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Neurobiology of Disease 21 (2006) 314 – 323

The therapeutic potential of neural stem/progenitor cells in murine

globoid cell leukodystrophy is conditioned by

macrophage/microglia activation

Serena Pellegatta,a Patrizia Tunici,a Pietro Luigi Poliani,a,b Diego Dolcetta,c Laura Cajola,a

Cristina Colombelli,a Emilio Ciusani,a Stefano Di Donato,a and Gaetano Finocchiaroa,*

aIstituto Nazionale Neurologico C. Besta, Department of Experimental Neuro-Oncology and Diagnostics, via Celoria 11, 20133 Milano, ItalybDepartment of Pathology, University of Brescia, Brescia, ItalycDIBIT-HSR, Milano, Italy

Received 25 March 2005; revised 15 July 2005; accepted 25 July 2005

Available online 30 September 2005

Twitcher (GALCtwi/twi) is the murine model of globoid cell leukodys-

trophy (GLD or Krabbe disease), a disease caused by mutations of the

lysosomal enzyme galactocerebrosidase (GALC). To verify the ther-

apeutic potential on twitcher of neural stem/progenitor cells (NSPC),

we transduced them with a GALC lentiviral vector. Brain injection of

NSPC-GALC increased survival of GALCtwi/twi from 36.1 T 4.1 to 52.2 T5.6 days ( P < 0.0001). Detection of GALC activity and flow cytometry

showed that NSPC-GALC and NSPC expressing the green fluorescent

protein were attracted to the posterior area of twitcher brain, where

demyelination occurs first. GALCtwi/twi microglia, also more abundant

in posterior regions of the brain, released significant amounts of the

cytotoxic cytokine TNF-alpha when matched with NSPC-GALC.

Thus, in murine GLD, and possibly in other demyelinating diseases,

NSPC are attracted to regions of active demyelination but have

limited survival and therapeutic potential if attacked by activated

macrophages/microglia.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Leukodystrophy; Neural stem cells; TNF-alpha; Microglia; Cell

therapy

Introduction

Twitcher is the authentic model in mice of globoid cell

leukodystrophy (GLD or Krabbe disease) (Suzuki, 1983). GLD

is a disorder involving the white matter of the central nervous

systems and the myelin of the peripheral nervous system.

Mutations in the gene for the lysosomal enzyme galactocerebro-

sidase (GALC) result in low enzymatic activity and decreased

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.07.016

* Corresponding author. Fax: +39 02 26681688.

E-mail address: finocchiaro@istituto-besta.it (G. Finocchiaro).

Available online on ScienceDirect (www.sciencedirect.com).

ability to degrade galactolipids, found almost exclusively in

myelin. The pathological changes observed, including the presence

of globoid cells and decreased myelin, appear to result from the

toxic nature of psychosine and accumulation of galactosylceramide

that cannot be degraded due to the GALC deficiency. The

histopathological hallmarks of the disease include extensive

demyelination of the central nervous system (CNS), gliosis and

the presence in the white matter of globoid cells, macrophages that

contain PAS-positive material.

Twitcher mouse, a natural mutant of the C57BL6J strain,

represents the murine model of the disease and reproduces the

same biochemical and histopathologic features. The symptoms

appear 3 weeks after birth and include the typical ‘‘twitching’’, a

severe loss of weight, head drop, gait incoordination and hindleg

weakness. Mice usually die around post-natal day 35 (Suzuki,

1995).

In twitcher, bone marrow transplantation (BMT) has been the

only therapeutical approach significantly delaying disease onset

and progression. BMT showed clinical and neuropathological

improvements with gradual disappearance of globoid cells,

limited remyelination and increased GALC levels in the CNS

due to the cross correction of enzymatically competent donor-

derived macrophages (Hoogerbrugge et al., 1988a,b, 1989;

Kondo et al., 1988; Seller et al., 1986; Suzuki et al., 1988;

Yeager et al., 1984, 1991, 1993). In late onset patients, some

benefit was documented from bone marrow transplantation

(Krivit et al., 1998). In early onset patients, severe limitations

to this treatment have been observed (Caniglia et al., 2002).

Transplantation of umbilical-cord blood in babies with infantile

Krabbe disease was found to alter favorably the natural history of

the disease only if performed before the development of

symptoms (Escolar et al., 2005).

The goal of our study was to evaluate a therapeutic approach

complementary to BMT, using engineered neural stem/progenitor

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323 315

cells (NSPC) as the main source of GALC. One rationale for this

approach is provided by the observation that hematopoietic stem

cells colonizing the brain do not differentiate as neurons or glial

cells but rather as microglial cells (Simard and Rivest, 2004;

Vitry et al., 2003). These microglial cells may facilitate the

catabolism of myelin debris but cannot substitute the oligoden-

drocytes that undergo apoptosis during GLD. Furthermore, NSPC

have demonstrated a promising regeneration potential in shiverer,

a model of dysmyelination due to the lack of the myelin basic

protein (Mitome et al., 2001; Yandava et al., 1999) and in

experimental allergic encephalomyelitis (EAE) (Pluchino et al.,

2003).

