Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain

Abstract

Filipin is an antibiotic polyene widely used as a histochemical marker for cholesterol. We previously reported cholesterol/filipin-positive staining in brain of β-galactosidase (β-gal) knockout (^−/−) mice (GM1 gangliosidosis). The content and distribution of cholesterol and gangliosides was analyzed in plasma membrane (PM) and microsomal (MS) fractions from whole-brain tissue of 15 week-old control (β-gal^+/−) and GM1 gangliosidosis (β-gal^−/−) mice. Total ganglioside content (μg sialic acid/mg protein) was 3-fold and 7-fold greater in the PM and MS fractions, respectively, in βgal^−/− mice than in βgal^+/− mice. GM1 content was 30-fold and 50-fold greater in the PM and MS fractions, respectively. In contrast, unesterified cholesterol content (μg/mg protein) was similar in the PM and the MS fractions of the βgal^−/− and βgal^+/− mice. Filipin is known to bind to various sterol derivatives and phospholipids on thin-layer chromatograms. Biochemical evidence is presented showing that filipin also binds to GM1 with an affinity similar to that for cholesterol, with a corresponding fluorescent reaction. Our data suggest that the GM1 storage seen in the β-gal^−/− mouse contributes to the filipin ultraviolet fluorescence observed in GM1 gangliosidosis brain. The data indicate that in addition to cholesterol, filipin can also be useful for detecting GM1.

Filipin is a polyene antifungal antibiotic produced naturally by the bacteria Streptomyces filipinensis (1–4)(Fig. 1). Early reports showed that filipin disrupted permeability of sterol-containing membranes in Neurospora crassa, causing cellular leakage (5, 6). Due to its fluorescence shift upon binding to cholesterol, filipin is visible under simple ultraviolet (UV) trans-illumination (7, 8). This property has made filipin a histochemical marker for unesterified cholesterol in numerous diseases, including Niemann-Pick Type C (NPC) (9–11), Alzheimer's disease (12), GM1 gangliosidosis (13), and Huntington's disease (14). Filipin has also been used extensively as a marker of cholesterol trafficking in subcellular membranes (15–17). Although most citations (about 1,000) to filipin in PubMed involve cholesterol binding, the specificity of the binding remains highly speculative (6).

Fig.1.

The structure of filipin III. Filipin III is the primary component of the filipin complex (53%), with I (4%), II (25%), and IV (18%) being closely related isoforms (53, 54).

Filipin fluorescence was previously reported in fibroblasts and in brain tissue from a murine model of GM1 gangliosidosis (11, 13, 15). These findings suggested that cholesterol was a secondary storage molecule to GM1 in this disease. GM1 gangliosidosis is an autosomal recessive glycosphingolipid storage disorder caused by mutations in lysosomal β-galactosidase activity (18–20). The loss or reduction of β-galactosidase activity results in a failure to cleave the terminal galactosyl residue of ganglioside GM1, leading to excessive central nervous system storage of GM1 and its asialo derivative, GA1 (21–25). Secondary storage materials are not uncommon in some lipid storage disorders (26–29). The presence of excess sphingolipid (sphingomyelin or lactosylceramide) alone is sufficient to induce cholesterol internalization and “trapping” (30, 31). The internalization of cholesterol in NPC1 knockout cells is dependent on the presence of ganglioside GM3 (32). Although abnormal cholesterol trafficking can be visualized in many lipid storage disorders, the colocalization of cholesterol and other storage materials is not clear (28, 33). The lipid raft hypothesis suggests that cholesterol and GM1 are organized with transmembrane proteins in distinct raft domains (34, 35), providing a model for extensive GM1/cholesterol colocalization seen in normal and disease states.

We previously described filipin-positive staining in brain tissue of β-galactosidase (β-gal) knockout (^−/−) mice, suggesting that cholesterol and GM1 were colocalized in lysosomes (13). Moreover, β-galactosidase gene therapy lessened or completely abrogated GM1 and GA1 ganglioside storage and filipin staining. However, the quantification of unesterified cholesterol showed no abnormal storage in the brains of β-gal−/− mice. No change in cholesterol content was observed after treatment with adeno-associated virus (AAV) gene therapy, which reduced ganglioside storage (13). If filipin staining is a reliable marker for membrane cholesterol content, then changes in filipin staining should correlate with measurable changes in the content of membrane cholesterol.

