Shengbin Huang,1,2 Yongfu Wang,1 Xueqi Gan,1,2 Du Fang,1 Changjia Zhong,1

Long Wu,1 Gang Hu,1 Alexander A. Sosunov,3 Guy M. McKhann,3 Haiyang Yu,2 andShirley ShiDu Yan1

Drp1-Mediated Mitochondrial AbnormalitiesLink to Synaptic Injury in Diabetes ModelDiabetes 2015;64:1728–1742 | DOI: 10.2337/db14-0758

Diabetes has adverse effects on the brain, especially thehippocampus, which is particularly susceptible to synapticinjury and cognitive dysfunction. The underlying mecha-nisms and strategies to rescue such injury and dysfunc-tion are not well understood. Using a mouse model oftype 2 diabetes (db/db mice) and a human neuronal cellline treated with high concentration of glucose, we dem-onstrate aberrant mitochondrial morphology, reducedATP production, and impaired activity of complex I.These mitochondrial abnormalities are induced by im-balanced mitochondrial fusion and fission via a glycogensynthase kinase 3b (GSK3b)/dynamin-related protein-1(Drp1)-dependent mechanism. Modulation of the Drp1pathway or inhibition of GSK3b activity restores hippo-campal long-term potentiation that is impaired in db/dbmice. Our results point to a novel role for mitochondriain diabetes-induced synaptic impairment. Exploration ofthe mechanisms behind diabetes-induced synaptic def-icit may provide a novel treatment for mitochondrial andsynaptic injury in patients with diabetes.

Mitochondrial dysfunction, synaptic damage, and resul-tant impairment in cognitive function are pathologicalfeatures of diabetes (1–11). Diabetes adversely affects thebrain and confers increased risk for depression and de-mentia (3,12,13). In neurons, synaptic mitochondria arevital for maintenance of synaptic function and transmis-sion through normal mitochondrial dynamics, distribu-tion, and trafficking, as well as energy metabolism andsynaptic calcium modulation. Imbalance in mitochondrial

dynamics contributes to oxidative stress– and hyperglycemia-induced alterations in mitochondrial morphology and func-tion (10,14,15). The molecular and cellular mechanismsregulating the precise distribution of mitochondria in neu-rons are poorly understood.

In addition to well-known adverse effects on thecardiovascular and peripheral nervous systems, the hip-pocampus is particularly susceptible to diabetes-inducedepisodic memory impairment (12) and synaptic plasticitydeficits (5,7,8,11,16–20). For example, rats rendereddiabetic by treatment with the pancreatic b-cell toxinstreptozotocin (STZ) (a model of type 1 diabetes) exhibitspatial learning and memory impairment (5,8). Similar def-icits have been reported in the insulin-resistant type 2 di-abetes mouse model (7,8,16). Clinical investigations alsodocumented impaired cognitive function in human subjectswith type 1 (caused by insulin deficiency) as well as type 2(mediated by insulin resistance) diabetes compared withage-matched nondiabetic subjects (21,22). In support ofthose findings, long-term potentiation (LTP) of synaptictransmission, believed to be a cellular mechanism of learn-ing and memory, is impaired in the hippocampus of diabeticanimal models (5,8,20). The decline of hippocampal LTPin diabetic individuals is partially due to increased corti-costerone levels and/or impaired insulin signaling (8,19).Given that mitochondrial function is abnormal in manytissues negatively affected by diabetes and that mitochon-dria are important for maintenance of synaptic plasticity,as this organelle provides energy and regulates intra-synaptic metabolic homeostasis, we hypothesized that

1Department of Pharmacology & Toxicology and Higuchi Biosciences Center,School of Pharmacy, University of Kansas, Lawrence, KS2State Key Laboratory of Oral Diseases, West China Hospital of Stomatology,Sichuan University, Chengdu, China3Department of Neurosurgery, College of Physicians and Surgeons, ColumbiaUniversity, New York, NY

Corresponding author: Shirley ShiDu Yan, shidu@ku.edu.

Received 12 May 2014 and accepted 15 November 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0758/-/DC1.

S.H. and Y.W. contributed equally to this work.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

1728 Diabetes Volume 64, May 2015

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mitochondria-dependent mechanisms contribute impor-tantly to diabetes-induced synaptic dysfunction such ashippocampal LTP deficit.

Mitochondria are dynamic organelles, which undergocontinuous fission and fusion. Fission events are regulatedby dynamin-related protein (Drp1), and fusion events areregulated by the large dynamin-related GTPases known asmitofusin (Mfn)1 and -2 as well as optic atrophy 1 (OPA1)(23–25). Alterations in mitochondrial dynamics signifi-cantly impact mitochondrial numbers and shape, respira-tory enzyme activity, and ATP production. Imbalances ofmitochondrial fission and fusion in diabetes result predom-inantly from upregulation of Drp1, which induces mito-chondrial division and dysfunction (impaired respirationand ATP production) in a variety of cell types, includingislet cells (26), hepatocytes (27), skeletal muscle cells(9,28), mononuclear blood cells (29), endothelial cells(30), and dorsal root ganglion neurons (15,31). Mitochon-drial dysfunction is also suggested as a cause for develop-ment of insulin resistance in skeletal muscle cells andhyperglycemia in type 2 diabetes (32,33). Comparedwith other cell types, neurons are highly susceptible tomitochondrial dysfunction because neuronal transmissionis regulated by energy homeostasis in synapses. In type 2diabetes, hippocampal synaptic plasticity is widely be-lieved to be impaired, but mechanisms for hippocampalmitochondrial damage and consequent neuronal dysfunc-tion are unknown. The current study sought to elucidatehow hippocampal mitochondria respond to diabetes-induced mitochondrial and synaptic alterations that re-sult in synaptic injury.

RESEARCH DESIGN AND METHODS

AnimalsThese studies were approved by the Animal Care and UseCommittee of University of Kansas-Lawrence in accor-dance with the National Institutes of Health guidelinesfor animal care. Leptin receptor heterozygous mice(animals are on C57BLKS background) were purchasedfrom The Jackson Laboratory. Heterozygous db/m micewere crossed with each other to generate three genotypesof mice: wild-type mice, leptin receptor heterozygous mice(db/m), and homozygous leptin-deficiency mice (db/db).The genotype of offspring was identified by PCR usingprimers (forward, ACA AGA ACA AAA AGC CTG AAACC, and reverse, CCA AAC TGA ACT ACA TCA AAC C)for genotyping as previously described (34). All db/m anddb/db male mice used for the described experiments were5–6 months of age unless otherwise stated. The investiga-tors were blinded to the mouse genotype and treatmentconditions in performing the immunoprecipitation, elec-tron microscopy, and immunohistochemistry experiments.

SK Cell Culture and TransfectionsHuman SK-N-SH cells were cultured on 24-well cultureplates at low density and incubated with DMEM with10% FBS and 5.5 mmol/L D-glucose. Cells were thentransfected with plasmids containing constitutively active

forms of hemagglutinin–glycogen synthase kinase-3b(HA-GSK3b) (HA-GSK3bS9A) or a dominant-negativeform of GSK3b (HA-GSKK85A) (Addgene), green fluores-cence protein (GFP)-tagged wild-type Drp1 or Drp1K38A

(provided by Dr. Yi-Ren Hong, Kaohsiung Medical Univer-sity Hospital, Taiwan), or empty control vectors (GFP orHA) using Lipofectamine 2000 (Invitrogen) according tothe manufacturer’s instructions. Forty-eight hours aftertransfection, cells were treated with indicated reagentsand assessed for changes in mitochondrial morphologyand function as well as molecular signaling pathways.

