Dr. Yohannes Haile Department of Medicine/Neurology

Supervisor: Dr. Fabrizio Giuliani Laboratory

I earned a B.Sc. majoring in Biology and minor in Chemistry from the University of Asmara-Eritrea, where I was born and raised. Because of my interest in research, upon graduation, I worked as a research assistant in the department of biology. I was involved in a research project dealing with hepatitis and HIV. Since 1998-2002, I worked as a biologist and assistant program manager in the head office of the ministry of health, National TB program, under the division of communicable diseases control (CDC) in Asmara-Eritrea. There, my duty was very broad and enabled me to develop many skills including supervision, teaching, research, conducting workshops, management, leadership, teamwork, preparation of budgets and action plans. This position also allowed me to enhance my communication skills with local and international experts/consultants coming through WHO. This was an ideal job with a conducive environment; however, pursuing M.Sc. and PhD study was always my dream. Consequently, in October 2002, I succeeded to secure two international scholarships (each for 2-years): One from the German Academic Exchange Service (DAAD) to pursue M.Sc. study at the University of Bremen, Germany. At the same time, I was awarded the second scholarship from the Netherlands Organization for International Cooperation in Higher Education (NUFFIC) for M.Sc. study at the University of Utrecht, the Netherlands. I opted the first and joined the University of Bremen from 2002-2004 where I did M.Sc. (with distinction), under the supervision of Prof. Rudolf Amann, in the Max-Planck Institute for Marine Microbiology (MPI). Using various molecular biology techniques, I identified the types and distributions of the microbes in the samples collected from the Namibian upwelling. These data were crucial for my supervisors to develop a bigger project which is still running.

Right upon completing M.Sc. in molecular microbiology from MPI at the University of Bremen, I joined the PhD program in neuroscience at the Center for Systems Neuroscience, within the University of Veterinary Medicine Hannover, Germany. This was a three years PhD program (2004-2007) where I tested and optimized the biocompatibility and bioresorbablity of a novel biomaterial (polysialic acid or polySia) for regenerative medicine as an alternative to the current neurosurgical treatment strategies. Under the supervision of Profs. Claudia Grothe and Rita Gerady-Schahn, I examined the role of polySia and polySia based hydrogel in the survival, proliferation and differentiation of embryonic progenitor cells, neonatal and adult Schwann cells, dorsal root ganglionic neurons and embryonic spinal motor neurons which are all possible candidates for reconstructive therapies. The findings suggested the biocompatibility and potential use of polySia as a scaffold material in nerve tissue regeneration. This project was very successful and generated 3 papers (I am a first author in two) published in Biomaterials which is a respected journal in the field. About 14 abstracts are published in different proceedings. These data were tremendously vital to renew the grant with generous funding and multiple positions at the end of the 3rd

year. While investigating whether polySia induced immunological reaction or cytotoxicity in rats, I developed/expanded an interest to explore the role of inflammation in neurodegeneration. For this reason, I pursued the study of inflammation-mediated neurodegeneration processes in neurodegenerative diseases, such as multiple sclerosis (MS) for my postdoctoral project.

Since January 2008, I have been working as a postdoctoral fellow in the laboratory of Dr. Fabrizio Giuliani (and co-supervised by Dr. Chris Bleackley) in the Department of Medicine at the University of Alberta. In the last four and half years, I investigated the mechanisms by which inflammation-mediated neuronal injury in neurodegenerative diseases, with special emphasis on multiple sclerosis. I investigated the role of inflammatory T cells (activated T cells: CD4, CD8), regulatory T cells and the effector molecule serine proteinase granzyme B (GrB) in mediating neuronal injury. I have produced relevant amounts of data showing that GrB is one of the most potent mediators of T cell-induced neuronal damage/death. These findings have generated four manuscripts in which I am the first author. Two of the manuscripts are published in the Journal of Leukocyte Biology and the Journal of Immunology while the others two are yet to be submitted. I published more than 20 abstracts produced out of these data. Moreover, I was a recipient of a poster presenter award at the Department of Medicine Research Day 2008 and one of my abstracts was selected for “Scientific Highlight” in the 61st

American Academy of Neurology meeting which was held, in 2009, in Seattle. Moreover, my work was given a platform presentation in the society for neurosciences (SFN) meeting in 2010 in San Diego. After unveiling the detailed mechanisms of inflammation or GrB-mediated neurodegeneration, I am studying the effect of GrB-inhibitors in an attempt to block T cell-mediated neuronal injury both in vitro and in vivo in the animal model of MS. Our abstract of these data is recently selected for an oral presentation in the upcoming International Society of NeuroImmunology (ISNI) congress which will be held in November 2012 in Boston, USA. While attempting to prevent neurodegeneration, I developed an interest to use stem cells to replace/restore the lost tissues, nerve and glial cells. Hence, on another project, I am characterizing the stem cell-like NTera2/D1 (NT2) cell lines, which are derived from human embryonic carcinoma cells. These are pluripotent stem cell lines which can be induced to differentiate into a range of cell lineages (eg. neurons and glial cells) in response to retinoic acid stimuli. Molecular, physiological and morphological characterization of neuronal cells derived from NT2 cells exhibited similar properties to human primary fetal neurons. This cell line can be employed as a model in the study of the biology of different neurodegenerative diseases. The manuscript is ready to be submitted to the journal of neuroscience. In addition, my interest is not limited to research but also I greatly enjoy teaching, mentoring and training students.

Finally, I would like to use this opportunity to thank my mentors, our collaborators and co-authors who contributed to our projects.

of July 3, 2012.This information is current as

Cell-Induced NeurotoxicityVulnerability of Human Neurons to T Granule-Derived Granzyme B Mediates the

Bleackley and Fabrizio GiulianiNicolas Touret, Thomas Simmen, Jian-Qiang Lu, R. Chris Yohannes Haile, Katia Carmine Simmen, Dion Pasichnyk,

http://www.jimmunol.org/content/187/9/4861doi: 10.4049/jimmunol.1100943September 2011;

2011; 187:4861-4872; Prepublished online 30J Immunol 

Referenceshttp://www.jimmunol.org/content/187/9/4861.full#ref-list-1

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The Journal of Immunology

Granule-Derived Granzyme B Mediates the Vulnerability ofHuman Neurons to T Cell-Induced Neurotoxicity

Yohannes Haile,* Katia Carmine Simmen,† Dion Pasichnyk,* Nicolas Touret,†

Thomas Simmen,‡ Jian-Qiang Lu,x R. Chris Bleackley,† and Fabrizio Giuliani*

Multiple sclerosis (MS) is considered an autoimmune disease of the CNS and is characterized by inflammatory cells infiltrating the

CNS and inducing demyelination, axonal loss, and neuronal death. Recent evidence strongly suggests that axonal and neuronal

degeneration underlie the progression of permanent disability in MS. In this study, we report that human neurons are selectively

susceptible to the serine-protease granzyme B (GrB) isolated from cytotoxic T cell granules. In vitro, purified human GrB induced

neuronal death to the same extent as the whole activated T cell population. On the contrary, activated T cells isolated from GrB

knockout mice failed to induce neuronal injury. We found that following internalization through various parts of neurons, GrB

accumulated in the neuronal soma. Within the cell body, GrB diffused out of endosomes possibly through a perforin-independent

mechanism and induced subsequent activation of caspases and cleavage of a-tubulin. Inhibition of caspase-3, a well-known

substrate for GrB, significantly reduced GrB-mediated neurotoxicity. We demonstrated that treatment of neurons with

mannose-6-phosphate prevented GrB entry and inhibited GrB-mediated neuronal death, suggesting mannose-6-phosphate

receptor-dependent endocytosis. Together, our data unveil a novel mechanism by which GrB induces selective neuronal injury

and suggest potential new targets for the treatment of inflammatory-mediated neurodegeneration in diseases such as MS. The

Journal of Immunology, 2011, 187: 4861–4872.

