rabbitwas fromThermo-Scientific. Peroxidase activity was detected using enhanced chemi-luminescence reagent (Thermo Scientific) and exposing mem-branes to GE-Amersham film.

ImmunofluorescenceCells were cultured on precoated glass coverslips and fixedwith

4% paraformaldehyde for 20 minutes. For BrdU incorporation,

Mitogenic Hedgehog Signaling and Mitochondria Form

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cells were pulsed with BrdU at a final concentration of 10 mmol/Lfor 3 hours prior to fixation. Immunofluorescence was performedby standard methods. Antibodies used for immunofluorescencewere Tom20 (Santa Cruz Biotechnology Inc.), PCNA (Merck)mouse, BrdU (BD Biosciences), F(ab0)2 Fragment Goat Ant-Mouse lgG (HþL; Jackson Immunoresearch Laboratories Inc.),and rabbit IgG (Life Technologies).

Mitotracker stainingA total of 100 nmol/L mitotracker (MitoTracker Red CMXRos,

Life Technologies) was prepared in requisite medium for CGNPs(�Shh) or MBCs, from a 1 mmol/L stock solution in DMSO.Cells were incubated for 45 minutes at 37�C and then fixedin 4% formaldehyde. After three washes in PBS, the coverslipswere mounted in ProLong Gold Antifade Mountant (Life

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Figure 1.Mitochondrial activity and biogenesis are reduced in CGNPs exposed to Shh. A, CGNPs were cultured for 48 hours on glass coverslips precoated with poly-DL-ornithine. Sonic Hedgehog and Cyclopamine were used where indicated. CGNPs were stained with mitotracker Red and incubated at 37�C for 45 minutes.Shh-treated CGNPs showed a decrease in their fluorescence intensity indicating depolarized mitochondria with a reduced MMP. Treatment with cyclopaminerestored the MMP to similar levels as vehicle. B, CGNPs �Shh were stained with Tom20 and visualized by confocal microscopy. Vehicle-treated CGNPs presenteddense, long networks of mitochondria although Shh-treated CGNPs showed more fragmented mitochondria. Treatment with cyclopamine restored the longfilamentous mitochondria. C, automated quantification, using the ImageJ software, of CGNPs�Shh, cyclopamine stained with mitotracker Red. D, measurement oftotal ATP produced by CGNPs. Shh-treated CGNPs produced less ATP as compared with vehicle-treated cells.

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Technologies). Quantification of mitotracker staining was carriedout using the ImageJ and normalized to number of nuclei stainedwith DAPI.

Image capturingConfocal images of stained primary cells and MBCs were

acquired using an Olympus FV1000 laser confocal microscopeand captured with the Olympus Fluoview Software (IntegratedCellular Imaging Core, Emory University). Quantificationof Western blots was carried out using the Adobe Photoshopsoftware.

Electron microscopyCells were cultured on precoated glass coverslips and fixed

overnight in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer(pH 7.4) at 4�C. For tissue sections, tumors or normal cerebellartissue was dissected out of a tumor bearing SmoA1 mouse andimmediately fixed in the above fixative. Cells/tissues were thenwashed with the same buffer and postfixed in 1% bufferedosmium tetroxide, dehydrated through a graded ethanol seriesto 100%, and embedded in Eponate 12 resin (Ted pella Inc.).Ultrathin sections were cut on a Leica UC6rt ultramicrotome(Leica Microsystems) at 70 to 80 nm, and counterstained with5% aqueous uranyl acetate and 2% lead citrate. Sections wereexamined on a JEOL JEM-1400 transmission electronmicroscopeequipped with a 2 K � 2 K Gatan US1000 CCD camera.

Aspect ratio (AR) measurementFor each cell treatment, approximately 120mitochondria were

measured to determine ARs using the ImageJ software (1.48f,Wayne Rasband, NIH).

ATP assayTotal ATP was extracted from CGNPs �Shh in 2.5% trichlor-

oacetic acid. ATP was measured using the ENLITEN ATP Assay kit(Promega) following the manufacturer's protocol.