Electrophysiological StudiesElectrophysiological recordings were performed on coro-nal hippocampal slices (400 mm in thickness) as previ-ously described (35). Hippocampal slices were recoveredat 37°C for at least 1 h and then maintained in an in-terface chamber at 29°C and perfused with artificialcerebral spinal fluid (124 mmol/L NaCl, 4.4 mmol/L KC,1 mmol/L Na2HPO4, 25 mmol/L NaHCO3, 2 mmol/LCaCl2, 2.0 mmol/L MgSO4, and 10 mmol/L glucose) con-tinuously bubbled with 95% O2 and 5% CO2. Field-excitatory postsynaptic potential (fEPSPs) was recordedfrom the CA1 region of the hippocampus by placingboth the stimulating and the recording electrodes in theCA1 stratum radiatum. Basal synaptic transmission (input-output curve) was assayed by plotting the stimulus voltage(V) against slopes of fEPSP to generate input-output rela-tions. A 30-min baseline recording was established usinglow-frequency stimulation (0.033 Hz; 0.1 ms impulse du-ration) and then adjusted intensity that induced fEPSPswith ;30% of the maximal fEPSP amplitude. LTP wasinduced by u-burst stimulation (four pulses at 100 Hz,with the bursts repeated at 5 Hz, and each tetanus, in-cluding three 10-burst trains separated by 15 s). In experi-ments using inhibitors, drugs were continuously perfusedover slices starting at least 1 h before LTP induction.Values of fEPSP slope were expressed as the percentagechange relative to their mean baseline amplitude.

Drug TreatmentsDrugs were prepared as stock solutions and were diluted tothe final concentration immediately before use. Incubationof hippocampal slices with drugs was performed in eithera recovery chamber or interface-recording chamber as needed.Drugs were added to culture plate wells. Final concen-trations and sources of the drugs were as follows:4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD8)(5 mmol/L; Sigma-Aldrich), mdivi-1 (10 mmol/L; Sigma-Aldrich), and glucose (50 mmol/L; Sigma-Aldrich). The finalconcentration of vehicle control DMSO was less than 0.5% inall experiments. For chronic in vivo administration of mdivi-1,the drug was dissolved in DMSO (10 or 25 mg/mL) asstock solution and further diluted in saline (DMSO:saline = 1:9) immediately before daily injection in diabeticor nondiabetic male mice at a dosage of 10 or 25 mg/kgi.p. Drug was administered once a day beginning at 19weeks of age and continued for 2 weeks. The same

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amount of DMSO dilution in saline was prepared andthen administered to age-matched male nondiabetic anddiabetic littermates as controls. One day after the end oftreatment, animals were subjected to either electrophysi-ological studies or biochemistry assays.

Mitochondria IsolationBrain mitochondria were isolated from whole brain cortexof mice or SK cultures as previously described (36).Briefly, samples were placed in 9 mL ice-cold mitochon-dria isolation buffer (225 mmol/L mannitol, 75 mmol/Lsucrose, and 2 mmol/L K2HPO4 [pH 7.2]) and homoge-nized (10 strokes) using a Douce homogenizer (KontesGlass Co.). Homogenate was centrifuged at 1,300g for5 min at 4°C. The resultant supernatant was then centri-fuged at 34,000g for 10 min after layering on 15% Percoll.After centrifugation, the homogenate was resuspendedand incubated for 5 min on ice in 20 mL mitochondriaisolation buffer with 0.02% digitonin and centrifuged at8,000g for 10 min. The pellet was washed twice in 1.5 mLmitochondria isolation buffer and centrifuged again at8,000g for 10 min. The final pellets were resuspended in200 mL mitochondria isolation buffer. Total protein con-centration of isolated mitochondria fraction was deter-mined by Bradford protein assay (Bio-Rad Laboratory).

Immunoprecipitation and Immunoblotting AssaysFor immunoprecipitation, hippocampal slices were lysedin nondenaturing lysis buffer containing 50 mmol/LTris HCl (pH 8), 150 mmol/L NaCl, 1 mmol/L EDTA,0.5% NP-40, and protease inhibitors (EMDMillipore). Aftercentrifugation at 12,000g for 10 min at 4°C, the superna-tant was precleaned with protein A/G-agarose beads(Thermo Scientific) for 2 h at 4°C, and 500 mg precleanedprotein extracts was incubated with antibody to GSK3b orDrp1 for 16 h at 4°C. Precipitated complexes were washedin lysis buffer and bound proteins analyzed by immuno-blots. For immunoblot experiments, hippocampal slices,cultured cells, or isolated mitochondrial/cytosol fractionswere homogenized in cell lysis buffer (Cell Signaling), andthen samples were separated in SDS-PAGE gels and trans-ferred onto membranes. Polyclonal rabbit anti–phospho-GSK3b (Ser9) (Cell Signaling), anti-GSK3b (3D10) (CellSignaling), anti–phospho-Drp1 (Ser616) (Cell Signaling),anti-Drp1 (Thermo Scientific), mouse anti–heat shockprotein 60 (HSP60) (Enzo), and mouse anti–a-tubulin(Cell Signaling) were used as the primary antibodies. Bind-ing sites of primary antibodies were visualized with horse-radish peroxidase–conjugated anti-rabbit IgG antibody(Life Technology) or anti-mouse IgG antibody (Life Tech-nology) followed by the addition of enhanced chemilumi-nescence substrate (GE Healthcare). Band relative opticaldensity was determined by NIH ImageJ software (publicdomain) and normalized with a-tubulin levels.

Confocal and Electron Microscopy to AssessMitochondrial MorphologyBrains were fixed by perfusion with 4% paraformalde-hyde, and after additional fixation for 72 h at 4°C, coronal

sections (25 mm) were cut with a Vibratome (LeicaVTS1000). After blockade with 5% goat serum in PBS, sliceswere incubated with primary antibody (anti–superoxide dis-mutase 2 [SODII], Enzo Life Sciences; anti–cytochromec oxidase [COXIV], Abcam; anti–microtubule-associatedprotein 2 [MAP2], Chemicon; or anti–Drp-1, BD Trans-duction Laboratories) overnight at 4°C. Secondary anti-body (Alexa Fluor 488–conjugated goat anti-rabbit IgGand/or Alexa Fluor 594–conjugated goat anti-mouse IgG[Invitrogen]) was applied for 1 h at room temperature.Nuclei were stained by DRAQ5 (5 mmol/L; Cell Signaling)for 5 min at room temperature. Blocking serum and pri-mary and secondary antibodies were applied in PBS with0.3% Triton X-100. Brain sections incubated with preim-mune serum or second antibody alone were used asnegative controls. Mitochondria in cultured cells werevisualized with MitoTracker red (100 nmol/L applied for30 min at 37°C before fixation; Molecular Probes). Brainslices and cell cultures were examined under confocal mi-croscope (Leica TCS SPE) with a 63 3 1.4 NA Apochromeobjective (Carl Zeiss MicroImaging, Inc.) at 488 nm, 594 nm,or 543 nm wavelengths, respectively. For quantification ofmitochondrial staining, four brain sections from each animal(five animals in each group) were randomly selected at dif-ferent rostro-caudal hippocampal levels. More than twentyrandomly selected hippocampal pyramidal neurons fromeach slice or SK cells in three independent experimentswere used for quantification of mitochondrial length anddensity using MetaMorph software as we previously de-scribed (37). For analysis of electron microscopic images,we used our previously published method (35,38). Briefly,animals were perfused with 4% paraformaldehyde and 0.1%glutaraldehyde, and brains were fixed in the same fixativebuffer overnight at 4°C. Small pieces of hippocampi wereembedded in LR white medium (Electron Microscopy Labo-ratory). Ultra-thin sections were counterstained with uranylacetate and examined using a JEOL 100S transmission elec-tron microscope. Mitochondrial parameters such as major/minor axis length, perimeter, area, and density were mea-sured using Metamorph software. The form factor wascalculated as perimeter2/4p 3 area. The investigatorswere blinded to mouse genotype and treatment condi-tions in performing morphological analysis and immuno-histochemical experiments.