Multiple sclerosis (MS) is an inflammatory autoimmunedisease of the CNS and is the most common cause ofnontraumatic chronic neurologic disability affecting

young adults in North America and Europe (1–3). MS has clas-sically been described as a demyelinating disorder characterizedby oligodendrocyte death and relative sparing of axons and neu-rons. Since the mid-1990s, neurodegeneration has been reportedas one of the main features of MS characterized by axonal losswithin lesions and involving both white and deep/cortical graymatter (4). In MS patients, magnetic resonance imaging and his-topathology of the brain revealed diffuse damage to the whitematter even in areas far from the inflammatory foci (5–7). Thus, ithas been suggested that the major cause of physical disability inMS patients is represented by axonal/neuronal degeneration, al-though the mechanisms that mediate this neuronal injury arepoorly understood (8).

In healthy individuals, lymphocyte traffic into the CNS is verylow and tightly controlled (9, 10). In contrast, under inflammatoryconditions, circulating T cells are activated and readily cross theblood-brain barrier, gaining access to the CNS in a significantnumber (10). Indeed, T lymphocytes are among the main con-stituents of the inflammatory infiltrates within MS lesions (11, 12).Earlier studies have shown a correlation among infiltrating lym-phocytes, axonal pathology, and neuronal death in particularwithin the progressive MS (13). More recently, it has been shownthat natalizumab, a mAb that prevents lymphocyte migrationacross the blood-brain barrier, significantly reduced light neuro-filament, a biomarker of axonal damage (14), in the CSF of re-lapsing remitting MS patients (15).Activated T lymphocytes secrete granzyme B (GrB), a serine

protease released from the granules of cytotoxic T cells, and inducetarget cell death by disrupting a variety of intra/extracellular pro-tein substrates (16–18). Caspases are among GrB substrates (19,20), and their activation leads to cell death by apoptosis. GrB-expressing cytotoxic T cells were observed in close proximity ofoligodendrocytes or demyelinating axons in acute MS lesions(21). Indeed, T cells activated by a soluble anti-CD3 Ab inducedsevere neurotoxic effect in both allogeneic and syngeneic systemsin vitro (22); however, the mechanisms of this T cell-mediatedneurotoxicity have not been explored. It has recently been shownthat recombinant GrB induces neuronal injury in vitro (23).However, recombinant GrB does not have the posttranslationalchanges occurring in the granule-derived GrB that are required toenter the target cell (24). Indeed, in the study by Wang et al. (23),the rate of neuronal injury was low (10–20%) and not comparableto what we observe in the T cell-mediated neuronal injury.In the current study, we show that MS active lesions, charac-

terized by the presence of infiltrating inflammatory cells, expressedhigh levels of GrB. In vitro, granule-purified human GrB andactivated T cells induced severe neurotoxic effects on humanneurons. Furthermore, in the murine system, T cells isolated fromGrB knockout BL6 mice were not able to induce killing of neurons

*Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7,Canada; †Department of Biochemistry, University of Alberta, Edmonton, AlbertaT6G 2H7, Canada; ‡Department of Cell Biology, University of Alberta, Edmonton,Alberta T6G 2H7, Canada; and xDepartment of Laboratory Medicine and Pathology,University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Received for publication April 4, 2011. Accepted for publication August 26, 2011.

This work was supported by the Multiple Sclerosis Society of Canada, the Universityof Alberta Hospital Foundation, the Alberta endMS Regional Research and TrainingCentre of the endMS Research and Training Network, and the Canadian Institutes ofHealth Research.

Address correspondence and reprint requests to Dr. Fabrizio Giuliani or Dr. R. ChrisBleackley, 9-101 Clinical Sciences Building, University of Alberta, Edmonton, ABT6G 2G3, Canada (F.G.) or Department of Biochemistry, University of Alberta,Edmonton, AB T6G 2H7, Canada (R.C.B.). E-mail addresses: giuliani@ualberta.ca(F.G.) or rbleackl@ualberta.ca (R.C.B.)

Abbreviations used in this article: Ac-IEPD-pNA, acetyl-Ile-Glu-Pro-Asp-paranitro-anilide; CM, conditioned media; EEA1, early endosome Ag 1; endoH, endoglycosi-dase H; GrB, granzyme B; HFN, human cortical fetal neuron; hGrB, human purifiedGrB; MAP-2, microtubule-associated protein-2; M6P, mannose-6-phosphate; M6PR,M6P receptor; MS, multiple sclerosis; NAWM, normal-appearing white matter; PFA,paraformaldehyde; RT, room temperature.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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derived from the syngeneic naive mice. Interestingly, GrB enteredneurons through the mannose-6-phosphate (M6P) receptor (M6PR)and induced cellular death by disrupting the cytoskeleton. Alto-gether, our data demonstrate that granule-derived GrB plays a sig-nificant role in T cell-mediated neuronal injury affecting neuro-degenerative diseases such as MS.

Materials and MethodsCulture of human T cells, RNA isolation, and real-timeRT-PCR

Human PBMCs were isolated from the blood of adult healthy volunteerdonors using the Ficoll-Hypaque centrifugation separation and suspended inserum-free AIM-V T cell culture medium (Life Technologies, Burlington,ON, Canada). T cells were plated, at a density of 200,000 cells/well in 200ml,on multiwell culture plates immobilized with either 5 mg/ml human anti-CD3 or anti-CD3/CD28 Ab (for induction of T cells activation) or on un-treated wells (for unactivated control cells) for 3 d. After 3 d in culture, cellswere collected and centrifuged to isolate cell pellets. In a separate ex-periment, frozen and paraffin-embedded tissue samples were collectedfrom three noninflammatory control cases (1 amyotrophic lateral sclerosis,1 Alzheimer’s dementia, and 1 cardiac arrest) and three MS patients,according to the guidelines approved by the local institutional ethics com-mittee. These samples were obtained from the brain bank of the MS clinic atthe University of Alberta. In the MS tissues, areas of active and chronicactive MS lesions as well as normal-appearing white matter (NAWM) wereidentified based on the presence or absence of infiltrating inflammatory cellsand demyelination in adjacent white matter sections, as described pre-viously (12). For RT-PCR, the frozen brain tissues and cells (separately)were lysed and homogenized in TRIzol (1 ml/106 cells; Invitrogen, Carls-bad, CA). Total RNA was collected from the aqueous phase and purifiedusing RNeasy mini kit, according to the stated protocol (Qiagen, Valencia,CA). The concentration and quality of RNA were measured using Nano-Drop 1000 Spectrophotometer (Thermo Scientific). The amount of 2 mgRNA for each reaction was used to synthesize cDNA. RNA was treatedwith DNase I (Promega, Madison, WI), and cDNA was synthesized usingoligo(dT) and Superscript II reverse transcriptase (Invitrogen), according tothe manufacturer’s recommended instructions. Semiquantitative RT-PCRwas carried out using Bio-Rad iQ SYBR green supermix on either iQ5 pro-gram or i-Cycler (Bio-Rad, Hercules, CA), according to the manufacturer’sprotocol. Primers for GrB (forward, 59-GCG GTG GCT TCC TGA TACAAG-39; reverse, 59-CCC CCA AGG TGA CAT TTA TGG-39) amplifi-cation were used at annealing temperature of 60˚C. All data were nor-malized to the average value of both human GAPDH (forward, 59-AGCCTT CTC CAT GGT GGT GAA GAC-39; reverse, 59-CGG AGT CAACGG ATT TGG TCG-39) and human b-actin (forward, 59-CCATCATGAAGT GTG ACG TGG-39; reverse, 59-GTC CGC CTA GAA GCA TTTGCG-39), and the results were expressed as relative fold increase.

