Occurrence of an anomalous endocytic compartmentin fibroblasts from Sandhoff disease patients

Brunella Tancini • Alessandro Magini •

Loredana Latterini • Lorena Urbanelli •

Virginia Ciccarone • Fausto Elisei • Carla Emiliani

Received: 12 June 2009 / Accepted: 16 September 2009 / Published online: 2 October 2009

� Springer Science+Business Media, LLC. 2009

Abstract Sandhoff disease (SD) is a lysosomal storage

disorder due to mutations in the gene encoding for the

b-subunit of b-hexosaminidase, that result in b-hexosa-

minidase A (ab) and b-hexosaminidase B (bb) deficiency.

This leads to the storage of GM2 ganglioside in endosomes

and lysosomes, which ends in a progressive neurodegen-

eration. Currently, very little is known about the bio-

chemical pathways leading from GM2 ganglioside

accumulation to pathogenesis. Defects in transport and

sorting by the endosomal–lysosomal system have been

described for several lysosomal storage disorders. Here, we

have investigated the endosomal–lysosomal compartment

in fibroblasts from SD patients and observed that both late

endosomes and lysosomes, but not early endosomes, have a

higher density in comparison with normal fibroblasts.

Moreover, Sandhoff fibroblasts have an intracellular dis-

tribution of terminal endocytic organelles that differs from

the characteristic perinuclear punctate pattern observed in

normal fibroblasts and endocytic vesicles also appear lar-

ger. These findings reveal the occurrence of an alteration in

the terminal endocytic organelles of Sandhoff fibroblasts,

suggesting an involvement of this compartment in the

disruption of cell metabolic and signalling pathways and in

the onset of the pathological state.

Keywords Lysosomal storage disease � Sandhoff

disease � Endocytic compartment � GM2 storage

Introduction

The endosomal–lysosomal system plays important roles in

cellular physiology. Beyond the well known function as

terminal degradative compartment necessary to maintain

the health of the cell, lysosomes are critical for many other

cellular processes, such as termination of signaling medi-

ated by cell-surface receptors [1] and degradation of

internalized peptides in antigen presenting cells [2].

Moreover, the intracellular membrane trafficking related to

the endosomal–lysosomal system plays pivotal roles in

diverse physiological and pathological processes, such as

secretion, plasma membrane repair, and endocytosis [3–5].

Mutations in the genes encoding for lysosomal hydro-

lases cause a group of severe diseases named lysosomal

storage disorders (LSD). These diseases are characterized

by accumulation within the lysosomes of partially unde-

graded catabolic products [6]. Currently, very little is

known about biochemical mechanisms leading from the

accumulation of these substrates to the disruption of cell

metabolic and signaling pathways, ending with the onset of

the pathology. Many recent studies have shown that several

LSD share pathological features with age-related neuro-

degenerative diseases such as Alzheimer’s and Parkinson

diseases, namely atypical central nervous system inflam-

mation [7], altered calcium homeostasis [8], and impaired

endosomal–lysosomal degradative capacity [9]. In addi-

tion, there is evidence that lysosomes in patients affected

by some LSD are characterized by remarkable changes in

terms of their chemical–physical and physiological prop-

erties [10–13].

B. Tancini � A. Magini � L. Urbanelli � V. Ciccarone �C. Emiliani (&)

Department of Experimental Medicine and Biochemical

Sciences, University of Perugia, via del Giochetto,

06126 Perugia, Italy

e-mail: emiliani@unipg.it

L. Latterini � F. Elisei

Department of Chemistry, University of Perugia, via Elce di

Sotto, 06125 Perugia, Italy

123

Mol Cell Biochem (2010) 335:273–282

DOI 10.1007/s11010-009-0277-0

Glycosphingolipid (GSL) storage disorders are a sub-

group of LSD caused by mutations in either hydrolase or

activator protein genes involved in catabolism of GSL. The

defective hydrolysis of these compounds leads to their

accumulation, mainly in neuronal endosomes and lyso-

somes. Fibroblasts from patients affected by a broad group

of GSL storage disorders have been reported to show

defects in lipid sorting and transport along the endocytic

pathway [14, 15]. GSL storage has been suggested to alter

membrane microdomain sphingolipid composition, thus

provoking the mistargeting and disrupted function of

membrane microdomain associated proteins that are

involved in signaling and transport processes [16]. The

alteration of the endocytic pathway has also implications

for therapeutic approaches toward GSL storage disorders.

In fact, enzyme replacement therapy and gene therapy

might fail because of abnormalities in enzyme targeting

within the cell, due to an impairment of the receptor-

mediated endocytosis pathway [17].