Mitochondrial Respiration Complex Activity and ATPMeasurementMitochondrial respiration complex activity was measuredin hippocampi or in SK cell homogenates as previouslydescribed (37,39). Briefly, hippocampi or cultured cellswere homogenized and sonicated in the isolation buffercontaining 250 mmol/L sucrose; 20 mmol/L HEPES, pH7.2; and 1 mmol/L EDTA. NADH-ubiquinone oxidoreduc-tase (COXI) enzyme activity was determined in 25 mmol/Lpotassium buffer containing KCl, Tris-HCl ,and EDTA(pH 7.4). The change in absorbance was monitored at 340nm wavelength every 20 s for 6 min using an Amersham

1730 Mitochondria and Hippocampal LTP Deficit Diabetes Volume 64, May 2015

Biosciences Ultrospect 3100 pro spectrophotometer. Forhomogenized samples (50 mg protein), the oxidationof NADH was recorded for 3 min after the addition of2 mg/mL antimycin, 5 mmol/L MgCl2, 2 mmol/L KCN, and65 mmol/L coenzymes Q1 to the assay mixture, and then2 mg/mL rotenone was added to the mixture. The absor-bance of samples was measured for another 3 min. En-zyme activities in complex II, complex III, complex IV, andcitrate synthase activity were determined as previouslydescribed (37). We also determined ATP content in hip-pocampus using an ATP Bioluminescence Assay Kit (Roche)according to manufacturer’s instructions with a Lumines-cence plate reader (Molecular Devices) with an integrationtime of 10 s.

Quantitative Real-Time PCRTotal RNA was extracted from hippocampal tissue usingTRIzol reagent (Invitrogen Life Technologies, Carlsbad,CA) according to the manufacturer’s protocol as describedin our previous study (40). cDNA synthesis was per-formed using random hexamer primers and the TaqManreverse transcription kit (Applied Biosystems, Foster City,CA). The TaqMan probes and primers Mm00802465_m1were used for the standard TaqMan PCR procedures inthe ABI Prism 7700HT Sequence Detection System (Ap-plied Biosystems). Each treatment was performed in qua-druplicate, and data were analyzed with the comparativeCt method (41) and normalized to GAPDH RNA of thecorresponding sample.

Serum Insulin and Corticosterone MeasurementAnimals were fasted for 6 h before obtaining blood samples.Blood samples were centrifuged at 2,500 rpm (4°C) for5 min. Serum was then collected and stored at280°C. ELISAkits for insulin and corticosterone were purchased fromAbnova. Manufacturer’s instructions for measurement of se-rum insulin and corticosterone concentrations were followed.

Data AnalysisData are presented as means 6 SEM. Statistics were an-alyzed using Statview software (SAS Institute). For com-parison between the two groups, a two-tailed independentStudent t test was used. For comparisons between multi-ple groups, one-way ANOVA was used followed by indi-vidual post hoc Fisher tests when applicable. P, 0.05 wasconsidered statistically significant.

RESULTS

Alterations in Neuronal Mitochondrial Morphology andFunction in Hippocampi of Diabetic MiceTo determine whether type 2 diabetes alters brainmitochondrial dynamics, architecture, and function, weexamined the changes in hippocampus, which is particu-larly susceptible to adverse effects of diabetes (hippocam-pal damage results in episodic memory impairment [12] andsynaptic plasticity deficits [5,7,8,11,16–20]). Using confocalmicroscopy and SODII (mitochondrial protein) immuno-staining, we first examined mitochondrial morphology inCA1 neurons in db/db hippocampus compared with

control db/m mice. SODII immunopositive mitochondrialdensity in db/db hippocampal neurons was significantlylower than that of db/m control mice (Fig. 1A and B).We next performed immunostaining with another mito-chondrial marker, COXIV, to further examine the mito-chondria density. As shown in Fig. 1C–D, the COXIVimmunofluorescence intensity was significantly decreasedin CA1 pyramidal neurons of db/db hippocampus com-pared with control db/m mice. To provide further evidence,we used electron microscopy to evaluate morphologicchanges in hippocampal mitochondria (Fig. 1E) such asdensity, mitochondrial major axis length (mitochondriallength), the ratio of mitochondrial major axis length tominor axis length (mitochondrial width), and perimetersand area for calculation of form factor (indicating mito-chondrial branching). The mitochondrial density (Fig. 1F),the length of mitochondria (major axes) (Fig. 1G), the ratioof mitochondrial length to width (Fig. 1H), and mitochon-drial form factor (Fig. 1I ) were significantly reduced withindb/db hippocampus compared with db/m control hippo-campus, suggesting decreased mitochondria branchingand increased fragmentation in diabetic hippocampus. Ithas been reported that changes in mitochondrial numbersand shape can lead to impaired respiratory enzyme activityand energy production in tissue of diabetic mice(15,27,30,42–45). Therefore, we next evaluated hippocam-pal mitochondrial function by measuring key enzyme ac-tivity associated with respiratory chain and ATP levels.Complex I activity was significantly reduced in db/db hip-pocampal mitochondria compared with age-matched non-diabetic littermates (Fig. 1J); complex II and IV activitieswere unchanged (Supplementary Fig. 1). Similarly, hippo-campal ATP abundance in db/db mice was significantlylower than in db/m control mice (Fig. 1K ). Our data dem-onstrate that mitochondrial shape changes were paralleledwith decreases of mitochondrial respiratory enzyme ac-tivity and ATP abundance in diabetic hippocampus.