We cloned and inserted into a lentiviral vector murine GALC

cDNA and successfully transduced NSPC with the lentiviral

vector, obtaining a high expression of the GALC transgene. We

then injected GALC-NSPC into the striatum of twitcher obtaining

a significant prolongation of survival, particularly when bilateral

injections of the cells were performed. Moreover, engrafted NSPC

showed the capacity to migrate in vivo from the injection site to the

demyelinated CNS areas, where they exert their therapeutical

effect. In these areas, however, their survival was limited and did

not allow a prolonged recovery.

Materials and methods

Animals

Heterozygous twitcher mice (GALCtwi/+) were obtained from

Jackson Laboratories (Bar Harbor, ME). Genotyping was per-

formed by PCR, using genomic DNA obtained from clipped tail on

PND 4–5, as described (Sakai et al., 1996). All experiments were

performed under the Institutional Guidelines for Animal Care and

Use. Animals were briefly anesthetized on wet ice and injected

with NSPC into the forebrain on PND 10 using these stereotaxic

coordinates: 1.5 mm lateral and 0.2 mm posterior to bregma; depth

2 mm. Cells were suspended in 1 Al of PBS and injected using a

Hamilton syringe.

Neuropathological analysis

Animals were deeply anesthetized by an ip overdose of

tribromoethanol and transcardially perfused with 10 ml of 0.1%

heparin/PBS followed by 4% para-formaldehyde/PBS. The whole

brains were post-fixed in the same fixative o.n., cryoprotected in

30% sucrose in PBS, embedded in OCT and snap frozen in cold

isopentane in liquid nitrogen. Tissue samples were cut on a cryostat

(Leica, Nussloch, Germany) in two ways: 5 Am cryostat sections

were mounted on superfrost slides and 30 Am thick floating

sections were collected in wells and stored at �20-C in a

cryopreservative glycol-based buffer. Hematoxylin and eosin

(H&E) staining was used to access basic histopathological

changes. Special histochemical staining were used to detect

demyelinated areas (Kluver–Barrera staining), PAS positive

globoid cells (Periodic Acid Shift staining) and axon damage

(Bielchowsky staining). The presence of gliosis and of macro-

phages/microglia was assayed by immunohistochemistry using,

respectively, the rabbit monoclonal anti-GFAP antibody (DAKO,

Cytomation, Denmark, working dilution 1:500) and the biotiny-

lated Griffonia Simplicifolia lectin I (Sigma Aldrich, St. Louis,

USA, working dilution 1:50). Briefly, sections were washed in

PBS and 0.3% PBS-Triton X-100, pre-incubated in 6% goat serum

and 0.5% BSA in 0.1% PBS-T and incubated overnight at 4-C with

the primary antibody. Slides were then washed in PBS and PBS–

Tween-20, incubated 30 min at room temperature in biotin-labeled

secondary goat anti-mouse (1:200, Dako, Denmark) antibody and

the signal was detected by streptavidin-conjugated horseradish

peroxidase (1:300, Dako, Denmark) and diaminobenzidine. Nuclei

were counterstained with hematoxylin.

Lentiviral vector generation

293T cells were plated at 5 � 106 cells per 10-cm plate for 24

h in Iscove’s medium with 10% fetal bovine serum (FBS), 25 U/

ml penicillin, 25 U/ml streptomycin and 200 mM l-glutamine

and refed with 10 ml of fresh medium 2 h prior to transfection.

The appropriate plasmids (transfer: pRRLsin-PPT-hCMV-GALC/

GFPpre; envelope: VSV-G; packaging: pCMV delta R8.74) were

added to a final volume of 1 ml of 0.1� TE (1�: 10 mM Tris pH

8.0, 1 mM EDTA pH 8.0 diluted 1:10 with deionized H2O) and

2.5 M CaCl2 mixing carefully. HEPES-buffered saline 2� (281

mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4, pH 7.12) was

added while aerating the solution with a pipette. The mixture

was added dropwise to 293T cells and swirled gently. Medium

was replaced with fresh growth medium 16 h post-transfection

and after an additional 24 h was collected, filtered through a

0.22 um pore size filter and concentrated by ultracentrifugation

(50,000 � g).

An ELISA assay (HIV-1 p24 Core Profile ELISA, Perkin-

Elmer Life Science, Boston, MA) was used to determine the

amount of p24 (a surface antigen expressed on viral envelope) as

a measure of virus concentration. This test, however, does not

allow to distinguish empty capsid from complete viral particles.