Human fetal neuron culture

Human brain tissue was obtained from 15- to 20-wk fetuses according to theguidelines approved by the local institutional ethics committee. Humancortical fetal neurons (HFNs) were isolated, as previously described (22).Briefly, brain specimens were washed in PBS, followed by removing themeninges and blood clots. The fragmented brain pieces were transferredinto 50-ml tube and digested in 4 ml 2.5% trypsin and 6–8 ml DNase I for30 min in 37˚C water bath. The activity of trypsin was inhibited by theaddition of 4 ml FBS. Cells were filtered in 125-mm mesh and spun for 5min at 1200 rpm. Following the final washing step, cells were suspended inculture medium (described below) and plated on 10 mg/ml poly-ornithine–coated T-75 flasks at a density of at least 70 3 106 cells/flask in 25 mlmedium. The cell culture was supported with MEM supplemented with10% FBS, 1% L-glutamine, 1% essential amino acids, 1% sodium pyru-vate, 0.1% dextrose, as well as 1% antibiotics. All medium componentswere obtained from Life Technologies. The neuron culture was incubatedat 37˚C with 5% CO2.

The purity of the neurons was maintained by suppressing the pro-liferation of astrocytes. The cells were treated with 5 mM 5-fluoro-2-deoxyuridine or 25 mM cytosine b-D arabinofuranoside (Arac-C; Sigma-Aldrich, St. Louis, MO) at interval of every 3–4 d starting from immediateseeding. Enriched neuronal cells (∼95%) were further trypsinized andplated (100,000 cells/well) onto 16-well Lab-tek (Nunc, Naperville, IL)slides for 3 d before coculturing with T cells (for neuronal killing assay).The maturity of HFNs has been previously described by assessing themorphological differentiation, neuron-specific biochemical expression, andphysiological properties of the neurons (25).

Mouse fetal neuron culture

Neurons were obtained from 15-d-old embryonic mice (strain: BL6).Pregnant mice were anesthetized and decapitated. The skin in the stomacharea was incised, and the embryos were taken out into the petri dish. Eachembryo was separated from the embryonic sack, and the head was cut andcollected in a separate petri dish. Under the microscope, the meninges werecarefully peeled off. The cortical parts of the two hemispheres were col-lected into a 15-ml falcon tube containing buffer HEPES, HBSS, andantibiotics. The tissues were washed twice with the buffer and incubated in1 ml trypsin for 10 min at 37˚C in the water bath. The activity of trypsinwas stopped by FBS, and the tissue was homogenized using pasture pi-pette. The cells were centrifuged for 5 min at 1400 rpm and suspendedin neurobasal medium supplemented with 1% sodium pyruvate, 25 mMHEPES, 1% antibiotic/mycotic, 1% glutamine, and 2% B-27 supplement.Cells were counted and plated in a concentration of 5 million cells/ml.

Isolation and culture of mouse T cells

Spleens were isolated from wild-type (strain: BL6) or GrB knockoutmale mice [strain: BL6.129Sgzmb(tm1Ley)]. The spleen was mechanicallydissociated, and T cells were isolated using the Ficoll-Hypaque centrifu-gation separation. They were then suspended in RPMI 1640 cell culturemedium supplemented with 1% glutamine, 1% HEPES, 1% antibiotic/mycotic, 10 mM 2-ME, and 5% mouse serum (Life Technologies).T cells were plated, at a density of 200,000 cells/well in 200 ml, onmultiwell culture plates either immobilized with 5 mg/ml mouse anti-CD3Ab (for induction of T cell activation) or on untreated wells for 3 d.

Immunocytochemistry

HFNs (100,000 cells/well) were cultured on poly-ornithine–coated 16-wellculture plates for 3 d. In parallel, T cells were cultured either on untreatedor anti-CD3/CD28–coated multiwells. After 3 d, the same amount (1:1) ofactivated T cells was applied into the neuron culture. Similarly, HFNs weretreated with GrB (100 ng/100 ml) purified from the human NK cell line,YT-Indy, prepared as previously described (26), and the coculture was keptfor 24 h. The control neuronal culture groups were treated with only AIM-V medium (without T cells) or unactivated T cells. Moreover, in the su-pernatant killing assay, supernatant either from unactivated or 3-d–acti-vated T cells was added, with or without GrB, into neuronal culture.

In caspase-inhibition study, HFNs were treated either with 200 mM Z-VAD-FMK (pan-caspase inhibitor) or 200 mM Z-DEVD-FMK (caspase-3inhibitor) (both from Kamiya Biochemical) for 2 h before addition of GrB.

Similarly, mouse neurons (100,000 cells/well) were cultured on poly-ornithine–immobilized 16-well culture plates for 3 d. Simultaneously,T cells from wild-type or GrB knockout mice were cultured either onuntreated or mouse anti-CD3–coated multiwells. After 3 d, 100,000T cells/well were applied into the neuronal culture and cocultured for 24 hsupplemented with neurobasal media (neurobasal medium supplementedwith 1% sodium pyruvate, 25 mM HEPES, 1% antibiotic/mycotic, 1%glutamine, and 2% B-27 supplement).

After 24-h coculturing, the cells were fixed with 4% paraformaldehyde(PFA) for ∼20 min, followed by 5-min washing with PBS. Neuronal cellswere permeabilized with 0.1% Triton X-100 for ∼10 min, and the non-specific binding was blocked with 5% goat serum in PBS for 1 h at roomtemperature (RT). The slides were carefully detached from the chamberwells, and 30 ml/well mouse primary mAb against human microtubule-associated protein-2 (MAP-2; diluted 1:1000 in blocking buffer; Sigma-Aldrich) was applied on each spot in the human neuronal culture system.However, in the mouse neuron staining, MAP-2 entailed nonspecificbackground because it was a mouse Ab. Therefore, in the mouse neuronalculture, we used rabbit mAb against mouse b-III tubulin (diluted 1:1000in blocking buffer). The culture was incubated for 45 min to 1 h at RTand followed by washing in PBS. To visualize neuronal cells, the culturewas stained with Alexa 594-conjugated secondary Ab (diluted 1:300 inblocking buffer) for 45 min to 1 h at RT in dark. The slides were thor-oughly washed in PBS, and the remaining plastic materials around thewells were carefully peeled off. After mounting in permaFluor mountmedium, MAP-2 (for human)- or b-III tubulin (for mouse)-stained neu-ronal cells were analyzed by fluorescence microscopy. Similarly, for in-filtrated lymphocyte analysis in the MS tissues, paraffin-embedded tissuesections were stained by immunohistochemistry with an anti-CD8 as wellas anti-CD3 mAbs. The myelination/demyelination status of axons wasevaluated using Luxol Fast Blue, according to the standard procedure.

For DAPI staining, cells were cocultured for 24 h, followed by fixationwith 4% PFA for 20 min. After washing for 5 min in PBS, DAPI (diluted1:1000 in PBS) was applied for 20–30 min in the dark and quantified underthe fluorescence microscopy.

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To evaluate the cytotoxic effect of T cells or GrB on neurons, six fieldsper well were randomly and manually counted using original magnification340 objective fluorescence microscopy. The mean value of the controlneuronal culture that was not exposed to T cells was treated as 100%. Theaverage number of MAP-2–, DAPI/MAP-2–, or b-III tubulin-positiveneurons/group was inferred as a percentage of the control neuronal cul-ture group. At least six wells were quantified from each condition. Everyexperiment was repeated at least three times.

Perforin inhibition study

It is well known that the lytic activity of perforin is dependent on thepresence of calcium ions to allow the formation of pores on the target cells(27). Therefore, to avoid the possibility of perforin contamination withinthe purified GrB, both purified GrB and supernatants from 3-d–activatedT cells were incubated with a Ca2+ chelating agent (0.4 M EGTA/0.8 MMgCl2) for 1 h before adding into neuronal culture, as previously described(28). HFNs were cultured for 3 d, followed by treatment either with pu-rified GrB or supernatant from activated T cells in the presence/absence ofthe Ca2+ chelating agent. The control groups were treated with AIM-Vmedia only. Similar experiments were conducted by preincubating acti-vated T cells with the Ca2+ chelating agent. After 24-h coculture, neuronalviability was evaluated by immunocytochemistry using anti–MAP-2 Ab.