Given that imbalance of mitochondrial fission andfusion plays a critical role in maintenance of morphology,distribution, and function, we next investigated whethermitochondrial dynamics properties were altered in di-abetes. Immunoblotting of hippocampal homogenatesrevealed that levels of the mitochondrial fission proteinDrp1 were increased in diabetic mice (Fig. 1L and O), butlevels of mitochondrial fusion proteins Mfn2 and OPA1 indiabetic db/db hippocampus were not significantly differ-ent from db/m control levels (P . 0.05 db/m vs. db/db)(Supplementary Fig. 2A). While the effect of Drp1 upreg-ulation/activation on mitochondrial fusion/fission issometimes still controversial, accumulated evidence sug-gests that the Drp1-dependent mitochondrial elongationor fragmentation is closely associated with its phosphor-ylation status at different amino acid sites. Among thosephosphorylated residues, phosphorylation of Ser616 wasreported to be associated with mitochondrial fission(46). Indeed, we observed an increase in Ser616 phosphor-ylation in parallel with an increase in total Drp1

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expression in hippocampus of db/db mice (Fig. 1M–O). Anelevated level of Drp1 was found in diabetes-affected CA1neurons (Supplementary Fig. 2B and C). Furthermore,quantitative real-time PCR assay revealed that the geneexpression of Drp1 was increased;1.3 fold in db/db ani-mals compared with that of db/m controls (SupplementaryFig. 2D). Because the mitochondrial translocation of phos-phorylated Drp1 at Ser616 is necessary to promote fission(46), we then determined the levels of Drp1 in mitochon-dria. Immunoblotting of the isolated brain mitochondriadisplayed increased levels of Drp1 (Fig. 1P and S) and ofphosphorylation of Drp1 (Fig. 1Q–S) in diabetes-affectedbrain compared with db/m controls. These results suggestincreased mitochondrial fission in diabetic mice, whichmay subsequently impair mitochondrial morphology andfunction.

Mitochondria Fission Is Increased in Diabetes CellCulture ModelNext, using a human neuronal SK cell line, we elucidatedthe role of Drp1 in mitochondrial alterations underdiabetic conditions. We first investigated whether therewas an increased mitochondrial translocation of Drp1 indiabetic conditions. Treatment with high glucose (50mmol/L D-glucose for 1 h) lowered the cytosol Drp1 level(Fig. 2A) but increased mitochondrial Drp1 protein(Fig. 2B) compared with that in control cells treatedwith L-glucose, a nonmetabolizable form of glucose (datanot shown). In addition, immunostaining of Drp1 dem-onstrated localization of Drp1 in mitochondria treatedwith high glucose as demonstrated by overlaying a Drp1image with the mitochondrial marker MitoTracker(Fig. 2C). These data suggested that high-glucose exposure

Figure 1—Mitochondrial alterations in hippocampus of db/db mice. A: Representative images of SODII staining of hippocampal neuronsfrom db/m (left) or db/db (right) mice. B: Measurement of mitochondrial density (data presented as the fold increase of the percentage ofarea occupied by mitochondria in cell body) using MetaMorph software. C: Representative images for COXIV (green) and MAP2 (red)staining in hippocampal pyramidal neurons from db/m and db/db brains. Nuclei were stained by DRAQ5 as shown in blue signals. D:Quantification of immunofluorescence intensity of COXIV in hippocampal pyramidal neurons from db/m and db/db mice. E: Electronmicroscope images of mitochondria (indicated by arrows) from db/m and db/db hippocampus. Measurements of mitochondrial density(F ), length (G), ratio of length to width (H), and form factor (I) from electron microscope images. J and K: Enzymatic activity of complex I (J)and ATP levels (K). L–O: Densitometry of immunoreactive bands for Drp1 (L) and Ser616 phosphorylated Drp1 (p-Drp1) (M) that normalizedto a-tubulin in hippocampus homogenates of indicated groups and phosphorylated Drp1/Drp1 (N). O: Representative immunoreactivebands for Drp1, phosphorylated Drp1, and a-tubulin. P–S: Densitometry of immunoreactive bands for Drp1 (P) and Ser616 phosphorylatedDrp1 (Q) relative to HSP60 (mitochondrial marker) in brain mitochondrial fractions isolated from db/m and db/db mice. The level ofphosphorylated Drp1/Drp1 was increased in db/db brain mitochondria (R). S: Representative immunoreactive bands for Drp1, phosphor-ylated Drp1, and HSP60 in mitochondrial fractions in groups of mice as indicated. Data are presented as fold increase relative to db/mmice. Scale bar = 10 or 50 mm in A or C for confocal images, respectively, and 500 nm for electron microscope images in E. N = 6–8 animalsper group.

1732 Mitochondria and Hippocampal LTP Deficit Diabetes Volume 64, May 2015

Figure 2—Drp1 activation is responsible for mitochondrial morphology changes in human neuronal SK cells under high-glucose con-ditions. A and B: The levels of Drp1 in cytosol (A) or in mitochondrial fraction (B) of indicated groups were quantified using ImageJ software.C: Representative images of MitoTracker red (100 nmol/L MitoTracker red was added to cultures 30 min prior to fixation), Drp1 (green),merged images, and an enlarged magnification (as indicated in white rectangle of merge image). Colocalization of Drp1 with MitoTrackerred is shown in yellow. The rightmost image is an enlargement of the boxed area as shown in the merged image. D: Representative imagesfor MitoTracker red staining to show mitochondrial morphology in the indicated groups of cells. E–G: Measurement of mitochondrial length(E ) and density (F ) using MetaMorph software and enzymatic activity of complex I (G) with mdivi-1 (+) or vehicle (-) treatment. H: SK cellswere transfected with construct encoding GFP vector alone (labeled as vector) or GFP-tagged Drp1K38A (DN-Drp1) and stained withMitoTracker red (100 nmol/L MitoTracker red was added to cultures 30 min prior to fixation). Representative images for GFP (left),MitoTracker red (middle), and the two merged (right) from SK cells transfected with the empty GFP vector (upper panel) or GFP-Drp1K38A

(DN-Drp1) (lower panel) construct in the presence of high-glucose concentration (50 mmol/L). I–K: Measurement of mitochondrial length (I),density (J), and complex I activity (K) in cells transfected with vector alone (-) or DN-Drp1 (+). L: SK cells were transfected with Drp1 siRNAor control siRNA, and then mitochondrial morphology was examined under confocal microscopy. Representative images for Drp1 immu-nostaining (left) (green), MitoTracker red (middle), and the two merged (right) from cells treated with control siRNA (upper panel) or Drp1siRNA (lower panel). M–O: Measurement of mitochondrial length (M), density (N), and complex I activity (O) in cells transfected with controlsiRNA (-) or Drp1 siRNA (+). Data are presented as fold increase relative to control siRNA-treated cells in the presence of high glucose.Scale bar = 10 mm. N$ 20 cells per group from two independent experiments. Mdivi-1, 10 mmol/L. H-GLu (-) or (+) denotes culture mediumcontaining 5.5 mmol/L (-) or 50 mmol/L (+).

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induce mitochondrial translocation of Drp1 in culturedSK cells, which was similar to what we observed in thetype 2 diabetes–affected animal brain (Fig. 1).

We also examined whether the high-glucose treatmentin vitro induced mitochondrial defects comparable withthose we demonstrated in vivo. Here, we observeddecreased mitochondrial length (Fig. 2D and E), density(Fig. 2D and F), and complex I enzyme activity (Fig. 2Dand G) in SK cells subjected to high-glucose conditions(50 mmol/L) for 1 h. Notably, the nonmetabolizablesugar, mannitol or L-glucose, used as an osmotic control,did not induce any noticeable alterations, meaning thatthe mitochondrial changes were specifically dependent onthe high glucose–induced diabetic insult (SupplementaryFig. 3). Importantly, high glucose–induced deleteriouseffects were significantly prevented by pretreatmentwith mdivi-1 (10 mmol/L), an inhibitor of Drp1 activity(47) (Fig. 2D–G). For determination of a specific effect ofDrp1 on mitochondria pathology, cells were transfectedwith inactivated Drp1 and evaluated for mitochondriamorphology and function under diabetic conditions. Inthis case, we transfected SK cells with GFP-tagged domi-nant negative mutant Drp1 (DN-Drp1) to determine theeffect of DN-Drp1 on high glucose–induced mitochondrialmorphology changes. Compared with the controlvector–transfected cells, inactivation of Drp1 signifi-cantly preserved mitochondria against deleterious effectsof high-glucose treatment (Fig. 2H–K). Additionally,knockdown of Drp1 with small interfering RNA (siRNA)protected mitochondrial morphology, dynamics, andfunction from adverse effects of high-glucose exposure(Fig. 2L-O). No significant difference of mitochondrialdynamics and function were found in cells transfectedwith DN-Drp1 (Supplementary Fig. 4A–C) or Drp1 siRNA(Supplementary Fig. 4D–F) compared with cells trans-fected with GFP vector alone or control siRNA underthe vehicle-treated normal glucose condition. These resultsindicate that Drp1 potentiates neuronal mitochondriaabnormalities under diabetic conditions in vitro as well.