The lentiviral titer was evaluated on HeLa cells plated at 5 � 104

cells per well on a 6-well plate 24 h prior to infection. HeLa cells

were infected with GFP lentiviral vector (two concentration,

corresponding to 1.5 and 15 ng of p24, respectively) in the

presence of Polybrene (4 Ag/ml) and refed with growth medium

24 h later. After 48 h, HeLa cells were evaluated for GFP

expression by FACSCalibur (Becton Dickinson, San Diego, CA,

USA).

Isolation of NSPC from mouse brain and lentiviral transduction

NSPC were derived from the brain of C57BL6J mice and

C57BL6J-EGFP mice on post-natal day 3 (PND 3) as previously

described (Benedetti et al., 2000) Briefly, the subventricular zone

(SVZ) was excised, triturated in wash medium containing EBSS,

EDTA and cysteine and centrifuged at low speed. The super-

natant was removed, and the pellet rewashed and digested with a

solution containing papain. After further centrifugation, the pellet

was resuspended in complete NS-A medium (EuroClone,

Wetherby, West York UK) and centrifuged at low speed. The

pellet was resuspended with the complete NS-A medium and

plated onto a 25 cm2 cell culture flask. After 4–5 passages,

NSPC were manually dissociated, plated at the density of 1 �106/flask 25 cm2 and infected with a lentiviral preparation

containing 50 ng of p24 in the presence of polybrene (4 Ag/ml

of NSA medium).

To assess the differentiation potential of NSPC, neurospheres

cultured for 7 days with EGF/bFGF/LIF or dissociated in the

presence of 3% FBS (differentiating conditions) were plated onto

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323316

poly-l-lysine coated chamber slides, fixed in 4% paraformalde-

hyde, washed with PBS, permeabilized in 0.2% NP-40 and blocked

with goat serum. Cells were incubated with primary antibodies

followed by FITC- or Cy3-conjugated secondary antibodies

(1:300). Slides were counterstained with a mounting medium

containing DAPI (Vector Laboratories) before examination by

fluorescence microscopy. The primary antibodies were: GFAP

(1:200; Dako, Denmark), beta-III tubulin (1:400, Covance,

Berkeley, CA) and nestin (1:50; Chemicon, Temecula, CA).

Analysis on dissociated neurospheres or differentiating NSPC

was performed using a FACSCalibur flow cytometer (Becton

Dickinson, San Diego, California, USA).

Enzyme assay in vitro and in vivo

To evaluate the efficiency of lentiviral vector transfection and

transgene expression, HeLa cells and NSPC were infected with

lentivirus-GALC, and GALC activity was assayed on cells

disrupted by sonication. To investigate GALC activity in vivo,

mice were perfused with saline solution to remove contaminating

blood before dissection of the brain. Small pieces of three brain

areas (anterior, middle, posterior) were disaggregated using the

Medimachine System (Becton Dickinson, San Diego, California,

USA). The cell suspension was recovered, and, after dilution

with water, homogenates were briefly sonicated and protein

content was determined using the MicroBCA reagents (PIERCE,

Rockford). The GALC assay was performed using [3H]galacto-

cerebroside (customer synthesis, Amersham, Wien, Austria)

mixed with non-hydroxy fatty acid galactocerebroside (Sigma

Chemical Co., St. Louis, MO; 796 ACi/mg, 1 mCi/ml). The

incubation was performed at 37-C for 2 h in a reaction mixture

containing Na-taurocholate, oleic acid and Na-acetate buffer

(pH 4.5).

In vivo distribution of the transfected NSPC

To look for GFP positive cells in the brain, the tissue sections

were mounted on a slide, nuclei were counterstained with a DAPI

containing mounting medium (Vector Laboratories) and examined

with a Zeiss Axioskop fluorescence microscope. The GFP

expression was also assayed by immunohistochemistry using a

rabbit polyclonal anti-GFP antibody (dilution 1:100, Santa Cruz

Biotechnology). Immunohistochemistry was performed with the

same procedure described above.

X-Gal Staining

A novel histochemical method to detect GALC was developed

modifying the radioactive enzyme assay described above using the

X-Gal substrate. To validate this method, HeLa cells untransduced

and transduced with lentivirus-GALC were plated onto chamber

slides, fixed with 4% paraformaldehyde for 15 min and incubated

3 h in humidity chamber at 37-C with an appropriate amount of

X-Gal staining solution, 20� KC [K3Fe(CN)6) + K4Fe(CN)6 �3H2O + PBS 1�], MgCl2 1 M, 40� X-Gal and 0.25 M sodium-

citrate buffer pH 4.5 with 0.7 mg/ml oleic acid (to avoid

background detection of lysosomal lactocerebroside and beta-

galactoside). GALC positive cells showed an intense blue-dotted

precipitate in their cytoplasm.

This method was also used to detect GALC activity in vivo.