Sandhoff disease (SD) is a GM2 gangliosidosis due to

mutations in the gene encoding for the b-subunit of the acidic

glycohydrolase b-hexosaminidase (Hex, EC 3.2.1.52)

[18–20]. This results in the deficit of both b-hexosaminidase

A (ab, Hex A) and b-hexosaminidase B (bb, Hex B) enzy-

matic activities. Lack of Hex A, that is the only isoform that

hydrolyzes GM2 ganglioside, elicits the intralysosomal

accumulation of this substrate mainly in the central nervous

system, leading to neurodegeneration and eventually to

death [18]. In order to gain insight into endosomal–lyso-

somal compartment features in SD we have characterized

some chemical–physical and functional aspects of the

endocytic compartment in a peripheral cell model of the

disease, i.e., fibroblasts from SD patients. Fibroblasts are a

consolidated and easily accessible source to study the path-

ogenic mechanisms that are responsible for neurological

disorders and have revealed a wealth of information on how

such disorders occur [21, 22]. In addition, fibroblasts are

usually used to set up gene transfer strategies and enzyme

replacement protocols for therapy. Here, we report that

Sandhoff fibroblasts show alterations of the endocytic

compartment. These abnormalities have been observed in

late endosomes and lysosomes, but not in early endosomes,

and may be involved in the pathological cascade of events

leading to the onset of the disease.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine

Serum, Trypsin, and Penicillin/Streptomycin were from

Seromed, Biospa. 2,20-Azino-bis(3-ethylbenzothiazoline)-6-

sulfonic acid (ABTS), acridine orange, bovine serum albumin,

fluorescein isothiocyanate (FITC)-dextran (PM 10000),

Imidazole, 4-methylumbelliferyl-b-galactoside (MUGal),

4-methylumbelliferone, monoclonal horseradish peroxidase

(HRP)-conjugated anti-rabbit and HRP-conjugated goat anti-

mouse secondary antibodies, HRP Type II, protease inhibitor

cocktail for mammalian cell extracts and sucrose were from

Sigma-Aldrich Fine Chemicals Co. Mouse monoclonal anti-

lysosomal associated membrane protein-2 (LAMP-2)(H4B4),

rabbit polyclonal anti-Rab7 (H-50), goat polyclonal anti-Rab7

(C-19), and rabbit polyclonal anti-Rab5A (S-19) antibodies

were from Santa Cruz Biotechnology, Inc. Rabbit polyclonal

antibody to anti-early endosome antigen 1 (EEA1) was from

Abcam. Alexa Fluor 488-conjugated donkey anti-rabbit,

Alexa Fluor 546-conjugated goat anti-mouse, and Alexa Fluor

546-conjugated donkey anti-goat secondary antibodies were

from Molecular Probes. Hybond-C Extra nitrocellulose and

Enhanced Chemilumiscence (ECL) Western Blotting detec-

tion reagents were from Amersham Biosciences. Protein assay

reagent and chemicals for SDS-PAGE were from Bio-Rad

Laboratories. All other reagents were of analytical grade.

Cell cultures

Fibroblasts from SD patients and healthy subjects matched

for sex and age were kindly provided by Dr. Mirella

Filocamo (Laboratorio di Diagnosi Pre/Post-natale Malattie

Metaboliche, Genova, Italy). All experiments were per-

formed using three different fibroblast samples from both

SD patients and healthy controls. All SD patients were

affected by the early onset form of the disease, character-

ized by neurological and mental deterioration with

regression of psychic and motor functions. The mutations

carried by the three genotypes, [c.299G [ T (r = 0)] ?

[c.512-1G[T (p.G171-L172del)], [c.300-2A[G (p.R101_

S148delfsX12)] ? [c.300-2A[G (p.R101_S148delfsX12)],

[c.850C [ T(p.R284X)] ? [c.1613 ? 2T [ G (p.E538ins

4X5)], have been previously characterized [23]. The activ-

ity of Hex A and Hex B isoenzymes in each cell sample

was measured after their separation by ion-exchange

chromatography and in SD fibroblasts it was less than 0.3%

as compared to control fibroblasts. Cells were cultured in

DMEM supplemented with 10% (v/v) heat-inactivated

fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin,

100 lg/ml streptomycin in a 5% CO2 incubator at 37�C.

Cell viability was determined by the Trypan blue method.