GSK3b Directly Regulates Drp1 in Diabetic ConditionIt is known that GSK3b is activated in db/db mice owingto dephosphorylation on Ser9 residue (48–50) and it canuse Drp1 as one of its substrates in an in vitro system(51–53). We thereby evaluated whether GSK3b could di-rectly regulate Drp1 in diabetes-affected brain. Immuno-precipitation of Drp1 followed by immunoblotting withGSK3b showed a GSK3b immunoreactive band. Con-versely, immunoprecipitation of GSK3b showed Drp1immunoreactive bands (Fig. 3A), suggesting the formationof a complex of Drp1 with GSK3b in a mouse model oftype 2 diabetes. GSK3b is a serine/threonine kinase, andphosphorylation on Ser9 residue (p-ser9 GSK3b) inacti-vates it (50). Most interestingly, p-ser9 GSK3b levels aredecreased in both diabetic human subjects and rodentmodels of diabetes (48,49), indicating increased GSK3bactivation. In the current study, we also observed an

increased GSK3b activation (reduced levels of p-ser9GSK3b) in db/db mice compared with db/m control litter-mates (Fig. 3B). Treatment with TDZD8 (5 mmol/L for1 h), an inhibitor of GSK3b (54), resulted in a significantincrease of p-ser9 GSK3b levels in db/db hippocampus(Fig. 3B), suggesting that TDZD8 treatment may be suf-ficient to inhibit GSK3b activation in diabetic hippocampaltissue. Further, in the presence of TDZD8, we observeda decreased Drp1 protein expression (P , 0.001, vehicle-perfused db/db hippocampal slices vs. TDZD8-perfuseddb/db slices [Fig. 3C]) and decreased Drp1 Ser616 phosphor-ylation (P , 0.05, vehicle-perfused db/db hippocampalslices vs. TDZD8-perfused db/db slices [Fig. 3C and D]) indb/db mouse hippocampal tissue.

To investigate direct effect of GSK3b on Drp1-mediatedmitochondrial alterations under diabetic conditions, hu-man neuronal SK cells were treated with GSK3b inhibitorTDZD8 or transfected with a dominant negative form ofGSK3b (DN-GSK3b) in the presence of high glucose.Clearly, pharmacological blockade (Fig. 3E–G) or geneticinhibition of GSK3b (Fig. 3H–I) prevented high glucose–induced Drp1 changes. In addition, transfection of SK cellswith the GSK3bS9A (constitutively active form of GSK3b)resulted in increased Drp1 expression and its phosphory-lation (Fig. 3J and K). Additionally, we performed tripleimmunostaining for mitochondria (red), Drp1 (green),and GSK3b (blue) to determine whether there was a colo-calization of GSK3b and Drp1 in mitochondria under a di-abetic condition. As shown in Fig. 3L, the high-glucosetreatment induced a colocalization (white) of GSK3b/Drp1 within mitochondria in SK cultures.

Since Drp1 plays a key role in maintenance of mito-chondrial structure and function and is directly regulatedby GSK3b, we next tried to determine whether modulationof GSK3b causes changes in mitochondrial morphologyunder diabetic conditions. Pharmacological blockade ofGSK3b with TDZD8 significantly suppressed mitochondrialfission (Fig. 4A and B) and increased mitochondrial density(Fig. 4A and C) and complex I activity (Fig. 4D) in thepresence of high-glucose treatment. Genetic inactivationwith DN-GSK3b also preserved mitochondria againsthigh-glucose insult (Fig. 4E–H). Neither pharmacological(Supplementary Fig. 4G–I) nor genetic (SupplementaryFig. 4J–L) inhibition of GSK3b had any significant ef-fect on the mitochondrial morphology and function invehicle-treated cells. As expected, activation of GSK3bby transfecting cells with GSK3bS9A elicited mitochondrialimpairments (Fig. 4I–L). These results demonstrate thatGSK3b-Drp1 interaction mediates mitochondrial alterationunder diabetic condition.

To validate that GSK3b acts as an upstream Drp1 sig-naling in diabetic conditions, we cotransfected SK cellswith GSK3bS9A with and without DN-Drp1 plasmids orDN-GSK3b with or without Drp1 plasmids. Transfectionwith DN-Drp1 prevented effects of GSK3bS9A-mediatedalterations in mitochondrial morphology and complex Ienzyme activity (Fig. 5A–D). However, transfection with

1734 Mitochondria and Hippocampal LTP Deficit Diabetes Volume 64, May 2015

DN-GSK3b did not completely restore Drp1 transfection–induced mitochondria impairments (Fig. 5E–H). These resultsindicate that Drp1 was required for GSK3b activation–mediated mitochondrial dysfunction. In addition, sup-pressing Drp1 did not change GSK3b activation thatwas induced by either high-glucose treatment in cells ortype 2 diabetic hippocampus. As showed in Supplemen-tary Fig. 5A and B, reduced GSK3b phosphorylation incells with high-glucose treatment was not restored bymodulation of Drp1 via transfection with DN-Drp1 orDrp1 siRNA. Similarly, administration of mdivi-1, a potent

cell-permeable inhibitor of Drp1 (47), to db/db mice failedto alter levels of p-ser9 GSK3b (P . 0.05, vehicle-treateddb/db vs. mdivi-1 treated db/db hippocampal slices [Sup-plementary Fig. 5C]). These data suggest that GSK3b isa likely upstream signaling molecule that controls Drp1activity under diabetic conditions.