RNA extraction and RT-PCRTotal RNA from CGNPs was extracted and purified using the

TRIzol reagent (Invitrogen) according to the manufacturer'sinstructions. cDNA was prepared from 1 mg of total RNA by usingHigh-Capacity cDNA Reverse Transcription Kit (Applied Biosys-tems) as per themanufacturer's instructions. qPCRwas performedusing the SsoAdvancedUniversal SYBRGreen Supermix (BioRad).RNA expression data were acquired using the C1000 TouchThermal Cycler and CFX96 Real-Time PCRDetection System. Thedata were analyzed for average results and standard errors arepresented. Primers for Mfn1, Mfn2, Drp1, Gli1, and GusB werepurchased from BioRad.

Results and DiscussionMitochondria are the primary site of energy production in the

eukaryotic cell. We have shown previously that Acetyl CoenzymeA Carboxylase (ACC), a key enzyme to convert Acetyl-CoA intoMalonyl-CoA, is altered by the action of Shh (19). This indicated acentral role of mitochondria in medulloblastoma metabolism asACC not only inhibits mitochondrial b–oxidation, but is also asubstrate for FASN. One of the first indicators of dysfunctionalmitochondria is theMMP (29). Therefore, to investigate the effectof Shh on MMP, we stained CGNPs with Mitotracker red, a

membrane potential-based mitochondrial dye. As mitochondriabecome depolarized, they lose their membrane potential andincorporate less dye into their membrane. Low intensity stainingwas observed in the Shh-treatedCGNPs as comparedwith vehicle-treated nonproliferating cells. To determine whether the canon-ical Shh pathway caused this effect, we treated the cells withCyclopamine, a Shh antagonist. The intensity of mitotracker wasrestored after this treatment indicating repolarization of mito-chondria (Fig. 1A and C). Stable variation in MMP has beenreported from different tumor cell subpopulations in vitro. Thesedifferences have been correlated with tumor invasiveness, metas-tasis, and general progression of disease (34). In general, a higherMMP is indicative of a rapidly proliferating cell, whereas lowerMMP would indicate initiation of apoptosis (35). Shh treatmentof CGNPs induces rapid proliferation in these cells and preventsthem from commencing differentiation. This is evident from thecyclin D2 expression in Shh-treated CGNPs (33). Thus, ourobservation of a decrease in MMP in Shh-treated CGNPs standsin contrast to previous reports of a higher MMP in proliferatingcells.

When mitochondria are subjected to stress, considerablechanges in their morphology become visible. Although mito-chondria in cells undergoing normal respiratory processes areinterconnected, long and tubular, those in cells under stress depicta fragmentedmorphology, with short and roundedmitochondria(26). To investigate whether the reducedMMPwe observe in Shh-treated CGNPs is associated with such morphologic changes, wecarried out immunofluorescence with Tom20, an outer mito-chondrial membrane protein, which is a commonmitochondrialmarker (36). Although vehicle-treated CGNPs demonstrated longinterconnected tubular mitochondria, Shh-treated cells presenteda fragmented pattern of mitochondria, with reduced Tom20staining (Fig. 1B, Supplementary Fig. S1B). Treatment with thesmoothened inhibitor cyclopamine restored the longfilamentousappearance of mitochondria (Fig. 1B). Because mitochondriaproduce most of the cell's ATP, the energy currency of the cell,we wanted to see if Shh treatment and associated reduction inMMP and mitochondrial fusion had an impact on the total ATPproduction in CGNPs. Indeed, we carried out an ATP quantifi-cation assay andobserved a twofold reduction in total ATP in Shh-treated cells, normalized to protein concentration (Fig. 1D).Taken together, these results indicate that Shh signaling in CGNPsreduces mitochondrial function, likely through effects on mito-chondrial biogenesis as shown in Fig. 1B.