Comparison between GrB constitutively secreted fromactivated T cells and human purified GrB used in neuronalkilling assay

T cells were activated for 3 d, and the conditioned media (CM; supernatants)from both activated and nonactivated T cells were analyzed on a 10% PAGEdenaturing gel. The gel was loaded with the following four groups (controland experimental groups): 15 ml AIM-V media; 15 ml unactivated T cellCM; 15 ml T-activated CM; or 21.43 ng human purified GrB (hGrB) fromYT-Indi cell lines. The proteins were transferred onto nitrocellulose over-night and probed against hGrB 2C5 Ab at a 1:500 dilution (Santa Cruz).

Measuring enzymatic activity of GrB constitutively secretedfrom activated T cells

hGrB and GrB secreted from T cells activated with either anti-CD3 ora combination of anti-CD3/CD28 were assayed for their enzymatic activity.GrB enzymatic activity was measured in RPMI 1640 reaction mix con-taining 50 mM HEPES (pH 7.5), 10% (w/v) sucrose, 5 mM DTT, 0.05%(w/v) CHAPS, and 300 mM acetyl-Ile-Glu-Pro-Asp-paranitroanilide(Ac-IEPD-pNA; Kamiya Biomedical). The plate was incubated for 6 hat 38˚C. Hydrolysis of Ac-IEPD-pNA was measured at 405 nm at time0 and every hour thereafter using a Multiskan Ascent spectrophotometer(Thermo Lab System).

Cleavage of GrB N-linked oligosaccharides byendoglycosidase H

The impact of endoglycosidase H (endoH) on GrB, either secreted fromactivated T cells (CD3 or CD3/CD28) (labeled CM), stored in their cells(labeled ⊄), or purified from YT-Indi, was assessed. Native cell lysate,CM, or purified GrB protein was first denatured in 5% SDS and 0.4 MDTT at 100˚C for 10 min, and then was incubated with recombinantendoH, according to the manufacturer’s instruction (BioLabs), anddigested at 37˚C for 2 h and 30 min. GrB glycoforms were resolved ona 14% SDS-PAGE, transferred onto a nitrocellulose membrane, and probedwith anti-human GrB 2C5 Ab diluted in 1:500 (Santa Cruz Biotech-nology).

GrB internalization and M6P blocking

PurifiedGrB was labeled using Alexa Fluor 488microscale protein-labelingkit (Invitrogen, Molecular Probes), according to the manufacturer’sinstructions. To assess whether GrB is acting on the surface or inside thecell, human neuronal cell cultures were treated with green fluorescence-tagged GrB (GrB488; 0.5–1 mg/ml) for either 3, 6, or 24 h at 37˚C.Similarly, some groups were treated with 25 mM M6P for 1–2 h beforeaddition of GrB. Following the incubation period, the media were removedand cells were washed three times with PBS, followed by 33 washing withMEM media plus 0.5% BSA (pH 2) at RT. Cells were washed again threetimes with PBS and then fixed for 30 min with 4% PFA at RT. Finally,slides were mounted and viewed under the confocal microscope at originalmagnifications 340–63.

In addition, the role of M6P in inhibiting GrB-induced neurotoxicity wasevaluated. After 3 d in culture, HFNs were treated with 1–25 mM M6P for∼2 h, followed by incubation with GrB (1 mg/ml) or activated T cells.

Control groups were treated with either GrB, activated T cells, or M6Palone, or remained untreated. After 24-h incubation, the viability of neu-rons was evaluated immunocytochemically using anti–MAP-2 Ab.

GrB488: colocalization with early endosome, intracellulardiffusion, and neuronal apoptosis

To evaluate whether GrB is taken up by early endosomes, HFNs werecultured for 3 d, followed by incubation (blocking) with donkey serum(1:10,000 in PBS) to block the unspecific binding. Neurons were treatedwith 0.5–1 mg/ml GrB488 for 15 min, 20 min, 30 min, 1 h, or 2 h. Afterwashing three times with PBS, neurons were fixed in 4% PFA and immunestained for early endosomal marker using mouse IgG1 anti-human earlyendosome Ag 1 (EEA1; 250 ng/ml or 1:1000 in PBS; BD Biosciences),following the protocol described above for immunocytochemistry. Theculture was further incubated for 20 min with DAPI (1:1000 in PBS) tostain the nucleus. The colocalization of GrB and EEA1 or DAPI wasassessed under confocal microscopy.

To analyze the time scale of GrB-induced cellular apoptosis, GrB488-treated neuronal cultures (coverslips) were washed with acidic media(MEM media + 0.5% BSA [pH 2] at RT), followed by final washing withPBS. The neurons were further incubated for 20–30 min with the followingapoptotic markers: annexin V 647 (1:200 in PBS) and propidium iodide(0.4 mg/ml). Nuclear fragmentation was evaluated by staining the neuronalnuclei with DAPI. After washing the culture with PBS, GrB488 in-ternalization and induction of apoptosis were evaluated by confocal mi-croscopy.

Western blotting

HFNs were cultured for 3 d. Simultaneously, human PBMCs were activatedusing anti-CD3 for 3 d. For caspase inhibition study, some groups of HFNcultures were treated with 200 mM Z-VAD-FMK (pan-caspase inhibitor)for 2 h. Neurons were then further treated with purified GrB (1 mg/ml),activated T cells, or unactivated T cells. Controls remained untreated, andHela cells were used as a positive control. After 24 h, T cells were care-fully washed, and proteins from HFNs were isolated and loaded into 10%electrophoresis gel. The protein was transferred into nitrocellulose mem-brane (1 h at 100 V). The nonspecific binding in the membrane wasblocked with 13 TBS/casein blocker (Bio-Rad). After several washes withPBS, the cleavage of the cytoskeletal protein was evaluated using Abagainst a-tubulin (1:3000).

Statistical analysis

Results were statistically analyzed as mean6 SD using GraphPad Prism 5.The groups were compared using one-factor ANOVA, followed by Tukeyposthoc test for normally distributed data. A two-tailed unpaired t test wasapplied to compare two groups with normally distributed data. The pvalues ,0.05 were considered significant. Asterisks correspond to *p ,0.05, **p , 0.01, and ***p , 0.001.

ResultsActive lesions of MS and activated T cells express GrB

Human brain tissue was obtained from three MS patients as wellas three noninflammatory disease control patients, and the lesiontypes were identified, as previously reported (12). Luxol Fast Bluestaining showed that MS active lesions are significantly demye-linated and are characterized by massive infiltration of in-flammatory cells (Fig. 1aA), whereas in the chronic active lesion,there is significant demyelination and decreased density of in-flammatory cells compared with the active lesion (Fig. 1aB). Nodemyelination or inflammatory infiltrates were observed in theNAWM as well as in the healthy control tissue (Fig. 1aC).Immunostaining for infiltrating T lymphocytes showed a signifi-cant number of CD8+ T cells in the active lesions compared withthe chronic active lesions. No CD8+ cells were identified in theNAWM (Fig. 1aD–aF, respectively) as well as no CD3+ T cells(data not shown). The presence of double GrB/CD3+ T cells inacute MS lesions has been previously reported (21). In this study,we assessed the expression of GrB in the different lesion types.RNAwas isolated from active and chronic active lesions as well asNAWM. Control tissue was obtained from normal subjects. RT-PCR revealed that MS active lesions expressed .100-fold change

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in GrB when compared with normal controls. On the contrary, theexpression of GrB in chronic active lesions was significantly lowerthan the active lesions, but significantly higher than the NAWM(p , 0.001; Fig. 1b). The difference in GrB expression betweenthe different lesion types correlated with the variation in density ofCD8+ T cells, as depicted in Fig. 1aD–aF. Similarly, anti-CD3–activated T cells expressed ∼40-fold change of GrB comparedwith the control unactivated T cells (Fig. 2a; p , 0.001). We have

previously shown that GrB is mainly expressed by CD8+ T cells,although CD4+ cells also express it, but to a lower level (29).