Mitochondrial Impairment Is Responsible for SynapticPlasticity DeficitWe further sought to investigate the role of Drp1 inmediating mitochondrial degeneration and energy supply

Figure 3—GSK3b/Drp1 interaction in diabetic hippocampus and SK cell line. A: Densitometry of immunoreactive bands. GSK3b (upperright panel) or Drp1 (upper left panel) after immunoprecipitation (IP) of Drp1 or GSK3b, respectively. Lower panels are protein input controls.NI, nonimmune IgG. B: Densitometry of immunoreactive bands for Ser9 phosphorylated GSK3b (p-GSK3b) relative to GSK3b in hippo-campus homogenates of the indicated groups. The representative immunoblots for phosphorylated GSK3b or GSK3b are shown in thelower panel. C and D: Quantification of the density of immunoreactive Drp1 and phosphorylated Drp1 bands normalized to a-tubulin (C).The lower panel of C shows representative immunoblots for phosphorylated Drp1, Drp-1, and a-tubulin in hippocampal homogenates fromdb/m and db/db with (+) or without (-) GSK3b inhibitor TDZD8. The phosphorylated Drp1/Drp1 was calculated (D) from hippocampal sliceswith or without GSK3b inhibitor TDZD8 (5 mmol/L, treated for 1 h). E–G: Densitometry of immunoreactive bands for p-GSK3b (E ), Drp1 andphosphorylated Drp1 relative to b-actin (F ), and ratio of phosphorylated Drp1 to Drp1 (G) in the SK cells with (+) or without (-) TDZD8treatment in the presence of high glucose (+) or culture medium (-). H and I: SK cells transfected with dominant negative form of GSK3b(DN-GSK3b) changed phosphorylated Drp1 and Drp1 expression levels (H) and phosphorylated Drp1–to–Drp1 ratio (I). The lower panel of Hshows representative immunoblots for the indicated protein in vector (-) or HA-DN-GSK3b–transfected (+) SK cells in the presence of high-glucose levels (+) or vehicle (-). J and K: SK cells transfected with constitutively active mutant of GSK3b (GSK3bS9A) changed phosphor-ylated Drp1 and Drp1 expression levels relative to b-actin (J) and phosphorylated Drp1–to–Drp1 ratio (K) in indicated groups. The lowerpanel of J shows representative immunoblots for the indicated protein in vector (-) or GSK3bS9A-transfected (+) SK cells. L: Representativeimages of triple immunostaining for mitochondria (MitoTracker red), Drp1 (green), and GSK3b (blue) in high glucose (H-Glu)–treated SKcells. The enlarged box area was demonstrated in the lower panel. Arrows pointing to spots denote colocalization of Drp1 with GSK3b andMitoTracker red. Data are presented as fold increase relative to vehicle-treated controls; N = 4–6 animals per group or culture wells fromthree independent experiments. Scale bar = 10 mm. H-GLu (-) or (+) denotes culture medium containing 5.5 mmol/L (-) or 50 mmol/L (+).(-) indicates the vehicle treatment or control vector transfection and (+) indicates the presence of the indicated drug treatment or indicatedconstruct transfection. IB, immunoblotting.

diabetes.diabetesjournals.org Huang and Associates 1735

deficiency in diabetes-affected hippocampus by assessingwhether suppression of Drp1 expression and activity indiabetic brain would have favorable effects. Treatment withDrp1 inhibitor mdivi-1 (25 mg/kg or 10 mg/kg once dailyfor 2 weeks) resulted in significantly increased mitochon-drial content (Fig. 6A and B), complex I enzymatic activity(Fig. 6C), and ATP levels (Fig. 6D) in db/db mouse hippo-campus. When the same mdivi-1 treatment was adminis-tered to db/m control animals, we found no significantchanges in mitochondrial density (Fig. 6A and B), complexI activity (Fig. 6C), or ATP levels (Fig. 6D). Electron mi-croscopy confirmed that mdivi-1 treatment (10 mg/kg)significantly preserved mitochondrial morphology bymaintaining mitochondrial length and shape (Fig. 6E–I).

Because mitochondrial morphology and function arecrucial for synaptic plasticity and function (55–58), wenext investigated mitochondria-dependent mechanismsof synaptic plasticity in hippocampus of the db/db mousemodel of type 2 diabetes. Using electrophysiological anal-ysis, we observed reduced LTP (Fig. 7C), whereas basal

neuronal transmission (input-output relationship [Fig. 7A])or short-term plasticity (pair-pulse facilitation [Fig. 7B])was not changed in db/db hippocampus. Treatment withmdivi-1 significantly reversed the decline in hippocampalLTP from diabetic hippocampus (P, 0.05, mdivi-1–treateddb/db mice vs. vehicle-treated db/db hippocampal slices[Fig. 7G and H]) without changes in basic neuronal trans-mission or pair-pulse facilitation (Fig. 7E and F). The treat-ment of nondiabetic db/m mice with mdivi-1 did notsignificantly affect hippocampal LTP (Fig. 7D and H), whichwas coincident with the unaffected mitochondrial structureand function (Fig. 6). Taken together, these results indicatea protective effect of inhibition of diabetes-induced Drp1activation on synaptic dysfunction.

Given that GSK3b activation modulated Drp1 signal-ing, we next evaluated whether treatment of hippocam-pus slices with GSK3b inhibitor TDZD8 or LiCl (4 mmol/L)recapitulated the protective effects of mdivi-1 on synap-tic function in vivo. Indeed, inhibition of GSK3b signif-icantly protected hippocampus LTP against diabetes insult

Figure 4—Inactivation of GSK3b prevents high glucose–induced mitochondrial morphology alterations and functional deficit. A–D: Rep-resentative images of MitoTracker red staining (A) and measurements of mitochondrial length (B), density (C), and complex I activity (D) with(+) or without (-) TDZD8 treatment (5 mmol/L for 1 h) in human SK cells under high-glucose conditions. E–H: Representative imagesof MitoTracker red staining (E ) and quantification of mitochondrial length (F ), density (G), and complex I activity (H) with empty (-) orHA-DN-GSK3b (+) plasmid transfection in human SK cells under high-glucose conditions. I–L: Representative images of MitoTrackerstaining (I) and quantification of mitochondrial length (J), density (K), and complex I activity (L) with empty (-) or HA-GSK3bS9A (+) plasmidtransfection in human SK cells under normal glucose conditions. Data are presented as fold increase relative to vehicle-treated SK cells.N $ 20 cells per group from three independent experiments. Scale bar = 10 mm. H-GLu (-) or (+) denotes culture medium containing5.5 mmol/L (-) or 50 mmol/L (+). (-) Indicates the vehicle treatment or control construct transfection and (+) indicates the presence of theindicated drug treatment or indicated construct transfection.

1736 Mitochondria and Hippocampal LTP Deficit Diabetes Volume 64, May 2015

(Fig. 7I–K) without changing basal synaptic function indb/m hippocampus. Since insulin (19) and corticosterone(8) signaling pathways were reported to mediate LTPimpairment in rodent models of diabetes, we exploredtheir reaction to mdivi-1 treatment. Consistent with theprevious report (8,19), we did find increased blood levels ofinsulin and corticosterone (P, 0.05 vs. db/m) in db/dbmice(Supplementary Fig. 6A and B). Mdivi-1 treatment did notsignificantly change insulin and corticosterone levels (P .0.05) (Supplementary Fig. 6A and B) or blood glucose con-tent in control or db/db mice (Supplementary Table 1).These findings suggest that the protective effect of mdivi-1on LTP restoration in diabetic hippocampus is not likelydue to insulin or corticosterone signaling pathways.

DISCUSSION

In the current study, we report a novel and pivotal role ofmitochondrial dysfunction in diabetes-induced synapticimpairment. Our data suggest a GSK3b/Drp1-dependentconnection between mitochondrial dysfunction in dia-betic neurons and LTP deficit. We believe that sucha link is rational because provision of neurons with

access to energy/metabolism homeostasis is the prerequisitefor synaptic transmission. Understanding mitochondrialalterations in animal models of diabetes will likely generatenovel approaches for the treatment of diabetes-associatedneurological disorders.