Mitochondria carry out the tricarboxylic acid cycle within theirlumen and utilize the NADH and succinate thus produced, todrive oxidative phosphorylation, also known as the electrontransport chain (ETC). This cycles electrons through five differentcomplexes and releases ATP and water outside the mitochondriainto the cytoplasm. Asmentioned above, the electron cycling andresulting proton gradient also gives rise to a difference in potentialacross the two mitochondrial membranes, known as MMP (29).All the complexes of ETC are located on extensions of the innermitochondrial membrane called cristae. As our next step, we usedtransmission electron microscopy to observe the ultrastructure ofmitochondria in Shh-treated CGNPs. Consistent with the highMMP and filamentous structure shown in Fig. 1, the vehicle-onlycells demonstrated elongated mitochondria with well-definedcristae structure although the Shh-treated cells consisted of round-ed, smaller-sized mitochondria with a reduced network of cristae(Fig. 2A). To further quantify the morphology of mitochondria,






we analyzed ARs of mitochondria from the electronmicrographs.For each cell treatment, about 120 mitochondria were measuredfrom three distinct cell preparations, to determine ARs using theImageJ software (1.48f, Wayne Rasband, NIH). The vehicle-trea-ted CGNPs had an average AR of 1.94 (SD ¼ 0.05), although theShh-treated CGNPs were at 1.51 (SD¼ 0.084), and the differencein AR was found to be significant (P ¼ 0.0013). The AR ofmitochondria in vehicle-treated CGNPs were therefore signifi-cantly higher than those of Shh-treated cells.

Changes in cristae structure could result in disruption of ETC,which accounts for the lowMMP and ATP observed in Fig. 1. Theshape and size ofmitochondria are largely governed by fusion andfission events. Such events help maintain functionality in mito-chondria when the cell undergoes metabolic or environmentalstress. Fission plays a role in quality control by removing thenonfunctional portion of a defective mitochondrion. Fusion, inturn, complementsmitochondria bymixing the contents of partlyfunctional mitochondria with those of fully functional ones. Thedynamin family of GTPases control the fusion and fission pro-cesses of mitochondria. Mitofusins 1 and 2 (Mfn1 and 2), which

are membrane-anchored dynamins, regulate fusion of outermembranes. Drp1, on the other hand, is recruited from thecytoplasm to the outer mitochondrial membrane to fission offmitochondria (37). To examine how these proteins were affectedby the action of Shh, we carried out Western blot analysis onlysates prepared from CGNPs cultured for 48 hours � Shh. Mfn1and 2 levels were downregulated in the presence of Shh and wererestored after treatment with cyclopamine (Fig. 2B and C). Drp1did not show significant change in the presence of Shh (data notshown).We, therefore, believe that suppression of fusion proteinsaccounted for the more fragmented appearance of Shh-treatedmitochondria in Figs. 1B and 2A. Of note, gene expressionanalysis of mitofusins and Drp1 did not show significant changein transcript levels between vehicle and Shh-treated CGNPs (Sup-plementary Fig. S1A), indicating a posttranscriptionalmechanismof regulation.

Because mitochondria manifested a fragmented appearanceafter treatment with Shh, we wanted to see if restoring theexpression of mitofusins could rescue the fused mitochondrialmorphology. Todo this, we carried out a gain-of-function analysis

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Figure 2.Shh treatment alters the morphology ofmitochondria. A, CGNPs � Shh were cultivatedon poly-DL-ornithine precoated plates andprocessed for transmission electronmicroscopy. Shh-treated CGNPs reveal swollenmorphology with aberrant cristae structure,whereas vehicle-treated cells show elongatedmitochondria with well-defined cristaestructure. B and C, representative Western blotanalysis of mitochondrial fusion proteins Mfn1and 2, respectively, in CGNPs� Shh. Both showa decrease in Shh-treated CGNPs, which isrestored after treatment with Cyclopamine, aShh antagonist. Enhanced Cyclin D2 leveldepicts enhanced proliferation brought aboutby the action of Shh. The relative bandintensities of Mfn1 and Mfn2 relative to B-actinweremeasuredusing theAdobePhotoshop andare depicted below each lane.