Continuous sucrose gradient

Microsome preparation and subcellular fractionation on

continuous sucrose gradient were performed as reported in

Yeyeodu et al. [24] with minor modifications. Briefly,

274 Mol Cell Biochem (2010) 335:273–282

123

Sandhoff and normal fibroblasts were scraped and resus-

pended on ice-cold 3 mM imidazole buffer, pH 7.4, con-

taining 0.25 M sucrose, 0.5 mM PMSF and 1 mM EDTA.

After an incubation on ice for 15 min, cells were homog-

enized with 25 strokes of a Dounce homogenizer (pestle A)

and centrifuged at 3,000g for 15 min. The supernatant was

recovered and centrifuged again on a Beckman Optima

Max Ultracentrifuge at 100,000g (TLA 100.3 rotor) for 1 h

at 4�C. Finally, the microsome pellet was resuspended,

layered on the top of a continuous sucrose gradient pre-

pared using 0.5 and 1.6 M sucrose (2.4 ml ? 2.4 ml) in

20 mM imidazole buffer, pH 7.4, and centrifuged at

170,000g for 70 min at 4�C on a Beckman MLS-50 rotor.

Fractions (0.260 ml) were collected from the top of the

gradient and aliquots used for Western blots and enzyme

assays.

Western blotting analysis

Samples were subjected to 12% SDS-PAGE under reduc-

ing conditions according to Laemmli [25]. Proteins were

transferred to nitrocellulose and the membrane probed with

primary antibody at the appropriate concentration and then

incubated with HRP-conjugated secondary antibody. Blots

were finally revealed using the ECL detection system.

Densitometric analysis was performed using the Image-

Master2D platinum software (Amersham Bioscience).

Enzyme assay

Triton X-100 (0.1%) was added to an aliquot of each frac-

tion gradient to solubilize cellular membranes. The activity

of the lysosomal enzyme b-galactosidase was determined

using the fluorogenic substrate MUGal 1.5 mM in 0.1 M

citric acid/0.2 M disodium phosphate buffer, pH 4.5 [26].

Fluorescence of the 4-methylumbelliferone product was

measured on a Perkin-Elmer LS B50 fluorimeter (excitation,

360 nm; emission, 446 nm). One enzymatic unit (U) is the

amount of enzyme that hydrolyzes 1 lmol of substrate/min

at 37�C.

Protein concentration was determined by the method of

Bradford [27] using bovine serum albumin as standard.

Fluorescence and confocal microscopy

Cells grown on glass coverslips were washed with phosphate-

buffered saline (PBS), fixed with 3.7% paraformaldehyde in

PBS for 10 min at room temperature, washed again with PBS

and then permeabilized and blocked in 0.2% Triton X-100/

PBS containing 3% bovine serum albumin (blocking solution)

for 1 h. Primary antibodies were diluted at the appropriate

concentration in blocking solution, added to cells and incu-

bated for 1 h. After washings, cells were incubated for 1 h

with the appropriate Alexa Fluor-conjugated secondary anti-

bodies diluted 1:2,000 in blocking solution. All incubation

steps were carried out at room temperature. Coverslips were

then washed with PBS containing 0.2% Triton X-100, rinsed

with water and mounted with Vectashield. For dextran loading

experiments, cells grown on glass coverslips were incubated

for 18 h in culture medium containing 2 mg/ml FITC-dex-

tran, rinsed with PBS, chased for 2 or 4 h in medium devoid of

the fluid-phase marker and then fixed and mounted as above.

For acridine orange staining, cells were grown on glass cov-

erslips and incubated for 10 min at 37�C in the presence of

2.5 lg/ml of acridine orange in the culture medium. After

washing with fresh DMEM, living cells were immediately

viewed by fluorescence microscopy. Cells were visualized

using a fluorescence light microscope (Nikon) through a 609

oil immersion objective and images were acquired with a

Sony camera. Confocal microscopy analysis of the samples

was carried out through a laser scanning confocal microscope

(Nikon, PCM2000). In this case, fluorescence images were

recorded using Ar- or He–Ne-lasers (kexc = 488 and 543 nm,

respectively) as light source and two acquisition channels

which differ for the spectral region selected by band-pass

filters, then visualized in different colors (false colors). Images

were obtained with a 609, 1.4 N.A. oil immersion objective

(512 9 512 pixels). Image processing was performed using

Adobe Photoshop software. For the quantification of the

colocalization all red- and green-colored pixels that were

found at identical positions in a confocal section were com-

pared to those red- and green-colored pixels that could not be

located to the same position. This analysis was performed for

at least 15 cells for each cell sample (45 cells for each

genotype).

Measurement of lysosomal pH

Sandhoff and normal fibroblasts were incubated for 18 h at

37�C in culture medium containing 2 mg/ml FITC-dextran

(mol wt 10 kDa), rinsed with PBS and then chased for

90 min in fresh medium devoid of the fluid-phase marker.