Perturbations in maintenance of mitochondrial mor-phology and function are involved in several adverseeffects of diabetes in many organs including pancreas(26,32), liver (27), skeletal muscle (9,28), and the vascularsystem (29,30). Brain is highly enriched with mitochondriaand strongly reliant on oxidative phosphorylation for en-ergy production. The current literature suggests a connec-tion between diabetes and mitochondrial biogenesis andfission within mouse dorsal root ganglia (15,31). One ofour recent publications suggests that mitochondrial dys-function is associated with synaptic deficit in an Alzheimerdisease mouse model superimposed with type 1 diabetes(36). However, it is still unclear whether dysfunctional syn-aptic plasticity (resulting in observed episodic memory im-pairment) links to mitochondrial defects in type 2 diabetes.

Staining of db/db mouse hippocampal pyramidal neu-rons for the mitochondrial marker SODII or COXIV

Figure 5—Effect of GSK3b on Drp1-induced mitochondrial morphology and function. A: Representative images showing MitoTrackerred staining in cells cotransfected with GFP empty vector (green) and HA-GSK3bS9A (HA-tagged GSK3bS9A [blue]) (upper panel) or withDN-Drp1 (GFP-tagged Drp1K38A [green]) and HA-GSK3bS9A (blue) (bottom panel), respectively. GFP (green) and HA (blue) costainingshowed cotransfection with both DN-Drp1 (or GFP empty vector) and GSK3bS9A plasmids (HA-positive staining). B–D: Measurement ofmitochondrial length (B), density (C), or complex I enzyme activity (D) in the indicated groups of cells. E: Representative images for GFP(green), MitoTracker red, and HA (blue) staining of cells transfected with GFP vector (upper panel), Drp1 (GFP-tagged wild-type Drp1[middle panel]), or Drp1 (GFP-tagged wild-type Drp1) with DN-GSK3b (HA-tagged) constructs (lower panel). Cells with both GFP and HA-positive staining were cotransfected with both Drp1 and DN-GSK3b plasmids. F–H: Measurement of mitochondrial length (F ), density(G), or complex I enzyme activity (H) among indicated groups. N$ 20 cells per group from two independent experiments. Scale bar = 10 mm.(-) Indicates the vector transfection and (+) indicates the presence of the indicated plasmid transfection.

diabetes.diabetesjournals.org Huang and Associates 1737

revealed a significant reduction of mitochondria com-pared with control(s). This reduction in neuronal mito-chondria is suggestive of a diabetes-mediated interferencewith mitochondrial biogenesis and fission. In addition,these mitochondrial morphology changes were confirmedby electron microscopy. Measurement of hippocampal mi-tochondrial functional capacity in this mouse model ofdiabetes indicated significantly reduced activity of mito-chondrial respiration complex I and lowered ATP content.Our results are consistent with findings of a similar studyprobing the skeletal muscle of patients with type 2 diabetes(33), in which there are impairments in energy metabolismand respiratory complex enzyme activity. Balance of mito-chondrial fission and fusion processes is crucial for main-tenance of mitochondrial morphology and function(59,60). In diabetic hippocampus, the mitochondrial fis-sion protein, Drp1, was significantly increased and itsphosphorylation was coincidentally elevated. On the otherhand, levels of Mfn2, OPA1, and other auxiliary mito-chondrial proteins such as HSP60 (data not shown)were not altered in diabetic hippocampus. Our findingsare consistent with previous investigations of diabeticneuropathy, which included reports of increased Drp1expression and mitochondrial fission in dorsal root

ganglion neurons of 6-month-old db/db mice (15,31). Incontrast to the greater numbers of mitochondria in dorsalroot ganglion neurons (31), we note that hippocampalneurons in 5- to 6-month-old db/db mice displayedsmaller numbers of mitochondria, while there was nosign showing mitochondrial decline among subjects youn-ger than 3 months (data not shown). Differences in sen-sitivity to hyperglycemia between hippocampal neuronsand dorsal root ganglion cells may account for the differ-ences in our findings compared with previous studies(31). In addition, increased mitochondrial fission mayeventually result in mitochondrial degeneration if the bal-ance of fission and fusion is not maintained. We furthertested whether mitochondrial enzyme (COXI) activity andATP content were changed at 3 months and 6 months ofage. We found that complex I enzyme activity signifi-cantly declined by 15% and 35% at 3 and 6 months ofages, respectively, though no significant alteration in ATPcontent was found at 3 months of age (SupplementaryFig. 7A and B). Similarly, the LTP significantly declinedby 16% in 3-month-old db/db hippocampus compared withthat of age-matched db/m control mice (SupplementaryFig. 7C–E). These data suggest age-dependent defects inmitochondrial and synaptic function. Pharmacologic or

Figure 6—Effect of Drp1 inhibitor mdivi-1 on mitochondrial changes in diabetic hippocampus. A: Representative images of SODII stainingof hippocampal neurons from the indicated groups using confocal microscopy. Scale bar = 10 mm. B–D: Quantification of mitochondrialdensity using MetaMorph software (B), complex I activity (C), and ATP levels (D) in the indicated groups of animals treated with vehicle (-) ormdivi-1 (+). Data are presented as fold increase relative to vehicle-treated db/m mice of indicated hippocampi. E: Representative electronmicroscope images of mitochondria (indicated by arrows). Scale bar = 500 nm. F–H: Quantification of mitochondrial length (F ), ratio oflength to width (G), form factor (H), and mitochondrial density (I) in the indicated groups of animals treated with vehicle (-) or mdivi-1 (+) fromelectron microscope images. N = 6–11 animals per group.

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genetic inactivation of Drp1 significantly preventedmitochondrial morphology and function changes in db/dbhippocampus or human SK cells under high-glucose condi-tions, indicating a role of Drp1 in diabetes-induced mito-chondrial morphology and functional changes.

There is at least one mechanism by which diabeteselevates Drp1 expression and phosphorylation resultingin mitochondrial alterations. To our knowledge, Drp1 isable to be phosphorylated at several sites by differentkinases. It was first identified that Drp1 can be phos-phorylated at Ser637 by protein kinase A (61,62), whichwas coincident with mitochondrial elongation. CaMK Ican also phosphorylate the Ser637 site to promote mito-chondrial fission (63). While the role of phosphorylatedDrp1 at the Ser616 site still remains paradoxical, increas-ing evidence points out that Ser616 Drp1 is related to themitochondrial fragmentation; for example, cyclin-dependentkinase 1/cyclin B–dependent phosphorylation at Ser616 ofDrp1 induced mitochondrial fission during mitosis(64). Furthermore, our recent studies suggest a role of

extracellular signal–related kinase 1/2 in oxidativestress–induced increased phosphorylation of Drp1 (37).In our present study, we observed mitochondrial degen-eration/fragmentation along with elevated levels of Drp1phosphorylation at Ser616 in diabetic conditions both invivo and in vitro. GSK3b is activated in db/db mice in vivoowing to dephosphorylation on Ser9 residue (48,49) andcan use Drp1 as one of its substrates in in vitro systems(51–53). We therefore focused on the role of GSK3b-mediated mitochondrial defects by modulating Drp1Ser616 phosphorylation. We have clearly demonstratedthat GSK3b activation was elevated in either type 2diabetes–affected hippocampus or high glucose–treatedhuman neuronal cell lines. While the underlying mecha-nisms for the alterations in GSK3b activation in in vivoand in vitro diabetic conditions are not clear, diabetes-induced Akt dephosphorylation (Supplementary Fig. 8Aand B) may contribute, at least in part, to the GSK3bactivation in addition to the aforementioned activationof other kinases. Indeed, we observe that pharmacological