Here we sought to test the hypothesis that GM1 ganglioside storage disrupts cholesterol localization within the plasma membrane (PM) and microsomes of GM1 gangliosidosis mouse brain. We found that even among subcellular (microsomal) fractions enriched in GM1 and GA1, there was no significant corresponding increase in unes­terified cholesterol content. Furthermore, we found that the filipin complex, which was reported previously to bind weakly to phospholipids (16), also binds gangliosides, especially GM1. These findings indicate that the filipin-positive staining seen in GM1 gangliosidosis mouse brain cannot be solely attributed to an increase in intracellular cholesterol. A preliminary report of these findings was presented at the 2010 meeting of the American Society for Neurochemistry (36).

MATERIALS AND METHODS

Mice

B6/129Sv mice heterozygous for the GM1 β-galactosidase gene (β-gal+/−) were obtained from Dr. Alessandra d'Azzo of Saint Jude Children's Research Hospital, Nashville, TN. These mice were derived by homologous recombination and embryonic stem cell technology as previously described (37). Homozygous β-gal−/− mouse pups were derived from crossing β-gal−/+ females with β-gal−/− male mice. Genotypes were determined by measuring β-galactosidase-specific activity in tail tissue using a modification of the Galjaard procedure (21, 38). All mice were propagated in the Boston College Animal Care Facility and were housed in plastic cages with filter tops containing Sani-Chip bedding (P. J. Murphy Forest Products Corp.; Montville, NJ). The room was maintained at 22°C on a 12 h light/12 h dark cycle. Food (PROLAB R/M/H/3000 Lab Chow; Agway, St. Louis, MO) and water were provided ad libitum. Nursing females were provided with cotton nesting pads for the duration of the experiment. All animal experiments were carried out with ethical committee approval in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee.

Brain subcellular fractions

Mice were euthanized by cervical dislocation, and excised brain tissue was immediately frozen for analysis. The procedure of Lomnitski et al. (39) was used to prepare brain subcellular fractions. Briefly, whole brains were homogenized in 4 ml of centrifugation solution [1 μg/μl aprotinin (Sigma Aldrich; St. Louis, MO), 1 mM PMSF, and 1 mM EDTA in 50 mM potassium phosphate buffer]. The homogenates were transferred to 5 ml polyallomer centrifuge tubes (Beckman; Palo Alto, CA) and centrifuged for 10 min at 1,000 g (4°C). The P1 (nuclear membrane) pellet was separated from the subsequent supernatant. The supernatant was collected and centrifuged again at 10,000 g (4°C), resulting in a P2 pellet enriched in PM and mitochondria. The supernatant was further centrifuged for 1 h at 150,000 g (4°C), resulting in a P3 pellet enriched in microsomes. Both the P2 and the P3 pellets for all samples were suspended in 1 ml dH₂O. A 500 µl aliquot of the whole-brain homogenate was collected before the first spin. From each whole-brain homogenate, P2 PM pellet suspension, and P3 microsomal (MS) pellet suspension from β-gal−/− and β-gal+/− mice, half of each homogenate was assayed for protein content, and the other half was used for the isolation and purification of lipids.

Protein quantification

Protein content from whole brain and individual membrane fractions were quantified using a Bio-Rad DC Protein Assay Kit (Bio-Rad; Hercules, CA). Serial 1:10 and 1:20 dilutions of each sample were aliquoted onto a Corning 96-well clear assay plate in triplicate. After the assay reagents were added, samples were incubated at room temperature for 15 min, and then read at 750 nm on an M5 SpectraMax Microplate Reader (Molecular Devices; Sunnyvale, CA). Protein concentrations were determined by fitting the samples to a standard curve using BSA.