Cells were scraped, resuspended in PBS and analyzed by

spectrofluorimeter to determine the lysosomal pH according

to Ohkuma et al. [28] with minor modifications. Briefly, cell

samples were sequentially excited at 457 and 488 nm and

the emitted fluorescence was measured in each case at

515 nm. The pH of the terminal endocytic organelles was

estimated comparing the ratio of the two fluorescence

intensities with a standard curve generated with FITC-

dextran-loaded microsomes at defined pH. Labeled

microsomes were prepared by loading of fibroblasts with

FITC-dextran as described above and subsequent centrifu-

gation of the post-nuclear supernatant at 100,000g for 1 h.

Aliquots of pelleted microsomes were resuspended in

Mol Cell Biochem (2010) 335:273–282 275

123

buffers of defined pH in the presence of 1 lM nigericin to

dissipate a potential pH gradient.

Fluid-phase endocytosis

Sandhoff and normal fibroblasts were incubated with pre-

warmed 3 mg/ml horseradish peroxidase (HRP) in DMEM

for 2 h at 37�C followed by extensive washing with PBS.

Cells were then trypsinized, pelleted and resuspended in

40 mM potassium phosphate buffer containing 0.5% (v/v)

Triton X-100, pH 6.8. After 30 min of incubation on ice,

cell lysates were centrifuged at 16,000g for 10 min and the

supernatants were used for HRP activity determination

using 2,20-Azino-bis(3-ethylbenzothiazoline)-6-sulfonic

acid (ABTS) as substrate [29, 30]. Briefly, 2–4 ll of

diluted extract were added to 1 ml of 0.7 mM ABTS in

100 mM potassium phosphate buffer, pH 5.0 at 25�C; then

30 ll of fresh prepared 0.3% H2O2 were added, samples

were mixed by inversion and the increase in absorbance at

405 nm was immediately recorded for 4 min. DA405 nm/min

was calculated using the maximum linear rate. The same

procedure was performed with untreated cells as negative

control. One unit (U) is the amount of enzyme that oxidizes

1 lmol of substrate/min at pH 5.0 at 25�C.

Results

Subcellular fractionation on continuous sucrose

gradient of Sandhoff fibroblast endocytic organelles

Continuous density gradients in the range of 0.3–1.6 M

sucrose were optimized to determine the buoyant density of

fibroblast endocytic organelles. Microsomal fractions from

both Sandhoff and normal fibroblasts were layered on the

top of the gradient and subjected to ultracentrifugation, as

described in the ‘‘Materials and methods’’ section. The

endocytic organelle buoyant density of Sandhoff and nor-

mal fibroblasts was compared by Western blotting analysis

of gradient fractions with organelle specific markers. Pro-

teins detected were Rab5 as early endosomal marker [31],

Rab7 as late endosomal marker [32], and LAMP-2 as

lysosomal marker [33]. Among density gradient systems

tested, the best separation profile was achieved using a

0.5–1.6 M sucrose gradient, although a very similar dis-

tribution pattern was also observed with a 0.7–1.5 M

sucrose gradient (data not shown). Results are reported in

Fig. 1. The comparison of normal and pathological orga-

nelle distribution profiles reveals differences between

Sandhoff and normal fibroblasts related to terminal

Fig. 1 Distribution of endocytic organelles in continuous sucrose

gradient. Normal (NF) and Sandhoff (SF) fibroblast microsomes were

resolved on a 0.5–1.6 M sucrose density gradient, and 0.260 ml

fractions were collected from the top. Proteins were separated by

SDS–PAGE and visualized by immunoblotting. Representative

Western blots (a) and densitometric analysis (b) showing the

distribution of LAMP-2, Rab7, and Rab5 proteins in the gradient

fractions are reported. The results are expressed as percentages of the

respective band volume in each fraction to the sum of all fractions.