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Figure 3.Manipulation of mitofusins rescues proliferation. A, Western blot showing induction in Mfn1 expression following adenoviral transduction of Mfn1. CGNPs weretransducedwith either GFP or Mfn1 expressing adenoviruses. Overexpression led to reduced cyclin D2 expression indicating reduced proliferation. The relative bandintensity of Mfn1 relative to B-actin was measured using the Adobe Photoshop and is depicted below each lane. B, the reduction in MMP brought aboutby Shh treatment of CGNPs, was restored to near-normal levels by ectopic expression of Mfn1 in CGNPs. C, the deformed and fragmented morphology ofmitochondria in Shh-treatedCGNPswas restored to their long,filamentous form following ectopic expression ofMfn1, as revealedby stainingwith Tom20. D, electronmicrographs showing ultrastructure of mitochondria in CGNPs� Shh and ectopically expressing Mfn1. The fragmented mitochondria visible in Shh-treated CGNPs,appear to be fusing together in Mfn1 overexpressing CGNPs and are longer in shape with well-defined cristae structure. E, Western blot showing induction inMfn2 expression following adenoviral transduction of Mfn2. The relative band intensity of Mfn2 relative to B-actin was measured using the Adobe Photoshop and isdepicted below each lane. F, ectopic expression of Mfn2 restores mitochondrial membrane potential, and the long, filamentous form of mitochondria, as revealed bystaining with Tom20 (G).






using adenovirus mediated over expression of Mfn1 (Fig. 3A).Indeed, the mitochondrial MMP and the long tubular morphol-ogy of mitochondria were restored following ectopic expressionof Mfn1 (Fig. 3B and C, Supplementary Fig. S2A). The ultrastruc-ture analysis also revealedmore elongatedmitochondria inMfn1overexpressing CGNPs (Fig. 3D). Similar observations weremadein CGNPs overexpressing Mfn2 (Fig. 3E–G, SupplementaryFig. S2C). Because Drp1 is primarily responsible for mitochon-drial fission, we carried out a loss-of-function analysis usingadenovirus-mediated downregulation of Drp1 (Fig. 4A). TheMMP was restored after Drp1 knockdown indicating repolariza-tion of mitochondria (Fig. 4B, Supplementary Fig. S3A). A reduc-tion in fission ofmitochondria was visible after Drp1 knockdown

as themitochondria start to becomemore fused (Fig. 4C).We alsonoted a decrease in cyclinD2 levels after restoration of mitofusinsor knockdown of Drp1, indicating a decrease in proliferation ofShh-treated CGNPs (Figs. 3A and E and 4A). To further confirmthis, we carried out BrdU labeling of proliferating cells afteradenovirus infections. Indeed, we observed a rescue in prolifer-ation after restoration of the mitofusins and Drp1 in CGNPs(Supplementary Figs. S2B, S2D, and S3B).

To determine if the abnormal mitochondrial morphology wasalso present in SmoA1 tumors, we analyzed electronmicrographsof tumor or adjacent normal cerebellar tissues. As expected, themitochondria of SmoA1 tumor sections were small, fragmentedwith ill-defined cristae although those in the normal tissue

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Figure 4.Manipulation of Drp1 rescues proliferation. A, Western blot showing reduction in Drp1 protein expression following adenoviral knockdown. CGNPs were transducedwith either scrambled-GFP or Drp1–shRNA expressing adenoviruses. Drp1 knockdown led to reduced cyclin D2 expression indicating reduced proliferation.B, the reduction in membrane potential brought about by Shh treatment of CGNPs, was restored to near-normal levels by knockdown of Drp1 in CGNPs. C, thedeformed and fragmented morphology of mitochondria in Shh-treated CGNPs was restored to their long, filamentous form following knockdown of Drp1, asrevealed by staining with Tom20.