Floating sections were collected in 24 well plates, rinsed 5 min in

PBS 1� (pH 4.5) at room temperature and stained with an

appropriate amount of 0.1% X-Gal solution. After incubation for 2

h in humidity chamber at 37-C, the floating sections were rinsed in

PBS 1� (pH 4.5) and counterstained with 0.1% neutral red for 10

min, at room temperature.

TNF-alpha assay

TNF-alpha release assay was performed by stimulating

CD11b+ macrophages (6 � 103) with NSPC (1.2 � 104). The

amount of TNF-alpha released into the culture supernatant was

determined by an ELISA assay kit (PeproTech EC Ltd., London,

UK).

CD11b+ cells were isolated from the brain of anesthetized

twitcher mice perfused with saline. The mechanical disaggrega-

tion of brain tissue was performed with a Medimachine using the

procedures suggested by the manufacturer (Becton Dickinson,

San Diego, CA, USA). The cell suspension was centrifuged for

10 min at 400 � g, the pellet was mixed with isotonic Percoll

in 50 ml centrifuge tubes and centrifuged for 10 min at 400 � g

(1 ml Percoll 100%, 2 ml Percoll 80%, 3 ml Percoll 40%, 2 ml

Percoll 30%).

The supernatant was discarded, while the layer between Percoll

80% and 40% was retained, resuspended, transferred to a conical

10 ml centrifuge tube and centrifuged 10 min at 2000 rpm.

Cells were magnetically labeled with CD11b MicroBeads

(Miltenyi Biotec, Bergisch Gladbach, Germany) and separated on

a column placed in the magnetic field of a MACS separator.

Magnetically labeled CD11b+ cells are retained in the column,

while the unlabeled cells run through. After removal of the column

from the magnetic field, magnetically retained CD11b+ cells are

eluted as the positively selected cell fraction.

To investigate in vitro the apoptosis in the presence of

TNF-alpha, NSPC were manually dissociated and plated at the

density of 2 � 105/well in 6-well culture plates in the

presence of different concentrations of murine recombinant

TNF-alpha (0.5, 1, 2, 4 and 6 ng/ml; Sigma Co., St. Louis,

MO). The apoptosis was evaluated by flow cytometry using

Annexin V-FITC (Bender MedSystems, Burlingame, CA):

apoptotic cells exhibit phosphatidylserine on the outside of the

plasma membrane. Changes in phosphatidylserine asymmetry

were analyzed by measuring Annexin V binding to the cell

membrane.

Results

NSPC transduced by lentivirus-GALC express high levels of GALC

in vitro

Our strategy for the treatment of twitcher has been based on

the implantation into the brain parenchyma of NSPC, engineered

to produce high levels of GALC. NSPC derived from the

periventricular region of C57BL6J syngeneic mice grow as

neurospheres in the presence of EGF and bFGF (Fig. 1A).

Under differentiating conditions (removal of EGF–bFGF and

addition of 3% rat serum), NSPC adhere to the plastic and

express glial or neuronal markers (Figs. 1B and C, respectively).

NSPC under differentiating conditions were characterized by flow

cytometry (Figs. 1D–G).The expression of nestin, a stem cell

marker, decreased, while that of neural markers increased.

Fig. 1. Characterization of mouse neurospheres. Neurospheres were obtained from neonatal brain as described under Materials and methods. Panel A shows

one neurosphere obtained from a GFP transgenic mouse, growing in EGF–bFGF and without serum. Panel B shows expression of an astrocytic (GFAP+,

green) and of an oligodendroglial (CNPase, red) marker after EGF–bFGF withdrawal and addition of 3% serum. Panel C shows expression of a neuronal

marker (beta-III tubulin, green) under the same culture conditions shown in panel B. Panels D–G show flow cytometry of neurospheres growing as

neurospheres (EGF–bFGF, no serum; shown in purple) or as adherent cells (no EGF–bFGF, 3% serum). Antibodies used are indicated in each panel.

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323 317

Overall, 76% of NSPC expressed neural markers (38% astrocytes,

27% neurons, 11% oligodendrocytes).

Two lentiviral vectors were prepared (see Materials and

methods). The concentration of p24 was 15 ng/ml for the lentiviral

vector expressing GFP and 24 ng/ml for the lentiviral vector

expressing GALC. Titer of lentivirus-GFP on HeLa cells was 2.3 �109 TU/ml, and the infectivity was 1.5 � 105 TU/ng p24. To

evaluate the efficiency of the lentivirus-GALC, NSPC and HeLa

cells were assayed for GALC activity after infection.

The assay was performed using [3H]galactocerebroside as

substrate, as reported previously (Chen and Wenger, 1993). In

HeLa cells, the activity increased about 57 folds as compared to

untransduced cells (237 nmol/h/mg protein vs. 4 nmol/h/mg

protein, respectively). In infected NSPC, GALC activity on

passage 9 increased about 4-fold as compared to wild-type cells

(417 nmol/h/mg protein vs. 108 nmol/h/mg protein, respectively).