Western blotting

The distribution of calnexin, an endoplasmic reticulum marker (40), among the subcellular fractions was determined by Western blot. Protein concentrations of cell fractions were estimated using the Bio-Rad DC protein assay as described above. Eighteen milligrams of total protein from each sample was loaded onto a 4–12% sodium dodecyl polyacrylamide gel (Invitrogen; Carlsbad, CA) and separated by electrophoresis. Gels were run for 30 min at 100 V, then 50 min at 150 V. Proteins were transferred for 16 h at 30 V to a polyvinylidene difluoride Immobilon-P membrane (Millipore; Billerica, MA). The membrane was blocked in 5% blotting-grade blocker nonfat dry milk (Bio-Rad) in 1× TBS with Tween 20 (TBS-T) for 3 h at room temperature. Blots were washed in TBS-T three times for 5 min each, and then probed for 16 h at 4°C with primary antibodies against calnexin (Millipore), diluted 1:750, and β-actin (Cell Signaling; Danvers, MA), diluted 1:4,000. Blots were washed again with TBS-T three times for 15 min, and then probed for 90 min at room temperature with secondary antibodies against mouse (Santa Cruz Biotechnology; Santa Cruz, CA), diluted 1:4,000, and rabbit (Cell Signaling), diluted 1:4,000. Protein bands were visualized with Pierce ECL Western blotting substrate (Fisher Scientific; Houston, TX).

Total lipid isolation

Total lipids were isolated and purified from cerebral cortex as previously described (19, 21). Frozen cortex samples were homogenized in centrifugation solution, separated into fractions as described above, and lyophilized for 24 h. After desiccation, the dried tissue was weighed and resuspended in water, and lipids were extracted overnight in CHCl₃-CH₃OH 1:1. Lipids were further separated into a neutral (F1) and acidic/ganglioside (F2) fraction via ion exchange chromatography over a Sephadex column. Neutral lipids (phosphatidylcholine, phosphatidylethanolamine, plasmalogens, ceramide, sphingomyelin, cerebrosides, and GA1) and cholesterol were eluted with CHCl₃-CH₃OH-dH₂O (30:60:8; v/v/v). Acidic phospholipids and gangliosides (including GM1) were eluted with CHCl₃-CH₃OH-0.8 M C₂H₃NaO₂ (30: 60: 8; v/v/v).

Sialic acid quantification

Gangliosides were further separated from acidic phospholipids and desalted as previously described (19, 21). Total ganglioside content was quantified before and after desalting using the resorcinol assay. Three aliquots of each ganglioside sample were dried under vacuum. A resorcinol-dH₂O, 1:1, v/v solution (resorcinol reagent-HCl-0.2 M resorcinol- dH₂O-0.1 M CuSO₄, 40: 5: 5: 0.125, v/v/v/v) was added to each sample, followed by submersion in a boiling water bath for 17 min. After cooling on ice, the reaction was stopped with butyl acetate-N-butanol, 85:15, v/v. Each sample was vortexed and centrifuged at 700 g for 2 min. The absorbance of the upper aqueous layer was recorded at 580 nm using a Shimadzu UV-1601 spectrophotometer (Shimadzu; Torrance, CA). Sialic acid values were fit to a standard curve using N-acetylneuraminic acid as a standard.

High-performance TLC

Lipids were spotted on 10 × 20 cm silica gel-60 high-performance TLC (HPTLC) plates using a Camag Linomat III auto-TLC spotter (Camag Scientific, Inc.; Wilmington, NC) as previously described (19, 21, 41). The amount of lipid per lane was equivalent to 1.5 μg sialic acid for gangliosides and 10 μg protein for neutral lipids/cholesterol. Gangliosides were separated by a single ascending run (90 min) in a solution of CHCl₃-CH₃OH-0.02% CaCl₂ (55:45:10; v/v/v), whereas neutral lipids/cholesterol were separated by an initial ascending run (up to 4.5 cm) with a solution of CHCl₃-CH₃OH-acetic acid-formic acid-dH₂O (35:15:6:2:1; v/v/v/v/v), dried for 15 min, and then run to the top with hexane-diisopropyl ether-acetic acid (65:35:2; v/v/v) (Fisher Scientific). Gangliosides were visualized by spraying the dried plates with the resorcinol reagent, followed by heating at 95°C. Neutral lipids and cholesterol were visualized by dipping the dried plates into 3% cupric acetate in an 8% phosphoric acid solution, followed by heating the plate to 140°C (41).