Activated T cells and GrB kill human neurons

To investigate whether expression of GrB has a neurotoxic effect,a neuronal killing assay was performed using MAP-2 as a marker ofneuronal viability, as previously described. Disappearance ofMAP-2immunoreactivity has been shown to be associated in vivo and

FIGURE 1. Identification of MS lesions and

measurement of GrB expression. a, Luxol Fast

Blue (LFB) staining in active, chronic active, and

NAWM lesions of MS (A–C respectively; original

magnification 320). The lower panels demon-

strate immunohistochemical staining for CD8+

T cells in active, chronic active, and NAWM

lesions (D–F; original magnification 340). b, RT-

PCR showing the expression of GrB in the nor-

mal control or active, chronic active, or NAWM

lesions of MS patients (*p , 0.05, ***p ,0.001). This difference in GrB expression may

attribute to the variation in density of infiltrated

T lymphocytes in the different lesion types. The

data in the graph are pooled from three in-

dependent experiments.

FIGURE 2. GrB expression and activated T cells

mediated neuronal killing in vitro. a, Expression of

mRNA for GrB in unactivated or anti-CD3–activated

T cells. b, Immunocytochemical staining using a neu-

ronal viability marker, MAP-2. Human neurons either

remained untreated control (A, B) or were cocultured

with unactivated T cells (C), cocultured with activated

T cells (D), or treated with GrB (E). Scale bars, 50 mm;

200 mm. c, Quantification of MAP-2–positive neurons

in the control group or the groups treated with unac-

tivated T cells, activated T cells, or hGrB. The per-

centage indicates the ratio between viable MAP-2–

positive neurons and untreated control. d, Quantifica-

tion of DAPI/MAP-2 double-positive (merged) cells in

the control group or the groups cocultured with unac-

tivated (Unact.) or activated (Act.) T cells. The per-

centages indicate the ratio between viable DAPI/MAP-

2–positive cells and controls. Asterisks indicate sig-

nificant difference between compared groups (*p ,0.05, **p , 0.01, ***p , 0.001). Data are represen-

tative of at least three individual experiments.

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in vitro with neuronal injury and death (22). Activated T cells werecocultured with HFNs for 24 h. Immunocytochemical analysis us-ing anti–MAP-2 Ab revealed that untreated control neurons (Fig.2bA, 2bB) and the group cocultured with unactivated T cells (Fig.2bC) remained viable, whereas a severe neuronal loss was noticedin the group cocultured with activated T cells (Fig. 2bD). Further-more, the addition of purified GrB (1mg/ml) to the neuronal cultureinduced dramatic neuronal killing (Fig. 2bE). Quantification ofMAP-2–stained neurons clearly showed that both activated T cellsand purified GrB induce the same extent of neuronal death (.60%),whereas the control groups did not produce any toxic effect (Fig.2c; p , 0.01). Furthermore, quantification of DAPI/MAP-2 double-positive (merged) neurons showed similar results (Fig. 2d; p ,0.001). Comparing the neurotoxic effects of CD4+ and CD8+ T cellsrevealed that the majority of neuronal death is inflicted by the latter,and correlates to the level of GrB expressed by CD8+ T cells (29).These findings suggest the relevance of GrB on neurotoxicity. Be-cause neuronal cultures had a purity of ∼95%, and there was nodifference between MAP-2– and DAPI-based quantification, weused MAP-2 immunoreactivity alone as a marker of neuronal via-bility in most of our experiments.

Activated T cells from GrB knockout mice fail to kill mouseneurons

To further confirm whether GrB induces neuronal killing, T cellswere isolated from the spleens of GrB knockout mice, activated

with anti-CD3, and cocultured with mouse embryonic day 15/16neurons. Control neuronal cultures were treated with activated orunactivated T cells isolated from wild type or treated with onlymedium without T cells. As shown in Fig. 3a, a viability assayusing anti–b-III tubulin Ab revealed that the neurons in thecontrol groups (Fig. 3aA–aC) remain healthy with unalteredmorphology. In contrast, significant neuronal loss was observed inthe group treated with activated T cells from control wild type(Fig. 3aD). Interestingly, activated T cells from GrB knockoutmice did not induce neurotoxicity (Fig. 3aE). Quantification ofb-III tubulin-stained neurons showed that activated T cells fromwild-type mice induce a significant neurotoxic effect (.50%)compared with activated T cells from GrB knockout mice, un-treated control, or control neurons cocultured with unactivatedT cells (Fig. 3b).

Supernatants from activated T cells do not induce killing ofhuman neurons

To assess whether GrB-mediated neuronal killing was inducedby constitutively secreted GrB or by the stored/degranulated GrB,supernatants from activated T cells were applied to HFN culturesand incubated for 24 h. Staining with anti–MAP-2 Ab showed thatthe supernatants do not induce any neurotoxic effect. On thecontrary, the addition of purified GrB to the supernatant of acti-vated T cells induced severe neurotoxicity, matching the effectof GrB alone (Fig. 4a; p , 0.05).

FIGURE 3. GrB knockout activated mouse T cells

do not induce neuronal killing. a, Immunocytochem-

istry, using anti–b-III tubulin Ab, showing embryonic

day 15 mouse neurons untreated control (A, B) or

cocultured with unactivated mouse T cells (C), cocul-

tured with activated mouse T cells from wild-type mice

(D) or activated mouse T cells from GrB knockout

mice (E). Scale bars, 50 mm (A), 200 mm (B–E). b,

Quantification of b-III tubulin-positive viable mouse

neurons from the groups shown in a. The percentage

indicates the ratio between viable b-III tubulin-positive

mouse neurons and untreated control (*p , 0.05,

**p , 0.01, ***p , 0.001). Mouse T cells were har-

vested from spleens of control wild-type or GrB

knockout mice. Data are representative of three in-

dividual experiments.

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To address the difference in neurotoxic effect between theconstitutively secreted and granule-derived GrB, Western blottinganalysis on T cell lysates and supernatants was performed. Theresults indicated that the secreted GrB differs in molecular massfrom the granule-purified human GrB (Fig. 4b, lanes 3, 4). Therewas no trace of GrB in the supernatant or lysate of unactivatedT cells (Fig. 4b, lanes 1, 2, respectively). GrB derived from theanti-CD3– or anti-CD3/CD28–activated T cell lysates had com-parable molecular mass to the granule-purified hGrB (molecularmass 32 kDa), whereas the secreted/released GrB had a highermolecular mass (molecular mass 35 kDa). To investigate whetherthe different molecular mass were related to a difference in gly-cosylation, the GrB proteins were enzymatically digested by

endoH that cleaves asparagine-linked mannose. Following di-gestion, the granule-stored GrB was cleaved into two distinctfragments of ∼30 and 27 kDa, whereas the secreted GrB in thesupernatant resulted in ∼35-, 32-, and 27-kDa fragments (Fig. 4c).These findings suggest two distinct isoforms of GrB: one storedwithin the granules and another constitutively secreted via a non-granule route. These GrBs differ in the amount of complexedmannose, and this is in agreement with previous reports (24).Moreover, the enzymatic activity of the supernatants obtainedeither from anti-CD3– or anti-CD3/CD28–activated T cells wascompared with purified granule-derived hGrB. Experiments usingGrB-ELISA showed high levels (beyond the kit’s detection limit)of GrB secreted by activated T cells into the supernatant (data not

FIGURE 4. Granule-derived GrB is required to mediate neuronal injury. a, Human fetal neurons were treated either with AIM-V T cell media (control) or

supernatants from unactivated and anti-CD3–activated T cells in the absence or presence of purified GrB or GrB alone in the T cell media (*p , 0.05). b,

Western blotting showing the relative molecular mass of GrB secreted into the supernatant obtained from activated T cells (lane 3; 35 kDa) and hGrB (lane

4; 32 kDa); lanes 1 and 2 are controls containing only cell culture media or supernatant attained from unactivated T cells, respectively. c, Comparison of

GrB in the CM or cell lysates (¢) of unactivated T cells, anti-CD3–activated T cells, anti-CD3/CD28–activated T cells, or hGrB. 2 or +, Indicates the

absence or presence of endoH. Addition of endoH-induced different fragments of GrB with molecular mass of 32 kDa (denoted by blue circle), 30 kDa

(blue asterisk), and 27 kDa (red asterisk). d, Measurement of GrB enzymatic activity against time. The enzymatic activity of human GrB (89 ng) was

compared with constitutively secreted GrB from unactivated T cell, anti-CD3–activated T cells (100% and diluted to 50 or 25%), or anti-CD3/CD28–

activated T cells (100% and diluted to 50 or 25%). e, Assessment of perforin inhibition, by chelating calcium using 0.4 M EGTA/0.8 M MgCl2, of

constitutively secreted or purified GrB-mediated neuronal killing (columns 3–5). The percentage indicates MAP-2–stained viable neurons in relation to the

controls (*p , 0.05). Data are representative of two to three individual experiments.