GALC activity in infected NSPC remained high after many

passages in culture. On passage 32 (about 11 months in culture),

the activity decreased about two-fold (from 417 nmol/h/mg protein

to 208 nmol/h/mg protein).

Effects of brain injection of NSPC on twitcher

Injection of NSPC-GALC modified the clinical evolution of the

twitcher phenotype. Comparison of treated and untreated twitcher

from the same progeny showed that the treatment was delaying the

disease onset of about 2 weeks. In agreement with this, the

pathological analysis performed on PND 17 in treated mice

showed very few macrophages and absence of reactive gliosis

when compared to brains of untreated mice (Figs. 2A–D). These

differences were progressively decreasing at further time points

(data not shown). Clinically, the delayed onset of the main

symptoms (twitching followed by increasing weakness of posterior

limbs) was followed by a rather rapid progression to death: in

Fig. 2. Effects of brain injection of NSPC-GALC in twitcher. (A–D) Neuropathological analysis of untreated vs. treated mice sacrificed on post-natal day 17

(PND 17). On PND 17, control mice showed abundant macrophage infiltration in the brain, particularly in posterior brain regions (A, cerebellum: lectin

macrophage staining, 40� original magnification) compared to the treated mice, where a very low number of macrophages could be detected (B, cerebellum,

lectin macrophage staining, 40� original magnification: the arrow shows one macrophage). In the control mice, reactive gliosis was frequently observed (C,

cerebellum, GFAP staining, 40� original magnification) compared to treated mice where GFAP immunostaining only showed normal GFAP reactivity (D,

cerebellum, GFAP staining, 40� original magnification). Scale bar: 50 Am in all the panels. (E) Kaplan–Meier survival analysis of twitcher mice treated by

NSPC injections. One or two injections of NSPC-GALC or two injections of untransduced NSPC were performed on PND 8, as described under Materials and

methods. Log rank test: NSPC-GALC 2 � 105 P = 0.008, NSPC-GALC 4 � 105 P < 0.0001, NSPC untransduced 4 � 105 cells P < 0.005, vs. no injection.

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323318

untreated animals, the twitching started around PND 21 and led to

death in about 2 weeks while in treated mice started after day 40

and led to death in about 1 week.

To evaluate the effects on survival of the intracerebral (IC)

injection of NSPC, three experimental conditions were tested. One

group of mice received IC injection of 2 � 105 NSPC-GALC into

the left hemisphere on PND 10 (n = 8). Another group of mice

received intracerebral injection of 2 � 105 NSPC-GALC into each

hemisphere (n = 7). A third group was injected IC with 2 � 105 of

wild-type untransduced NSPC into each hemisphere (n = 4).

Uninjected twitcher (n = 29) were used as controls and survived

36.1 T 4.1 days. The Kaplan–Meier analysis of survival is shown

in Fig. 2E.

Unilateral injection of NSPC-GALC led to a 17.5% increase in

survival (42.4 T 5.0 days, P = 0.008 vs. controls). Bilateral

injection of NSPC-GALC increased survival of 44.6% (52.2 T 5.6

days, P < 0.0001 vs. controls), while bilateral injection of

untransduced NSPC increased survival of 22.4% (46.5 T 2.5 days;

P < 0.005 vs. controls). We also evaluated the contribution to

survival of intraperitoneal injection (ip) of NSPC-GALC. Two

mice were injected ip with 1 � 107 NSPC-GALC and survived

41.5 T 0.7 days; (P = 0.1 vs. controls). To evaluate the fate of

NSPC after systemic injection, we injected twitcher mice (n = 3) ip

with 1 � 107 NSPC-GFP. The fraction of NSPC cells evaluated by

flow cytometry in the brain was rather low (3%; see Supplementary

Table 2).

Transduced NSPC injected intracerebrally increase GALC activity

of twitcher mice

To investigate the functional contribution of NSPC-GALC

injection, GALC activity was tested in GALCtwi/twi mice after

injection on PND 10 of 2 � 105 NSPC-GALC into each

hemisphere (n = 3). Uninjected GALCtwi/twi and GALCtwi/+ mice

Table 1

GALC activity was expressed as nmol/h/mg protein (mean T SD)

GALC assay in vivo

Brain area GALC+/+ mice GALCtwi/+ mice GALCtwi/twi mice GALCtwi/twi mice treated

PND 17 Anterior 4.13 T 0.10 1.25 T 0.06 0 0.88 T 0.02

Middle 3.16 T 0.13 1.24 T 0.04 0 1.48 T 0.08

Posterior 3.42 T 0.22 1.51 T 0.11 0.11 T 0.19 1.83 T 0.06

PND 24 Anterior 4.05 T 0.07 1.17 T 0.04 0 0.85 T 0.06

Middle 3.63 T 0.07 1.74 T 0.54 0 1.30 T 0.05

Posterior 3.07 T 0.09 1.09 T 0.03 0 0.27 T 0.09

PND 31 Anterior 3.52 T 1.02 1.02 T 0.23 0 0.60 T 0.04

Middle 3.38 T 0.32 1.15 T 0.09 0 1.06 T 0.07

Posterior 3.97 T 0.92 1.43 T 0.08 0 0.01 T 0.01

The three brain areas are divided and shown in Fig. 4.