GM1 and cholesterol quantification

A Personal Densitometer SI with ImageQuant software (Molecular Dynamics; Sunnyvale, CA) was used to estimate the concentrations of gangliosides and cholesterol on HPTLC. The total ganglioside content was normalized to 100%, and the percent distribution of each band was used to determine the sialic acid concentration for individual gangliosides. GM1 content was expressed as μg/mg protein. For neutral plates, the band density value for cholesterol in each sample lane (equivalent to 10 μg of protein) was fit to a standard curve and used to determine cholesterol concentration expressed as μg/mg protein.

Filipin UV visualization

Purified GM1 and cholesterol (from mouse brain) were successively spotted with increasing nanomole amounts on an HPTLC plate and separated by the neutral solvent systems described above. For UV visualization of whole-brain homogenates and subcellular fractions, the equivalent of 20 mg of protein was spotted for both neutral lipids and gangliosides. HPTLC plates were sprayed with a solution of filipin-PBS [6.0 mg filipin complex dissolved in 300 ml dimethyl formamide (Sigma Aldrich) added to 49.7 ml PBS]. The filipin-stained plates were shielded from light and placed in a Shel Lab General laboratory incubator (Sheldon Manufacturing; Cornelius, OR) at 37°C for 90 min. The plates were then washed three times in dH₂O, and visualized via UV transillumination as previously described (16). UV fluorescence at 365 nm was determined using a Camag TLC Scanner 4 (Camag Scientific, Inc.) with winCATS software. After UV visualization, gangliosides were visualized by spraying the dried plate with the resorcinol reagent and heating at 95°C. Cholesterol was visualized by dipping the dried plate into 3% cupric acetate in an 8% phosphoric acid solution, followed by heating the plate to 140°C, as described above (41).

RESULTS

To determine whether the fluorescence from the filipin complex corresponded to cholesterol content, we evaluated the content and distribution of lipids in whole brain and in subcellular fractions from 15 week-old normal (β-gal+/−) and GM1 gangliosidosis (β-gal−/−) mice. Calnexin, an endoplasmic reticulum marker, was enriched in the MS fraction, compared with whole brain and PM (Fig. 2). No significant differences were found between the +/− and −/− mice for brain weight or protein content in PMs or microsomes (Table 1). The total sialic acid content in β-gal−/− mouse brain was significantly higher than in normal mouse brain (Table 1). Sialic acid concentration (μg/mg protein) was similar in microsomes and PM of +/− mice (Table 1). Total sialic acid content for −/− mice was 2-fold greater, 3-fold greater, and 7-fold greater in whole brain, PM, and MS fractions, respectively, compared with that of +/− mice (Table 1). Most of the increase in total sialic acid concentration in the −/− mice was due to an increase in GM1 content (Fig. 3). GM1 content was 50-fold greater in the microsomes of −/− mice than in those of +/− mice (Fig. 3, Table 1).

Fig. 2.

Calnexin is enriched in P3 (MS) fractions of normal and GM1 gangliosidosis mouse brain. Equivalent protein aliquots from each subcellular fraction were separated by electrophoresis, transferred to immobilon-P membranes, and incubated with antibodies against calnexin (upper bands) and β-actin (lower bands). Representative samples are from one β-gal+/− or β-gal−/− brain.

TABLE 1.

Protein, cholesterol, and ganglioside content in subcellar fractions of GM1 gangliosidosis mouse brain

Genotype	Fraction	Wet Weight	Proteina	Sialic Acidb	GM1c	Cholesterold
		mg	mg/ml	μg/mg protein	μg sialic acid/mg protein	μg/mg protein
	Whole brain	403 ± 20	5.9 ± 0.1	3.8 ± 0.2	0.5 ± 0.1	85 ± 38
β-gal+/−	P2		2.4 ± 0.4	7.8 ± 0.3	0.7 ± 0.1	131 ± 16
	P3		0.7 ± 0.2	7.4 ± 0.9	1.0 ± 0.1	117 ± 30
	Whole brain	414 ± 4	6.0 ± 0.1	7.8 ± 0.3e	5.2 ± 0.2e	80 ± 9
β-gal−/−	P2		2.7 ± 0.1	22.4 ± 0.7e	17.0 ± 0.7e	150 ± 15
	P3		1.2 ± 0.2	52.5 ± 4.4e	49.0 ± 4.0e	163 ± 18

Values are expressed as the mean ± standard error of the mean. N = 3 mice per group.