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shown). Thus, the supernatants were diluted in media into 50 or25%, and hydrolysis of the GrB substrate Ac-IEPD-pNA wasmeasured at 405 nm. Only the higher concentrations of hGrB(.178 ng) showed more activity than the supernatants. Consti-tutively secreted GrB from activated T cells cleaved IEPD-pNAsubstrate in vitro, meaning significantly higher GrB activitycompared with supernatant derived from unactivated T cells (Fig.4d). These findings demonstrate that the difference in neurotoxiceffect between secreted and granule-derived GrB is not related toa difference in their enzymatic activity, but possibly associatedwith posttranslational modifications (30). In addition, an assaywas performed to test whether perforin, a pore-forming protein,played a role in the GrB-mediated neuronal killing. Previousreports have shown that the lytic activity of perforin is dependenton the presence of calcium ions (27, 28, 31–33). Granule-purifiedGrB does not contain perforin; however, to exclude any possiblecontamination with this protein, both purified GrB and/or super-natant from activated T cells were incubated with calcium chelator(0.4 M EGTA and 0.8 M MgCl2) for 1 h before adding them tothe neuronal culture. Quantification of MAP-2–positive neuronsshowed that perforin inhibition does not prevent neuronal killingeither in the neuronal culture treated with purified GrB or in theculture treated with supernatant plus GrB (Fig. 4e; p , 0.05).Similarly, perforin inhibition (Ca2+ chelation) did not preventGrB-mediated events (e.g., caspase 3 activation, a-tubulin cleav-age) or activated T cell-mediated neuronal death (data not shown).These findings suggest that GrB-mediated neuronal killing onhuman neuronal cells is independent of perforin.

Purified GrB internalization and neurotoxicity are blocked byM6P

Our study showed that GrB-mediated neuronal injury is in-dependent of perforin. Thus, we investigated the possible entry siteof GrB. Based on our findings that the secreted and granule-derivedGrB differ in the amount of mannose, and considering previous

reports on the role of M6PR as a receptor for GrB (30) and itsselective expression on neurons but not on astroglia (22, 30, 34),we investigated the role of this receptor on GrB-induced neuro-toxicity. As illustrated in Fig. 5a, unlike the untreated controls inwhich neurons show a regular morphology (Fig. 5aA, 5aA9), there issignificant amount of GrB internalized into the neuronal cells in thegroup treated with the fluorescent labeled GrB488 (see arrows in Fig5aB9 indicating internalized GrB). This internalization was com-pletely blocked when the neurons were preincubated with 25 mMM6P (Fig. 5aC9). Unlike the GrB-treated group (Fig. 5aB, 5aB9), theneurons preincubated with M6P (Fig. 5aC, 5aC9) had normalmorphology similar to untreated neuronal control cultures (Fig. 5aA,5aA9). Further immunocytochemical analysis using anti–MAP-2staining revealed no changes in neuronal density and morphologyin the M6P plus GrB- or M6P alone-treated groups compared withcontrol untreated neurons (Fig. 5bA, 5bC, 5bD). In contrast, a se-vere neuronal loss was observed in the GrB alone-treated group(Fig. 5bB). Quantitative analysis showed that the viability ofneurons in the GrB-treated group is only 16.84 6 11.25%, andM6P pretreatment significantly increases neuronal viability to66 6 26.76% (Fig. 5c; p , 0.001). However, in the T cell–neuroncoculture, pretreatment of neurons with M6P did not preventneuronal loss, although there was a trend toward neuroprotectionat 25 mM M6P (Fig. 5d). This suggests that activated T cells usealternative mechanisms to deliver GrB or to induce target celldeath.

Purified GrB is taken up by early endosomes and diffused inthe cytoplasm of neuronal cells independent of perforin

To assess whether the internalized GrB is initially stored in theendosomes, neurons were treated with GrB488 for 15 min to 2 h.Immunostaining revealed that GrB488 is essentially colocalizedwith the endosomal marker EEA1 within 15 min of GrB treatment(see Fig. 6aA–aE), and then gradually diffuses out into the cyto-plasm in the absence of any lytic agent (Fig. 6aF–aJ).

FIGURE 5. GrB internalization induces neuronal

apoptosis and is blocked by M6P. a, Human fetal

neurons were either untreated controls (A or A9), treated

with 0.5 mg/ml GrB488 (B or B9, arrows indicate in-

ternalized GrB), or blocked with 25 mM M6P and then

treated with 0.5 mg/ml GrB488 (C or C9). A, B, and C

are phase-contrast images of their respective micro-

graphs. b, Immunocytochemistry using anti–MAP-2 Ab

shows untreated control neurons (A), GrB-treated neu-

rons (B), 25 mM M6P + GrB-treated neurons (C), or 25

mM M6P alone-treated human neurons (D). c, Quan-

tification of the MAP-2–positive viable human neurons

shown in B. d, Human fetal neurons were untreated

(control), or cocultured with activated T cells (lane 2),

or pretreated with 1 or 10 or 25 mM M6P and then

cocultured with activated T cells (lanes 3–5, re-

spectively). Quantification of the MAP-2–positive

neurons revealed that pretreatment of neurons with

M6P does not prevent activated T cell-mediated neu-

ronal death. Instead, there is a slight trend at the con-

centration of 25 mM M6P, but it was not statistically

significant. The percentage of MAP-2–positive neurons

is expressed in relation to the control (*p , 0.05,

***p , 0.001). Data are representative of three indi-

vidual experiments.

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We further evaluated GrB-induced cellular apoptosis. Immu-nostaining showed colocalization of GrB488, annexin V 647, andpropidium iodide in single neurons (see arrows in Fig. 6bA, 6bA9).Moreover, some DAPI-stained cells showed nuclear fragmentationsurrounded by GrB (data not shown), suggesting an apoptotic celldeath. This GrB-mediated neuronal apoptosis was rapid and oc-curred within 40 min; however, not all of the neurons were dyingat the same time, and this can be related to the fact that the cultureis a mixture of heterogeneous neuronal cells with potentiallydifferent sensitivity to GrB cytotoxicity.