Three different fibroblast samples from both SD patients and healthy

controls were used and three independent experiments for each cell

sample were performed. Very similar results were obtained for each

genotype. Here, representative results of one experiment with SD and

normal fibroblasts are reported

276 Mol Cell Biochem (2010) 335:273–282

123

endocytic vesicles distribution. In fact, although lysosomes

were found to be typically distributed in two major peaks in

both cell types as already described [24], their buoyant

density profiles were dissimilar: light lysosomes appeared

mainly localized in the gradient fractions 3–4 in both

Sandhoff and normal fibroblasts, while dense lysosomes

appeared mainly localized in the gradient fractions 11–12

in control fibroblasts and 16–17 in Sandhoff fibroblasts

(Fig. 1-LAMP-2). Moreover, late endosomes detected by

the Rab7 marker protein resulted mainly distributed in the

gradient fractions 4–5 and 9–10 in control fibroblasts and

in the fractions 6–7 and 10–11 in Sandhoff fibroblasts

(Fig. 1-Rab7). The buoyant property of early endosomes

was also assessed and in this case the distribution of the

early endosome marker Rab5 resulted very similar in

Sandhoff and normal fibroblasts, as determined by Western

blot analysis of gradient fractions. In fact, as shown by

densitometry profiles, early endosomes were broadly dis-

tributed in the fractions ranging from 4 to 11 in both cell

types (Fig. 1-Rab5).

As the observed difference could be due to abnormali-

ties in the delivery of the protein marker LAMP-2, a

membrane protein that is targeted to lysosomes by clathrin-

coated pits, the gradient distribution of lysosomes was also

assessed by evaluation of the enzymatic activity of

b-galactosidase, a soluble lysosomal glycohydrolase that is

targeted to lysosomes by a mannose 6-phosphate receptor-

dependent pathway [34]. The density gradient pattern

obtained with b-galactosidase assays overlapped that

observed with anti-LAMP-2 antibody, thus confirming that

in Sandhoff fibroblasts the population of dense lysosomes

is denser than in normal fibroblasts (Fig. 2).

Analysis of endocytic compartment

by immunofluorescence

Immunofluorescence microscopy was used to specifically

label endocytic organelles of Sandhoff and normal fibro-

blasts. Early endosomes were labeled using EEA1 (early

endosome antigen 1) as marker [35] and fluorescence

microscopy analysis (Fig. 3-EEA1) showed homogeneously

dispersed granules in the cytoplasm of both normal and

pathological fibroblasts. The lysosomal compartment

stained with anti-LAMP-2 antibodies evidenced the char-

acteristic perinuclear punctate pattern in normal fibroblasts,

whereas Sandhoff fibroblast lysosomes appeared as aggre-

gate structures mainly localized in a restricted region of the

cytoplasm (Fig. 3-LAMP-2).

Confocal microscopy analysis was then used to further

characterize the late endocytic compartment in normal and

pathological cells. It was previously reported that in normal

fibroblasts the lysosomal membrane protein LAMP-2 par-

tially colocalized with the late endosome marker Rab7 [36].

Sandhoff and normal fibroblasts were stained for LAMP-2

and Rab-7 and representative images are shown in Fig. 4,

panel A. We confirmed that in normal fibroblasts LAMP-2

partially colocalize with Rab7. However, in Sandhoff

fibroblasts the extent of Rab7 and LAMP-2 signals over-

lapping was less consistent. The quantification of the colo-

calization degree (see ‘‘Materials and methods’’ for details)

Fig. 2 Distribution of b-galactosidase in continuous sucrose gradi-

ent. Normal (NF) and Sandhoff (SF) fibroblast microsomes were

resolved on a 0.5–1.6 M sucrose density gradient and 0.260 ml

fractions were collected from the top. b-galactosidase distribution in

the gradient fractions was determined by enzyme activity assay. The

results are expressed as percentages of the respective enzymatic

activity in each fraction to the total activity. Three different fibroblast

samples from both SD patients and healthy controls were used and

three independent experiments for each cell sample were performed.

Very similar results were obtained for each genotype. The results

shown the means ± SD of all the experiments performed with each

genotype

Fig. 3 Intracellular distribution of early endosomes and lysosomes in

normal and Sandhoff fibroblasts. Normal (NF) and Sandhoff (SF)

fibroblasts were fixed, permeabilized and immunostained with

antibodies to anti-EEA1 and anti-LAMP-2 to label early endosomes

and lysosomes, respectively. The labeled compartments were then

visualized by fluorescence microscopy. The images show represen-

tative cells, based on experiments with three independent cell samples

of both SD patient and human normal fibroblasts

Mol Cell Biochem (2010) 335:273–282 277

123

revealed that about 82% of Rab7 could be colocalized with

LAMP-2 in normal, while this percentage decreased to 49%

in Sandhoff fibroblasts. Very poor colocalization was

observed between the early endosome marker EEA1 and

the late endosome marker Rab 7 in both cell types (Fig. 4,

panel B), thus confirming that the early endosomes com-

partment did not differ between pathological and normal

conditions.