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323 319

(n = 3 each) and normal controls GALC+/+ mice (n = 3) were also

tested. Enzyme assays were performed at three time points (PND

17, 24, 31) and in three brain areas: anterior, middle (where cells

had been injected) and posterior (see Fig. 4). GALC activity was

almost undetectable in GALCtwi/twi as compared with GALCtwi/+

and GALC+/+ mice (see Table 1). GALC activity in treated

GALCtwi/twi increased to levels similar to those of GALCtwi/+ mice

on PND 17. On PND 24, GALC activity decreased drastically in

the posterior area of the brain and became negligible on PND 31.

Activity in the middle and the anterior regions also decreased on

PND 24 and 31, but less significantly.

Fig. 3. Migration and differentiation of NSPC in twitcher brain. Panel A shows mig

shows injected NSPC away from the injection site and ramified glial morphology. P

group of NSPC expressing GFP. Panel E shows the merging of the two figures wi

marker GFAP. Panel F shows the histochemical staining with LacZ of a group of

for LacZ and immunohistochemistry for GFAP that overlap in one cell indicated b

panel G.

Investigations on the fate of NSPC after brain injection into

GALCtwi/twi mice

To investigate the fate and the migrating capacity of NSPC after

brain injection in twitcher, we looked for the presence of NSPC-

GFP by histology and flow cytometry at different time points

(PND 17, 24, 31). GALCtwi/twi (n = 20) and GALC+/+ (n = 20)

mice were injected with 4 � 105 NSPC-GFP.

NSPC-GFP migrated to different areas, also distant from the

injection site, acquiring a differentiated phenotype. Histological

analysis revealed the presence of cells not only in the site of

ration of NSPC along the needle track and surrounding one vessel. Panel B

anel C shows an area of active gliosis (red staining for GFAP) and panel D a

th few yellow cells, suggesting that part of the NSPC express the astrocytic

NSPC-GALC along the needle track. Panel G shows histochemical staining

y the asterisk. Panel H shows a magnified picture of the same cell shown in

Fig. 4. Flow cytometry of GFP-NSPC after injection into twitcher brain. The figure shows the amount of GFP in three brain areas (shown in the cartoon) and at

the three time points indicated. A much larger fraction of NSPC are present in the posterior area of the brain in twitcher mice than in controls on PND 17 ( P <

0.00001). In anterior and middle area on PND 17, the amount of GFP is significantly larger in normal that in twitcher mice ( P < 0.0001 and P = 0.001,

respectively).

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323320

injection, but also in the subventricular zone and the corpus

callosum (Figs. 3A and B).

We investigated further the fate of transplanted cells by

immunohistochemistry with antibodies for nestin and for glial,

microglial and neuronal markers. The results provided evidence for

GFAP expression while staining for other markers was negligible

(Figs. 3C–E and data not shown).

Three brain areas were considered for flow cytometry of GFP-

positive NSPC: anterior, middle (where cells had been injected)

and posterior, including the brainstem (Fig. 4). GALCtwi/twi (n =

20) and GALC+/+ (n = 20) mice were injected with 4 � 105 NSPC-

GFP. Interestingly, the amount of GFP-positive cells in the

posterior area was much higher in twitcher (n = 4) than in normal

mice (n = 4) on PND 17 (P < 0.00001). In twitcher and normal

mice on PND 24 (n = 8) and 31 (n = 4), the amount of GFP-

positive cell decreased in all areas.

Microglia/macrophages from twitcher brain release TNF-alpha

when matched with NSPC expressing GALC

Pro-inflammatory cytokines play a pathogenic role in twitcher:

in particular, TNF-alpha promotes the apoptotic death of oligoden-

drocytes (D’Souza et al., 1995). To test if TNF-alpha could also

play a role in the death of NSPC injected into twitcher brain, we

Table 2

Release of TNF-alpha by CD11b+ cells purified from Twitcher

1^ assay 2^ assay

TNF-alpha ng/ml TNF-alpha ng/ml

CD11b+ alone 0.56 T 0.02 0.44 T 0.01

CD11b+/GL261 0.64 T 0.01* 0.87 T 0.04

CD11b+/NSPC-GFP 1.41 T 0.04*** 0.91 T 0.02**

CD11b+/NSPC-GALC 2.15 T 0.04**** 0.95 T 0.02**

CD11b+/NSPC-GALCtwi/twi 0.47 T 0.09 0.54 T 0.02

CD11b+/NSPC-GALC+/+ 1.12 T 0.03* 0.65 T 0.00*

* P = 0.01.