GrB-induced neuronal death is caspase dependent

To investigate the substrate/target of GrB during neuronal apo-ptosis, HFNs were treated with either activated T cells or GrB.Control groups were cocultured with unactivated T cells. A positivecontrol was performed using HeLa cells (35). In both experimental(using HFNs) and positive control (HeLa cells) groups, Westernblotting analysis showed that the cytoskeletal protein a-tubulingets cleaved when HFNs are cocultured with activated T cells ortreated with hGrB. This indicates that activated T cells releasegranule-derived GrB, which mediates neuronal killing by desta-bilizing the cytoskeletal protein of the neuronal cells (Fig. 7a[lanes 3, 4], 7a9 [lanes 4, 5]). The bands in the control groups,such as the untreated HFNs, HeLa cells, or neurons coculturedwith unactivated T cells, remained uncleaved (Fig. 7a, 7a9). Toaddress whether GrB acts directly on the substrate, a-tubulin, orinduces apoptosis indirectly through caspase activation, HFNswere incubated with the pan-caspase inhibitor Z-VAD-FMK priorto the coculture or treatment with purified GrB. As previouslydescribed, both activated T cells and GrB induced a-tubulincleavage (Fig. 7b, lanes 3, 5). This cleavage was absent when theneurons were pretreated with Z-VAD-FMK (Fig. 7b, lanes 2, 6),suggesting that the a-tubulin cleavage is caspase dependent. To

further elucidate the role of caspase-mediated neuronal killing,another set of experiments was conducted using caspase-3 in-hibition. When neuronal cultures were treated with GrB alone,almost 80% of neurons were killed (Fig. 7cB, 7cE, column 2; p ,0.05). In contrast, neuronal cultures pretreated with Z-VAD-FMKor the caspase-3 inhibitor significantly reversed this severe neu-ronal loss (Fig. 7cA, 7cC, 7cD, 7cE, columns 3, 4). These datashow that GrB-induced neuronal apoptosis is mainly mediated bycaspase-3 activation.

DiscussionOur study addresses the detailed mechanisms of GrB-mediatedneuronal injury that might contribute to neurodegenerative pro-cesses of MS (Fig. 8). It is well documented that T cells expressand release GrB upon activation or following the encounter witha foreign Ag. Indeed, active lesions of MS patients are charac-terized by the presence, among others, of infiltrating inflammatoryT cells. In this study, the expression of high levels of GrB in theactive lesions of MS is possibly a consequence of the presence ofinflammatory T cells, mainly CD8+ T cells, within the tissue. GrB-expressing T cells were found in close apposition to demyelinatedaxons in the parenchyma of acute MS lesions (21). Similarly, ina rat model of spinal cord injury and cerebral ischemia, it hasbeen shown that neuronal death occurs in close proximity to GrB-positive cells (36, 37). In our study, we show that GrB-expressingactivated T cells induced neuronal killing (60%) comparable togranule-purified human GrB. Furthermore, activated T cells iso-lated from wild-type mice were significantly neurotoxic, whereasactivated T cells from GrB knockout mice failed to induce anyneuronal injury. Our findings are in agreement with a previousreport by others who have shown that GrB-deficient CTLs wereunable to induce DNA fragmentation in target cells (17). Althoughit is known that there are significant differences between human

FIGURE 6. Micrographs showing the uptake of GrB by endosomes. a, Human fetal neurons were treated with 0.5–1 mg/ml GrB488 for 15 min (A–E) and

for 30 min (F–J). Immunostaining for colocalization of GrB and EEA1 showed that the internalized GrB is initially stored in the endosomes (see 15 min; D

and E), and then gradually released out of the endosomes without the addition of any lytic agent, and diffused into the cytoplasm (see 30 min; H–J). A and F

are phase-contrast images, B and G stained for early endosomal marker (EEA1 red), C and H are GrB488 (green), D and I are merged EEA1 + GrB488, and

the nuclei in E and J are stained with DAPI (blue). b, Time evaluation of GrB488-induced human neuronal apoptosis using apoptotic markers. A9, GrB

diffused in the soma of neurons (green); axons and cell membrane positive for annexin V 647 (red) and propidium iodide-stained nuclei (blue) (see arrows

in A9; A is a phase-contrast image of A9). (Scale bar, 50 mm.) Neuronal apoptosis occurred within 40 min from GrB488 treatment.

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and murine granzymes, our results support the evidence that GrBis responsible for the induction of neuronal injury in the mousesystem (38).More recently, Wang et al. (23) have shown that anti-CD3/

CD28–activated T cells release GrB into the supernatant, whichinduces mild cytotoxicity (∼20%) on human neurons. In contrast,we found that supernatants from activated T cells could not induceneuronal injury, whereas cell-to-cell contact-dependent neuronalkilling induces severe (60–80%) neuronal death. Using a transwellsystem in which neurons and activated T cells are coculturedin two separate compartments divided by a porous membrane,allowing only the exchange of soluble factors, we have previouslyreported that no neurotoxicity occurs. In addition, we have shownthat neurons are highly susceptible to activated T cell-mediatedcytotoxicity independently of MHC class I or II expression and inthe absence of added Ag (22). It is well known that the expressionof MHC-1 on healthy and fully differentiated neurons within theCNS is minimal or negligible (39, 40). Furthermore, most in-filtrating T cells within MS lesions are Ag nonspecific, and it hasbeen shown that T cells are able to induce collateral bystanderaxonal damage and/or neuronal death within the CNS. Thesefindings underline the importance of cell–cell contact in deliver-ing the “kiss of death” to the target neurons and induce effectiveneurotoxicity. This interaction may occur independent of Agspecificity (41–43).Moreover, we observed that GrB constitutively secreted in the

activated T cell supernatant has higher molecular mass (35 kDaversus 32 kDa) and different glycosylation compared with thegranule-derived GrB released following degranulation at the im-mune synapse. It was also recently reported that CTLs releasemainly inactive GrB (GrB zymogen) through the constitutivepathway (44), which might explain the low neuronal killing in-duced by the supernatant of activated T cells. Interestingly, in ourstudy, the activity of the constitutively secreted GrB was compa-rable to that of granule-purified GrB. However, endoH, whichcleaves asparagine-linked mannose oligosaccharides, digested theconstitutively secreted GrB into three different fragments (∼32,30, and 27 kDa), whereas granule-derived GrB produced twofragments of 30 and 27 kDa, consistent with previous reports (24).

These data suggest that the two GrBs differ in their post-translational modification and mannose content. To effectivelyexert its cytotoxic effect, GrB needs to enter the target neuronalcell. It has been shown that mannose is essential for GrB entry intosome target cells and that the M6PR is the death receptor for GrB.M6PR is highly expressed on the surface of neurons, but not onglial cells (30, 45). Indeed, neurons are sensitive to CTL-mediatedkilling (46). On the contrary, no T cell- or GrB-mediated cyto-toxicity has been described in glial cells such as astrocytes andoligodendrocytes (22, 23). These observations suggest M6PR asthe potential candidate receptor for GrB entry into neuronal cells.In this study, we show that blocking the M6PR with its high-affinity ligand M6P effectively inhibited granule-purified humanGrB internalization and prevented neuronal killing (Fig. 5a–c),confirming that the main route of purified GrB entry into neuronsis through M6PR. Therefore, unlike the constitutively secretedGrB, which contains highly complex carbohydrates, possession ofM6P enabled the granule-purified GrB to enter to the target cellvia M6PR and induce apoptosis. In contrast, Wang et al. (23) haveshown that M6P is not able to prevent GrB-mediated neuronalkilling. However, they found that the killing is prevented by per-tussis toxin, suggesting the mediation of a Gi-coupled receptorsuch as M6PR. This apparent discrepancy can be related to thelow dose of M6P used (1 mM compared with 25 mM in our study)because, as previously reported, M6P inhibits GrB-mediated cel-lular killing in a dose-dependent manner (30). Similarly, in ourstudy, both purified GrB- and T cell-based neurotoxicity assaysshowed that low concentration of M6P (1–10 mM) did not protectneurons from death, and any concentration of M6P higher than 25mM displayed different levels of toxicity on the cells. We havepreviously reported that half-maximal inhibition of target cellfragmentation was only evident with M6P at unphysiologicalconcentrations (.25 mM) (30). In addition, in Wang et al. (23),the use of recombinant GrB without posttranslational modificationand subsequent addition of M6P can also explain these differ-ences. Moreover, although M6PR may interact with several non-M6P–containing ligands, they have much lower affinity for thereceptor (47). Furthermore, the constitutively secreted 35-kDaGrB, usually found in the supernatant of activated T cells, lacks

FIGURE 7. a-Tubulin is a GrB substrate in human

fetal neurons and its cleavage is caspase dependent. a,

Western blotting showing the cleavage of a-tubulin by

GrB. Neurons were treated with GrB (lane 3) or un-

treated (lane 1). HeLa cells were used as a positive

control (35). a9, Cleavage of a-tubulin in the presence

of either activated T cells or purified GrB (lanes 4, 5,

respectively). Control lanes 1–3 show neurons alone or

neurons cocultured with unactivated T cells or activated

T cells alone where cleavage of a-tubulin is absent. b,

Cleavage of a-tubulin by activated T cells (lane 3) or

GrB (lane 5) and positive control (lane 7); the cleavage

was absent when neurons were treated with 200 mM Z-

VAD-FMK (pan-caspase inhibitor) prior to treatment

with activated T cells (lane 2) or GrB (lane 6). Lanes 1

and 4, Control untreated neurons or cocultured with

unactivated T cells. c, Micrographs showing MAP-2–

stained untreated control neurons (A), GrB-treated

neurons (B), pan caspase-inhibitor + GrB-treated (C),

or caspase 3 inhibitor + GrB-treated neurons (D) and

quantification (based on percentage of MAP-2–positive

control neurons) of the groups stated above (E). *p ,0.05. Scale bars, 50 mm. Data are representative of

three individual experiments. The asterisks indicate the

lanes where a-tubulin is cleaved as a result of treatment

with GrB or activated T cells.