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Figure 5.Mitochondrial morphology is altered in SmoA1 mouse medulloblastomas. A, scanning electron micrographs of medulloblastoma tissue and normal cerebellumobtained from NeuroD2-SmoA1 transgenic mice. Note the swollen, rounded, smaller-sized mitochondria with aberrant cristae structure in SmoA1 tumor tissue ascompared with elongatedmitochondriawithwell-defined cristae in normal SmoA1mouse cerebellum. B, representativeWestern blot analysis of fusion (Mfn1, 2) andfission (Drp1) proteins in NeuroD2-SmoA1 mouse medulloblastoma tissue and adjacent, normal cerebellum. Mfn1 and 2 are reduced although Drp1 is induced,indicating increased fragmentation of mitochondria in medulloblastoma tissue. C, SmoA1 mouse medulloblastoma cells (MBCs) were cultured in vitro for 48 hoursand processed for electron micrography. MBCs displayed gross abnormality in the structure of mitochondria that were swollen with blebbing and completedisruption of cristae. D and E,Western blots showing induction inMfn1 and 2 protein expression following adenoviral transduction. Overexpression of Mfn1 or 2 led toreduced cyclin D2 expression indicating reduced proliferation. The relative band intensities of Mfn1 and Mfn2 relative to B-actin were measured using the AdobePhotoshop and are depicted below each lane. F,Western blot showing reduction in Drp1 protein expression following adenoviral knockdown. Drp1 knockdown led toreduced cyclin D2 expression indicating reduced proliferation. G, because MBCs are derived from a mouse model that constitutively express Shh, we expected theMMP to be lower in untreated cells. InMBCs thatwere transducedwithMfn1 orMfn2 adenovirus, however, theMMPwas higher comparedwith GFP transducedMBCs,indicating a restoration of MMP in these cells. H, aberrant mitochondrial morphology in MBCswas restored to long, filamentous form following ectopic expression ofMfn1 or 2, as revealed by staining with Tom20. I and J, similar results were observed after Drp1 knockdown in MBCs stained with mitotracker or Tom20.






displayed elongated mitochondria with well-defined cristae (Fig.5A). To further investigate this, we carried out Western blotanalysis of mouse cerebellar tumor tissue as well as adjacentnormal tumor tissue. The levels of Mfn1 and 2 proteins wereremarkably reduced in the tumor tissue as compared with thenormal tissue. The levels of Drp1, on the other hand, werestrikingly higher in the tumor tissue relative to the normal tissue(Fig. 5B). We also examined micrographs of SmoA1 MBCs cul-tured in vitro. The mitochondria in these cells were severelyaffected with swollen cristae and nonexistent mitochondriallumen (Fig. 5C). To study the effects of mitofusin overexpressionin MBCs, we prepared in vitro cultures of SmoA1 tumor cells andcarried out adenovirus-mediated overexpression of Mfn1 and 2,and downregulation of Drp1. Similar to our observations inCGNPs, we observed reduced levels of CyclinD2 upon ectopicexpression of Mitofusins 1 and 2 (Fig. 5D and E) or adeno-mediated knockdown of Drp1 (Fig. 5F). The MMP of SmoA1tumor cells in vitro appeared to have been rescued by overexpres-sion of Mfn1, 2 or knockdown of Drp1 (Fig. 5G and I Supple-mentary Fig. S4A and S4B). Immunofluorescence using Tom20initially revealed a near absence of structurally intact mitochon-dria in Shh-treated CGNPs. This was rescued after overexpressionof Mfn1 and 2 and knockdown of Drp1 (Fig. 5H and J).

It is noteworthy from our results that restoration of mitochon-drial biogenesis could rescue the nonproliferative phenotype ofShh-treated CGNPs as well as MBCs. It has been observed thatalterations in energy metabolism can lead to cell-cycle exit inDrosophilaneural stem cells (38).More recently, it has been shownthat manipulation of a-ketoglutarate levels in embryonic stemcells can affect self-renewal and differentiation, adding to agrowing body of evidence that alteration of metabolic regulationcan impact upon cell fate (39). Mitochondria are well known tohouse and initiate cascades of caspases leading the cell to apo-ptosis (32). However, under our conditions of cell culture, and inthe developing cerebellum, Shh stimulation drives CGNP prolif-eration, not apoptosis, suggesting a novel role for Shh in affectingcellular metabolism by altering mitochondrial form and biogen-esis, resulting in impaired oxphos and reduced MMP.

Previous studies have implicated Shh signaling in heightenedactivity of FASN and other enzymes contributing to increased FAS