Uptake of FITC-dextran

Sandhoff and normal fibroblasts were incubated with FITC-

dextran for 24 h (pulse), then the fluid-phase marker was

chased for 2 or 4 h. The longest period of chase was carried

out to ensure that internalized FITC-dextran molecules had

reached the final destination. After the treatment, cells were

analyzed by fluorescence microscopy and representative

results are reported in Fig. 5. It clearly appeared (panel A)

that pathological cells, as well as their normal counterpart,

were able to internalize FITC-dextran molecules. However,

the intracellular distribution of fluorescent granules was

different in Sandhoff fibroblasts with respect to normal cells,

as perinuclear punctate structures were observed in normal

fibroblasts, while in Sandhoff fibroblasts FITC-dextran loa-

ded vesicles appeared as aggregates mainly localized in a

restricted region of the cytoplasm. Furthermore, the endo-

cytic vesicles of Sandhoff fibroblasts appeared brighter than

those of normal cells, suggesting that a larger amount of

FITC-dextran had been loaded. No differences were

observed between 2 and 4 h of chase (data not shown).

Confocal analysis revealed that endocytic vesicles

reached by dextran were lysosomes in normal fibroblasts as

well as in Sandhoff fibroblasts. In fact, in both cell types

the fluorescent fluid-phase marker showed a high degree of

colocalization with the lysosomal marker LAMP-2 (Fig. 5,

panel B). Again, Sandhoff fibroblast vesicles loaded with

FITC-dextran appeared more fluorescent compared to

normal fibroblast vesicles.

As it is known that the FITC-dextran fluorescence

properties are dependent on the pH, the lumenal pH of the

Fig. 4 Immunofluorescence

microscopy colocalization of

endocytic organelles in normal

and Sandhoff fibroblasts. Normal

(NF) and Sandhoff (SF)

fibroblasts were fixed,

permeabilized and

immunostained with: antibodies

to anti-LAMP-2 and anti-Rab7 to

label lysosomes and late

endosomes, respectively,

(panel A) or antibodies to anti-

Rab7 and anti-EEA1 to label late

endosomes and early

endosomes, respectively

(panel B). Cells were visualized

by confocal microscopy. Right

columns display the merged

images. The images show

representative cells, based on

experiments with three

independent cell samples of both

SD patient and human normal

fibroblasts

278 Mol Cell Biochem (2010) 335:273–282

123

terminal endocytic compartment of normal and pathologi-

cal fibroblasts was measured to verify if the observed dif-

ferences in vesicle fluorescence brightness could arise from

a difference in the pH of the acidic environment of these

organelles. The apparent lysosomal pH, estimated by a

fluorimetric method as described in the ‘‘Materials and

methods’’ section, resulted to be 5.13 (SD ± 0.01) in

normal fibroblasts and 5.15 (SD ± 0.03) in Sandhoff

fibroblasts, thus indicating that pathological cells have a

normal lysosomal pH value, in good agreement with data

available for other mammalian cell types (Fig. 6) [12].

Acridine orange is a lysosomotropic fluorescent probe

that shifts from green to orange light emission upon pro-

tonation at low pH. Staining with acridine orange con-

firmed both the normal acidic pH and the perinuclear

aggregation of terminal endocytic vesicles observed by

FITC-dextran uptake in Sandhoff cells (Fig. 7). It is worth

underlying that the aggregate appearance of pathological

fibroblast organelles appeared to be consistent with their

higher density with respect to the control, as resulted by

sucrose density gradient experiments.