Figure 7—Effect of Drp1 inhibitor mdivi-1 or GSK3b inhibition on diabetes-impaired LTP in hippocampal CA1 region. A and B: Basalsynaptic transmission (A) and paired-pulse facilitation (PPF) (B) were not significantly affected by diabetes. C: Hippocampal LTP issignificantly lower in db/db mice compared with db/m control slices. D–F: Hippocampal LTP (D), basal synaptic transmission (E), andpaired-pulse facilitation (F ) in db/m mouse brain administered with vehicle or mdivi-1 (10 and 25 mg/kg i.p injection daily for 2 weeks) weresimilar. G: Diabetes-impaired hippocampal LTP was restored by mdivi-1 administration. Upper panel is representative of fEPSP tracesindicating the neurotransmission responses before (gray) and after (black) u-burst stimulation from mdivi-1–treated/nontreated animals.Vertical bar = 1 mV, horizontal bar = 5 ms. H: LTP levels of indicated animal groups were calculated by averaging the last 10 min of fEPSPslope. I–K: Blocking GSK3b activation with TDZD8 (5 mmol/L) or LiCl (4 mmol/L) suppressed LTP reduction in db/db hippocampus (I) butdid not change the baseline or the LTP levels in db/m controls (J). LTP levels of indicated animal groups were calculated by averaging thelast 10 min of fEPSP slope and are illustrated in K. TBS, u-burst stimulation. Dashed lines in H and K indicate the baseline level. TBS,u-burst stimulation; V/S, voltage/second. N = 8–14 slices of 5–8 animals per group.

diabetes.diabetesjournals.org Huang and Associates 1739

inhibition of GSK3b successfully prevented Drp1 phos-phorylation at Ser616 in the diabetes-affected mousebrain and high glucose–treated human cell line. Theseresults further support the role of GSK3b in diabetes-induced mitochondrial defects via Drp1-mediated abnor-mal mitochondrial fission, though our results would notexclude the possibility of the involvement of extracellularsignal-regulated kinases 1/2 in high glucose–inducedDrp1 Ser616 phosphorylation and mitochondrial fragmen-tation (65). Furthermore, we demonstrated that geneticactivation of GSK3b without high-glucose treatment canalso promote mitochondrial fragmentation. The inactiva-tion of GSK3b prevents high glucose–induced mitochon-drial alterations. Taken together, these data suggest thatGSK3b is one of the mechanisms underlying diabetes-induced mitochondrial abnormalities.

GSK3b most likely acts as an upstream signaling mech-anism for Drp1 upregulation in diabetes-induced mito-chondrial dysfunction. Phosphorylation of Ser9 on GSK3bin db/db hippocampus was decreased, resulting in higherlevels of the active form of GSK3b. Inhibition of GSK3bactivity was associated with suppressed phosphorylationof Ser616 on Drp1, a critical step enabling Drp1 GTPaseactivity for mitochondrial fission (46). Currently, thereare no inhibitors available for prevention of Drp1 Ser616

phosphorylation; however, treatment with mdivi-1 (inhib-iting the GTPase activity of Drp1) maintained mitochon-drial morphology and function, though GSK3b activationwas unaltered. GSK3b not only acts as an upstream signal-ing mechanism for Drp1 but also directly interacts withDrp1, which, in turn, is responsible for phosphorylationof Drp1 Ser693 and leads to decreased GTPase activityand an enhancement of mitochondrial fusion/elongation(53). We also determined whether an inactive form ofGSK3b (p-ser9 GSK3b) interacts with Drp1. We foundthat pull-down GSK3b (active form) yielded Drp1 immuno-reaction bands in db/db animals, but no interaction wasfound between p-ser9 GSK3b and Drp1 (data not shown).

Using a human neuronal cell line, we directly modulatedGSK3b or Drp1 activation by transfection with differenttargeted vectors. Inactivation of GSK3b prevented GSK3b-involved activation of Drp1 signaling and changes in mito-chondrial morphology and function under diabetic conditions.However, inactivation of Drp1 did not alter GSK3b acti-vation even though it preserved mitochondrial morphol-ogy under high-glucose conditions, suggesting a role forGSK3b in diabetes-induced activation of Drp1 signalinginvolved in alterations in mitochondrial morphology.

Another major novel finding of the current study isthat diabetes-induced synaptic transmission deficit, atleast in part, depends on GSK3b/Drp1-mediated mito-chondrial abnormalities. Consistent with the results ofother researchers (5,7,8,11,16–20), we observed LTP re-duction in the hippocampal CA1 region in db/db mice.Because complete absence of Drp1 in mice is lethal, wewere not able to generate diabetic db/db mice with Drp1knocked out at this point; however, we successfully

restored diabetes-induced mitochondrial alterations bytreating mice with the Drp1 inhibitor, mdivi-1. Treatmentwith mdivi-1 restores mitochondrial function along withthe improvement in LTP in the diabetic hippocampus.Notably, a recent investigation demonstrates that mdivi-1also inhibits the rapidly activating delayed-rectifier po-tassium channel (66) in murine cardiomyocyte cell lines.While the inhibitory effect of mdivi-1 on potassium chan-nels has not yet been tested in neurons, mdivi-1 mightcause an elevated excitatory activity in the central nervoussystem if a potential effect on potassium ion channelexists. This requires further investigation. The beneficialeffect of inhibiting Drp1-mediated aberrant mitochon-drial and synaptic function by mdivi-1 treatment is un-likely owing to an insulin- or corticosterone-dependentmechanism (8). Indeed, corticosterone levels are increasedin diabetic animals compared with nondiabetic litter-mates; however, inhibition of Drp1 using mdivi-1 failedto prevent diabetes-induced corticosterone increases butdid ameliorate diabetes-induced hippocampal LTP deficits.

In summary, our study offers new insights into the roleof GSK3b/Drp1 interaction in diabetes-induced mitochon-drial and synaptic dysfunction. We clearly demonstratedalterations in mitochondrial structure and function indiabetes-affected hippocampus. Impaired structural orfunctional capacity of mitochondria is attributable totype 2 diabetes–induced impairment of synaptic plasticity.Inhibition of GSK3b/Drp1-mediated deficits in mitochon-drial architecture and resultant functional changes amelio-rate LTP deficit. Blockade of GSK3b activation or inhibitionof mitochondrial division significantly improves mitochon-drial and synaptic function under diabetic conditions.These studies suggest targets for the development of inter-ventions to prevent or treat diabetes-induced mitochon-drial deficits and accompanying neurodegeneration.

Funding. This study was supported by grants from the National Institute onAging (R37AG037319 and R01AG044793) and National Institute of NeurologicalDisorders and Stroke (R01NS065482).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. S.H., X.G., and D.F. performed experiments andstatistical analysis. Y.W. supervised and analyzed all the experimental data, wrotethe manuscript, and performed experiments and statistical analysis. C.Z. and L.W.performed experiments. G.H. prepared constructs. A.A.S. and G.M.M. conductedelectron microscope experiments. H.Y. performed statistical analysis. S.S.Y. directed,designed, and supervised this study and wrote the manuscript. S.S.Y. is the guar-antor of this work and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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1742 Mitochondria and Hippocampal LTP Deficit Diabetes Volume 64, May 2015