(19, 20). Here, we report a decrease in MMP coupled with adecrease in the overall ATP production by primary CGNPs treatedwith exogenous Shh to drive proliferation, and in MBCs bearingconstitutive Shhpathway activity.Due to the site-specificnature ofoxphos inside the mitochondria, any changes induced by theaction of Shh on mitochondrial structure would lead to alteredmitochondrial activity. Therefore, the effect of Shh on mitochon-drial biogenesis gains special importance. The mitochondria ofShh-treated cells presented a fragmentedmorphology, which wasconfirmed in the ultrastructure of cells. The proteins regulatingfusion and fission events, primarily responsible for the tubular orfragmented morphology of mitochondria, were also deregulatedunder the effect of Shh signaling. Restoration of the mitofusinsand knockdown ofDrp1, however, restored the normalmorphol-ogy of themitochondria (Fig. 6), andwas associatedwith reducedlevels of cyclin D2, an indicator of proliferation. Because we didnot observe any changes at the transcript level of themitofusins orDrp1, we can speculate that themechanismof Shh effects on theseproteins could either be accelerated degradation or reducedtranslation of the biogenesis proteins. Given the significance ofthe mTOR pathway in regulation of mitochondrial biogenesis, itis worthwhile also to speculate on these lines, especially becauseour labhas previously demonstrated that Shh activity is associatedwith enhanced mTOR pathway activation (40, 41).

Our findings illustrate the changes incurred in the morphologyof mitochondria and the consequent impact on cellular prolifer-ation due to the mitogenic action of Shh signaling. With impor-tant roles for mitochondria in apoptosis already established, thisstudy highlights mitochondrial morphology as a potential ther-apeutic target whose restoration can alter the metabolic profile oftumor cells to that of nontransformed, nonproliferating cells, thuspotentially suggesting a novel treatment paradigm.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Malhotra, A.M. KenneyDevelopment of methodology: A. Malhotra, A.M. Kenney

Respirative phenotype

Interconnected mitochondria Enlarged cristae compartment

Nonproliferating CGNPs

Glycolytic phenotype

Shh treated CGNP

Mfn2Drp1

Mfn1

Deformed cristaeFragmented mitochondria

H+H+

H+

H+

H+H+H+

H+

Filamentous shape restored

Modified from:Alirol & Mar�nou, 2006

Figure 6.Model for the action of Shh onmitochondrial structure and function.In an untreated cell with a respirativephenotype, the environment is aerobicand the mitochondria are long andtubular with an enlarged cristaecompartment. In a Shh-treated cell,the cellular environment becomesacidic due to enhanced glycolysis(20, 26) and themitochondria becomefragmented with a reduced cristaecompartment. This morphology,however, can be rescued by therestoration of mitochondrialbiogenesis genes: Mitofusins 1,2 andDrp1. This also rescues the cell to itsoriginal nonproliferative phenotype.


Malhotra et al.




Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Malhotra, A. Dey, N. PrasadAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Malhotra, A. Dey, N. Prasad, A.M. KenneyWriting, review, and/or revision of the manuscript: A. Malhotra, A. Dey,A.M. KenneyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Malhotra, A. DeyStudy supervision: A.M. Kenney

AcknowledgmentsThe authors acknowledge the assistance from Neil Anthony (Integrated

Cellular Imaging (ICI) core, Emory University) during image acquisition andanalysis. The authors thankHongYi from the ElectronMicroscopy core at EmoryUniversity for help with preparation of samples and Transmission ElectronMicroscopy. Funding for the Electron Microscope is provided by the NIH grant(1 S10RR02567901). This work has been supported by grants to A.M. Kenneyfrom the NIH (NINDS R01 NS061070), CURE Childhood Cancer Foundation,

Pediatric Cancer Research Foundation, and Winship Cancer Institute (P30center grant: CA138292). Imaging studies were supported by a pilot grant toA. Dey from the Integrated Cellular Imaging (ICI) core at Emory University.

Grant SupportAnna Marie Kenney: NINDS (R01 NS061070), CURE Childhood Cancer

Foundation, Pediatric Cancer Research Foundation, and Winship Cancer Insti-tute (P30 center grant- CA138292). A. Dey: A pilot grant for Imaging studies,Integrated Cellular Imaging (ICI) core at Emory University and the YoungInvestigator Award from Alex's Lemonade Stand Foundation for childhoodcancer.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 19, 2015; revised September 28, 2015; accepted September 28,2015; published OnlineFirst October 7, 2015.

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Malhotra et al.




2016;14:114-124. Published OnlineFirst October 7, 2015.Mol Cancer Res Anshu Malhotra, Abhinav Dey, Niyathi Prasad, et al. and MedulloblastomaSuppressing Mitofusins in Cerebellar Granule Neuron Precursors Sonic Hedgehog Signaling Drives Mitochondrial Fragmentation by

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