** P = 0.001.

*** P < 0.0001.

**** P < 0.0001 vs. CD11b+/NSPC–GALCtwi/twi.

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323 321

first purified macrophages/microglia from twitcher brain on PND

29 using immunopurification with CD11b antibodies bound to

microbeads. Starting from 0.7 to 1 � 105 cells/twitcher, we

obtained 2–3 � 103 cells that, according to flow cytometry

analysis with anti-CD11b antibodies, contained 88% of positive

cells (not shown). CD11b+ cells, however, could not be obtained

from normal brains using the same procedure. Twitcher CD11b+

cells released significantly more TNF-alpha when matched

with NSPC-GALC or NSPC-GFP cells than with twitcher NSPC

(Table 2).

To evaluate the potential effect of TNF-alpha on injected

NSPC, we investigated in vitro the apoptosis of NSPC in the

presence of TNF-alpha using FITC-labeled Annexin V and flow

cytometry (see Materials and methods). The results indicate that

TNF-alpha may cause the apoptosis of NSPC (see Supplementary

Table 1), suggesting a direct role of this cytokine in limiting the

survival of NSPC transplanted into twitcher brain.

Discussion

Our experiments have demonstrated that the brain injection of

neurospheres, a mixture of cells containing, in addition to neural

stem cells, neuronal and glial progenitors in different states of

differentiation (Bez et al., 2003; Suslov et al., 2002), may

ameliorate the clinical course of twitcher, the murine model of

infantile globoid cell leukodystrophy (GLD). In particular,

injection of GALC-NSPC increased twitcher survival to 52 days.

This gain was lower than that obtained by bone marrow

transplantation (74 days; Yagi et al., 2004 and our unpublished

observations), similar to that obtained by substrate restriction

strategies (56.7 days survival with l-cycloserine; LeVine et al.,

2000) or transfer to a different background (52 days; Biswas et al.,

2002), and higher than adenovirus-mediated gene transfer of the

GALC gene (42 days; Shen et al., 2001). After injection, the cells

are attracted by regions of active demyelination that in twitcher

begins posteriorly in the CNS and spreads subsequently to the

anterior part (Taniike and Suzuki, 1994). In that environment,

NSPC do not survive long, and our data support the idea that

cytokines released by microglial cells, like TNF-alpha, are partially

responsible for such fate. This, overall, appears to limit the efficacy

of this therapeutic approach to the treatment of GLD.

What are the reasons underlying the therapeutic efficacy of

NSPC transplantation and those explaining its limited success?

Three issues can be considered: the enzymatic activity of GALC,

the differentiation of NSPC and their survival after transplantation.

The GALC enzyme is partially secreted, and its re-uptake can

complement GALC deficiency also in surrounding cells, as

indicated by experiments performed on cells overexpressing GALC

and cell lines from GLD patients (Luddi et al., 2001; Rafi et al.,

1996). Specifically, we could show that NSPC overexpressing

GALC were complementing effectively the GALC deficiency of

cells from twitcher or from GLD patients (Torchiana et al., 1998).

These experiments provided a rationale for our therapeutic

approach to twitcher, relying on brain injection of NSPC obtained

from neonate mice. The injection into each cerebral hemisphere of

NSPC obtained from normal mice and showing robust levels of

GALC activity was able to prolong significantly the survival of

twitcher (Fig. 2). NSPC transduced by GALC lentivirus (NSPC-

GALC) showed a four-fold increase of GALC activity when

compared to untransduced NSPC. When GALC-NSPC were

injected, a more prolonged survival was obtained, but the difference

with untransduced cells did not reach the significance (Fig. 2),

suggesting that GALC activity of injected cells is not a limiting

factor avoiding a more prolonged survival of twitcher.

GALC activity also appears to be maintained in vivo for more

than 3 weeks, as suggested by histochemical evaluation with the X-

Gal substrate (Fig. 3). Furthermore, in vitro assay of GALC

activity shows that the CMV-driven expression of the GALC

transgene, provided by the lentivirus vector, remains high after

many weeks and passages in culture (see Results) at difference

with LTR-driven GALC expression after retroviral transduction

(Torchiana et al., 1998). Thus, NSPC transduced by lentiviral

vectors expressed high levels of metabolically active GALC, and

this activity remained at high levels for many weeks.