Fluid-phase endocytosis

The pH measurements excluded the occurrence of an

alteration in the acidic environment of the endocytic

organelles as possible cause of Sandhoff fibroblast vesicles

higher fluorescence. Therefore, to explain the nature of the

more intense fluorescence of pathological fibroblasts with

respect to controls, the function of the endocytic pathway

was assessed by determining the uptake of the fluid-phase

marker horseradish peroxidase (HRP) into both normal and

Sandhoff cells. Cells were incubated with 3 mg/ml of HRP

for 2 h and then the amount of accumulated HRP in the

cells was quantified by enzymatic assay. As reported in

Fig. 8, the intracellular accumulation of HRP was found to

be about twice in Sandhoff fibroblasts as compared to

Fig. 5 Fluorescence microscopy characterization of endocytic vesi-

cles in normal and Sandhoff fibroblasts. Normal (NF) and Sandhoff

(SF) fibroblasts were loaded for 18 h with FITC-dextran followed by

2 h chase in the absence of the fluid-phase marker. The cells were

then fixed and the labeled vesicles were visualized by fluorescence

microscopy (panel A). For confocal microscopy measurements (panel

B), FITC-dextran loaded cells were fixed, permealized and immuno-

stained with antibodies to anti-LAMP-2 to label lysosomes. The right

columns display the merged images. The images show representative

cells, based on experiments with three independent cell samples of

both SD patient and human normal fibroblasts

Fig. 6 Apparent lysosomal pH measurement. Normal (NF) and

Sandhoff (SF) fibroblasts were loaded with FITC-dextran as described

in ‘‘Materials and methods’’ section. The fluorescence intensity ratio

of cell samples were compared with a standard curve generated with

FITC-dextran-loaded microsomes at defined pH. Three independent

cell samples of both SD patient and human normal fibroblasts were

used and three independent measurements for each standard curve

point were performed

Fig. 7 Lysosome acridine orange staining. Normal (NF) and Sand-

hoff (SF) fibroblasts were fixed, permeabilized and stained with the

fluorescent day acridine orange to visualize the lysosomes by

fluorescence microscopy. The images show representative cells,

based on experiments with three independent cell samples of both SD

patient and human normal fibroblasts

Mol Cell Biochem (2010) 335:273–282 279

123

controls. This experiment indicated that the uptake of the

fluid-phase cargo was increased in pathological cells and

might explain the more intense FITC-dextran associated

fluorescence of Sandhoff fibroblasts.

Discussion

Glycosphingolipidoses are a group of severe LSDs caused

by mutations in either hydrolase or activator protein genes

involved in catabolism of GSLs. Among glycosphingoli-

pidoses, SD is a GM2 gangliosidosis characterized by

severe neurological symptoms caused by mutation of the

gene encoding for the b-subunit of the lysosomal hydrolase

b-hexosaminidase. The resulting deficit of Hex A activity

elicits the intralysosomal accumulation of GM2 ganglio-

side, mainly in the central nervous system, leading to

neurodegeneration and to death. In particular the most

common form of SD is the infantile form, which follows a

rapid neurodegenerative course leading to death by

2–4 years of age [18]. To date, there is no cure or effective

treatment for this severe disorder.

The endosomal–lysosomal compartment of peripheral

cells from patients affected by LSD shows altered prop-

erties and functional abnormalities. In fact, changes in

chemical–physical properties of endocytic organelles, such

as pH and size, and abnormal transport along the endocytic

pathway have been reported [10–13]. Moreover, even if

each gangliosidosis has a unique biochemical defect with

respect to sphingolipid degradation and is characterized by

the accumulation of different lipids, SD shares with these

disorders common alterations in lipid endocytic trafficking

that are secondary to the metabolic deficit [14, 15].

Fibroblasts have been used as a peripheral cell model in

many studies on neurodegenerative disorders and many

alterations have been observed related to how such disor-

ders occur. In this study, we have shown that Sandhoff

fibroblast endocytic compartment differs from its normal

counterpart because terminal endocytic organelles (in

particular lysosomes) are denser and form aggregate

structures, mainly localized in a restricted region of the

cytosol. The aggregate appearance of pathological fibro-

blast organelles is consistent with their higher density as

revealed by density gradient analysis. Fluorescence

microscopy of FITC-dextran uptake reveals that fluorescent

loaded vesicles in pathological cells are mainly localized in

a restricted perinuclear area of the cytosol, while in normal

fibroblasts fluorescent structures show the characteristic

lysosomal perinuclear punctate pattern. The anomalous

distribution pattern of endocytic vesicles is consistent with

the cytosolic distribution of lysosomes that is observed by

fluorescence microscopy of LAMP-2 stained cells. Con-

focal analysis also shows that FITC-dextran-loaded vesi-

cles colocalize to a high extent with LAMP-2 positive

structures, thus demonstrating that these vesicles are indeed

lysosomes. In addition, fluid-phase endocytosis measures

reveal that the endosomal uptake is increased in patho-

logical cells. This is coherent with the more intense fluo-

rescence of FITC-dextran-loaded vesicles of Sandhoff

fibroblasts in comparison with normal fibroblasts. Fur-

thermore, the measure of lysosomal pH excluded an

increase in fluorescence emission due to environmental

alkalinization. Therefore, pathological cells are able to

internalize fluid-phase marker molecules and deliver them

to lysosomes. Moreover, endocytotic activity appears

increased in pathological cells.