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the M6P-targeting motif, and this explains its failure to acquiremannose and enter the target cell (24) and to induce significantneuronal death. Overall, these findings would explain the di-vergence in the rate of GrB-mediated cell death between Wanget al. (23) (10–15%) and our study (60–80%).As described above, granule-purified GrB internalization into

neuronal cells is dependent on M6PR. In contrast, M6P pre-treatment of neurons did not prevent T cell-mediated neuronaldeath. This lack of protection could be related to the contemporaryrelease of perforin and activation of a membrane-repair response, asrecently described by Thiery et al. (48). This recent study supportsprevious reports showing membrane receptors as being not cru-cially important for the delivery of GrB into the target cell duringthe T cell-mediated cellular death; and the binding of GrB to thetarget cell can be facilitated by the expression of surface moleculessuch as heparan sulfate (49, 50). Similarly, other reports showedM6PR-independent target cell death using M6PR knockout mice, aswell as employing various cell lines that overexpress/lack M6PR(51). Nevertheless, this does not imply that M6PR is not abso-lutely essential for the endocytosis of GrB; rather, it suggests thatM6PR is not the only port of entry for GrB directly released bythe T cells into the target neurons. Indeed, we have previouslyreported that CTL-mediated DNA fragmentation is significantlyhigher on M6PR-overexpressing compared with M6PR-deficientcells, and the cellular apoptosis increases with the augmentation ofthe ratio between effector and target cells. In addition, it is pos-sible that high concentrations of GrB are released at T cell–neuroninterface such that some uptake can occur in a M6PR-independentfashion or GrB could possibly antagonize M6P on the receptor(30). Moreover, lymphocytes may possess multiple lytic mecha-nisms to induce apoptosis on the target cell (22, 52).Perforin has been reported as a key mediator in the CTL-induced

axonal/neuronal damage in various mice models of autoimmunediseases (53–57). In contrast, our study showed that GrB in-ternalization into neurons as well as purified GrB and T cell-mediated neuronal killing are independent of perforin (Figs. 4e,6a). Following its internalization through M6PR, GrB is imme-diately taken up by early endosomes. Previous works, in other celltypes, demonstrated that internalized GrB does not induce deathuntil endosomes are disrupted by perforin or adenovirus withsubsequent release of GrB within the cytoplasm (48, 58, 59). In-terestingly, in this study, GrB was released out of the earlyendosomes in the absence of any lytic agents, diffused in thecytoplasm, and rapidly induced neuronal apoptosis. Using annexinVas an early apoptotic marker, we showed colocalization of annexinV 647, GrB, and propidium iodide. DAPI staining revealed nu-clear fragmentation surrounded by GrB-positive endosomes. Inaddition, GrB-mediated responses such as caspase 3 activation anda-tubulin cleavage were not affected by perforin inhibition. Thesefindings suggest a perforin-independent cell death following in-ternalization of GrB, although we cannot exclude the possibility ofa concomitant effect on the extracellular matrix. It has been shownthat a perforin-independent GrB-mediated smooth muscle celldeath occurs through cleavage of extracellular matrix and anoi-kosis (60). These findings suggest that GrB-mediated target cellapoptosis could be cell specific and eventually pursue differentpathways depending on the cell type. The inability of preventingT cell-mediated neuronal death by inhibiting perforin does notdecrease the relevance of this pore-forming protein; instead, ithighlights the existence of other routes and mechanisms such asM6PR, contributing to the entry of GrB and subsequent inductionof neuronal cell apoptosis. Nevertheless, the release of GrB fromthe endosomes without addition of perforin or any other lyticagent is absolutely novel and requires further investigations.

The mechanisms that lead to diffuse neuronal and axonal injuryin MS have not been thoroughly explored (61). In the cerebralcortex of chronic MS patients, axonal and neuronal degenerationoccurs in the absence of parenchymal inflammatory infiltratinglymphocytes (7, 62), and it strongly correlates with clinical dis-ability (63, 64). However, the mechanisms of neuronal loss in MSare not yet defined, although a possible role is played by a “dyingback” mechanism consisting of a retrograde degeneration ofneuronal soma following axonal injury (8, 46, 65). Gray matteratrophy characterizes the chronic stages of the MS neuronal in-jury. In contrast, acute MS neuronal injury is represented by ax-onal transection and formation of axonal spheroids suggestive ofa cytoskeletal disruption (6). In the animal model of MS, exper-imental autoimmune encephalomyelitis, T cells mediated thedisruption of the microtubule network within neurons (66). In-deed, the release of GrB within the target cell initiates apoptosisby cleaving a variety of protein substrates that have either direct orindirect consequences in DNA fragmentation and cell death (16,17, 67). A microtubule component, a-tubulin, was recently shownto be a new substrate for GrB in HeLa cells (35). In this study, toour knowledge, we showed for the first time that a-tubulin iscleaved by GrB in human neurons. This cleavage could explainthe disruption of the fast axonal transport and subsequent neuritetransection and spheroid formation at the proximal end (6, 8).It is widely accepted that GrB induces apoptosis by activating

caspases in target cells (19). In our study, activated T cells or GrB-induced death was blocked by the pan-caspase inhibitor Z-VAD-FMK, suggesting that this GrB-mediated cleavage of a-tubulin isdependent upon caspase activation. Caspase-3 inhibitors reversedthe severe neuronal killing induced by GrB, confirming the pre-vious reports that this caspase is a key substrate for GrB-mediatedcell death and is highly expressed within neuronal cell bodies andmediates the death of the soma (16, 23, 67, 68).In conclusion, we demonstrate that neurons are highly suscep-

tible to T cell-mediated cytotoxicity because of their selectiveexpression of M6PR, which allows GrB internalization. Inside theneuron, GrB releases out of the endosomes independent of perforin

FIGURE 8. Schematic diagram summarizing the mechanism of GrB-

mediated neuronal apoptosis. Anti-CD3–activated or CTL release granule-

derived GrB into the target cell (neuron). GrB enters into neurons through

M6PR and accumulates within the endosomes. GrB diffuses out from the

endosome and activates caspase-3. The activation of caspase-3 results

in either a direct cellular apoptosis or cleavage of a cytoskeletal protein

(a-tubulin) and subsequent neuronal apoptosis.

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and cleaves both caspases and a-tubulin with subsequent neuronaldeath (Fig. 8). Inhibiting the activity of GrB together with tar-geting the binding site of GrB to M6PR or blocking activation ofcaspases can offer potential novel approaches for future therapiesof neuroinflammatory diseases of the CNS such as MS.

AcknowledgmentsWe thank Dr. Chris Power’s laboratory and Kristofor Ellestad at the Uni-

versity of Alberta for technical assistance and Dr. Von Wee Yong at the

University of Calgary for revising the manuscript. We also thank Jasmine

Snyder at the University of Alberta for reading the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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