Limitations to address the second issue, the differentiation of

NSPC after brain injection in twitcher, were caused by the lack of

specific and sensitive antibodies for GALC detection at immuno-

histochemistry. Four different peptides were used for rabbit

immunization, but the signal obtained by the respective antibodies

was weak on immunoblotting and immunocytochemistry and even

weaker on immunohistochemistry. To gain hints on NSPC

behavior after transplantation into the brain of twitcher, we used

X-Gal staining to detect GALC activity and immunohistochemistry

with GFP antibodies identifying differentiated NSPC expressing

GFP. These experiments showed that NSPC can migrate long

distance from the injection site, traveling from the striatum through

the corpus callosum (see Fig. 3B) and that after transplantation

they preferentially express astrocytic markers, like GFAP (see Figs.

3C and E). The pro-inflammatory cytokine interleukin-6 (IL-6) that

is significantly expressed in twitcher brain (LeVine and Brown,

1997) may induce NSPC to undergo astrocytic differentiation

(Figs. 3G and H) selectively through the JAK/STAT pathway in

vitro (Bonni et al., 1997). These findings are in agreement with our

results and suggest that NSPC injected into twitcher brain may

undergo an IL-6-driven astrocytic differentiation. Although not

directly proven by our experiments, it is likely that such inhibition

of oligodendroglial differentiation is a limiting factor of the

therapeutic potential of injected NSPC, making impossible the

substitution of oligodendrocytes undergoing apoptosis in murine

and human GLD (Nagara et al., 1986; Taniike et al., 1999).

Together with IL-6, TNF-alpha is the other important pro-

inflammatory cytokine that is expressed in the brain of twitcher and

particularly in the pons and in the medulla (LeVine and Brown,

1997). Globoid cell positivity for TNF-alpha was also found at

autopsy in the brain of three patients with GLD (Itoh et al., 2002).

TNF-alpha is one key player in the pathogenesis of inflammatory

S. Pellegatta et al. / Neurobiology of Disease 21 (2006) 314–323322

brain diseases. Transgenic mice overexpressing TNF-alpha

develop spontaneous inflammatory disease (Probert et al., 1995),

and TNF-alpha may directly induce apoptosis in oligodendrocytes

(Akassoglou et al., 1998; D’Souza et al., 1995). More recently, a

relevant action of this cytokine has been demonstrated on NSPC:

TNF-alpha inhibits NSPC proliferation and induces their migration

(Ben-Hur et al., 2003). Our results are in agreement with these

observations, showing that NSPC after brain injection migrate

preferentially to posterior regions of the CNS, where demyelination

is more active and TNF-alpha expression is higher. The stimulation

of NO release caused by psychosine, the toxic metabolite that is

abundant in GLD (Giri et al., 2002), could boot further TNF-alpha

cytotoxicity since NO co-operates with TNF-alpha to cause

neuronal death and demyelination (Blais and Rivest, 2004).

We also found that CD11b+ microglia/macrophages obtained

from twitcher brain release significantly more TNF-alpha when

matched with NSPC overexpressing GALC (Table 2). TNF-alpha

stimulates NSPC expression of the co-stimulatory molecule CD80

(Imitola et al., 2004). CD80 has a critical role in T cell activation

also in the brain, as shown by EAE models (Chang et al., 1999).

T cells, however, are not significantly increased in the brain of

twitcher (this, together with the absence of major alterations of

the blood–brain barrier (Kondo et al., 1987) appears as a major

difference with the pathogenesis of demyelination of EAE), and

CD8+ cells found in the brain of twitcher have a CD3�/Mac1+

phenotype, suggesting that they are part of the monocyte/

macrophage lineage (Itoh et al., 2002). This mechanism therefore

does not appear involved in the progressive disappearance of

NSPC cells that we have found after brain injection in twitcher.

Recent work, however, indicates that CD80 cross-linking

enhances apoptosis of NSPC through a caspase-3-mediated

process (Imitola et al., 2004). It appears plausible that CD80

cross-linking of NSPC and microglia, which also express CD80

during activation (De Simone et al., 1995; Satoh et al., 1995;

Stein et al., 2004), is one mechanism causing apoptosis of NSPC

grafted into twitcher brain.

Overall, the data indicate that the inflammatory environment

associated to neurodegeneration can hamper significantly the

survival of transplanted NSPC in twitcher. Microglia activation

and TNF-alpha seem to play a central role in this process. These

observations suggest that to be of therapeutic relevance the use of

NSPC in GLD should be considered in the context of immune

suppressive treatments able to prolong the survival of transplanted

cells.

Acknowledgments

We thank Luigi Naldini and Antonia Follenzi for sharing

plasmids and protocols for lentiviral preparation, Ilaria Visigalli for

help with in vivo experiments, Carolina Frassoni for help with

confocal analysis and Elena Torchiana for help and suggestions on

the isolation of CD11b+ cells. This work was partially supported

by grants to GF from TeleThon and from the Istituto Superiore di

Sanita’.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version. at doi:10.1016/j.nbd.2005.07.016.

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