Many studies have reported that altered expression

levels of Rab-GTPase proteins could affect endocytic

compartment organelle characteristics and functionality

[36–38]. In particular, it has been shown that the overex-

pression of the small GTPase Rab7, a protein involved in

the control of aggregation and fusion of late endosomes–

lysosomes, increases the size of lysosomes and the extent

of perinuclear aggregation [36]. In this study, we have

observed that the cytoplasmic distribution and the larger

size of lysosomes in Sandhoff fibroblasts are reminiscent of

those described for Rab7 overexpressing cells. However,

Rab7 expression level analyzed by Western blotting

resulted similar between Sandhoff and normal fibroblasts

(data not shown). The overexpression of Rab5, which

regulates fusion of early endosomes, has also been shown

to determine the formation of enlarged endocytic com-

partments [37]. However, Western blotting analysis of

Fig. 8 Uptake of the fluid-phase marker horseradish peroxidase.

Normal (NF) and Sandhoff (SF) fibroblasts were incubated with

3 mg/ml of HRP in DMEM for 2 h at 37�C. The amount of HRP

internalized by the cells was quantified by enzyme activity assay.

Three different fibroblast samples from both SD patients and healthy

controls were used and three independent experiments for each cell

sample were performed. Results show the means ± SD of all the

experiments performed with each genotype. ** P \ 0.01 (SF vs. NF)

according to unpaired two-tailed Student’s t test

280 Mol Cell Biochem (2010) 335:273–282

123

Rab5 detected the same expression level between Sandhoff

and normal fibroblasts (data not shown). This demonstrates

that perinuclear aggregation of lysosomes is not due to

alterated expression of these Rab-GTPase proteins.

Furthermore, the normal level of Rab5 is consistent

with the fact that fluorescence microscopy and density

gradient fractionation have not revealed any significant

differences in the early endosome compartment between

pathological and control fibroblasts. Based on these

results we have inferred that early endosomes are normal

in Sandhoff cells and we have excluded that the observed

alterations of the endocytic compartment function might

arise from alteration of these organelles. Confocal anal-

ysis shows that in Sandhoff fibroblasts the degree of

Rab7/LAMP-2 colocalization is lower than in normal

fibroblasts. The density gradient centrifugation analysis

also supports confocal microscopy evidences. As a matter

of fact, densitometric profiles of normal and pathological

cells obtained by Western blotting analysis of gradient

fractions with anti-Rab7 and anti-LAMP-2 antibodies

overlap to a minor extent in Sandhoff than control

fibroblasts.

Provided results underline some peculiar differences

between the terminal but not the early endocytic com-

partment in Sandhoff fibroblasts as compared to normal

fibroblasts. In particular, the aggregate formation, the

anomalous cytosolic distribution of lysosomes and the

reduced extent of Rab7/LAMP-2 colocalization suggest

the occurrence of an alteration in the dynamic properties

of terminal endocytic organelles, as indicated for other

GSL storage diseases such as Niemann-Pick C [15, 16].

Our findings suggest that in Sandhoff cells an abnormal

lipid membrane composition due to the lysosomal GSL

accumulation might alter the distribution of proteins

controlling endosomal membrane dynamics, thus per-

turbing the functionality of the endocytic pathway. As the

integrity of endocytic compartment is important for axo-

nal and dendritic transport, this event might critically

impair cell function at central nervous system level.

Although more experiments are needed to verify if the

observed events occur in cell types other than fibroblasts

such as neurons, which are particularly affected in

Sandhoff disease, our results provide insight into the cell

response to the enzymatic deficiency leading to the GM2

ganglioside lysosomal storage. Fibroblasts are not

strongly compromised by the accumulation of GM2, so

the observed endosomal–lysosomal alteration highlights

an aspect of the cellular dysfunction causing the disease

not directly related to the substrate accumulation. In

addition, the integrity of endocytic compartment may be

critical for the effectiveness of therapeutic approaches

such as enzyme replacement and gene therapy. As a

matter of fact, the observed abnormalities could represent

an obstacle to the efficacy of these therapeutic strategies

[17], as their success requires the effective transport of

the therapeutic enzyme into lysosomes by an endocytosis

mechanism.

Acknowledgments This work was supported by COFIN-PRIN

(Cofinanziamento-Progetto di Ricerca di Interesse Nazionale) and

FIRB (Fondo per gli Investimenti della Ricerca di Base) grants to

C.E. This work was also supported by Fondazione Cassa di Risparmio

di Perugia, Grant 2008.021.375 to C.E. We thank the ‘‘Diagnosi

PrePostnatale Malattie Metaboliche’’ Laboratory (G.Gaslini Institute)

for providing us with specimens from the ‘‘Cell line and DNA bank

from patients affected by Genetic diseases’’ Biobank- Telethon

Genetic Biobank Network (project no. GTB07001A). We thank

Dr. Maria Ragano Caracciolo for the valuable comments and critical

reading of the manuscript.

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