^aProtein values were determined using the Bio-Rad assay as described in Materials and Methods.

^bSialic acid values were determined using the resorcinol assay as described in Materials and Methods.

^cGM1 content was determined through densitometric scanning of HPTLC plates as seen in Fig. 3.

^dCholesterol content was determined through densitometric scanning of HPTLC plates as seen in Fig. 4.

^eSignificantly different from the β-gal^+/− value in the same fraction at P < 0.01 (using Student's t-test.)

Fig. 3.

High-performance TLC of gangliosides. Gangliosides were isolated and purified for each fraction from control (β-gal+/−) and GM1 gangliosidosis (β-gal−/−) mouse brains. 1.5 μg sialic acid were spotted per lane. Quantification of GM1 content is displayed in Table 1.

The HPTLC of the neutral lipids shows that phosphatidylethanolamine was the major neutral phospholipid of the PM fraction, whereas phosphatidylcholine was the major neutral phospholipid of the MS membrane fraction (Fig. 4). Asialo GM1 (GA1) accumulated in β-gal−/− mouse brain, with the greatest concentration found in the MS fraction (Fig. 4). No significant differences were found in cholesterol content between +/− and −/− mice in whole brain, PM, or microsomes (Table 1).

Fig. 4.

HPTLC of cholesterol and neutral lipids. Neutral phospholipids and cholesterol were isolated and purified for each fraction from control (β-gal+/−) and GM1 gangliosidosis (β-gal−/−) mouse brains. The equivalent of 10 μg of protein was spotted for each neutral lipid sample. Notably, asialo GM1 (GA1) is enriched in the MS fraction of the β-gal−/− mouse brain. Quantification of cholesterol content is displayed in Table 1.

To quantitatively assess the fluorescent reactivity of filipin, we spotted HPTLC plates with equivalent nanomole amounts of both cholesterol and ganglioside GM1 from mouse brain. Cholesterol exhibited strong UV fluorescence (area units/nmol), as expected, when incubated with filipin complex on HPTLC (Fig. 5A). A best-fit line revealed a linear correlation between nanomole cholesterol content and detectable fluorescence (Fig. 5B); no quenching was observed at the concentrations measured (16, 42). We found that GM1 ganglioside also elicited detectable fluorescence with the filipin complex after a 90 min incubation (Fig. 5A). UV fluorescence also increased, corresponding with a linear increase in GM1 content at higher nanomole amounts (Fig. 5B). Notably, the fluorescence observed from filipin/GM1 binding was equal to or greater than the fluorescence observed from filipin/cholesterol binding (Fig. 5B).

Fig. 5.

Filipin complex binds to ganglioside GM1 on HPTLC. HPTLC plates containing equivalent nanomole amounts of unesterified cholesterol and GM1 ganglioside were separated by dual solvent systems and incubated with filipin complex for 90 min. A: UV transillumination revealed reactivity with both lipids. The presence of GM1 was subsequently confirmed by the resorcinol reagent. B: Quantitation of UV fluorescence at 365 nm. R² values suggest a linear correlation between the nanomole of GM1 or cholesterol with filipin fluorescent detection.

To determine whether our lipid quantification corresponded with fluorescence, we applied filipin to HPTLC of neutral lipids and gangliosides isolated from the subcellular fractions in β-gal+/− and β-gal−/− mouse brain. Cholesterol and phosphatidylcholine were both visible by UV transillumination after incubation with filipin in the neutral lipids purified from MS fractions of +/− mice (Fig. 6A). GA1, cholesterol, and phosphatidylcholine were detectable in MS fractions of −/− mice (Fig. 6A). There was no difference in the fluorescent signal (area units/mg protein) of cholesterol in MS fractions in −/− and +/− mice (Fig. 6A, Table 2). There was no detectable fluorescent signal from the MS gangliosides of +/= mice. (Fig. 6B, Table 2). Conversely, there was a prominent fluorescent signal corresponding to GM1 in the MS gangliosides of −/− mice (Fig. 6B, Table 2).

Fig. 6.

Filipin reactivity with P3 (MS) phospholipids, cholesterol, and gangliosides. Equivalent (20 mg) protein aliquots from the isolated neutral lipid (A) or ganglioside (B) MS fractions of control (β-gal+/−) and GM1 gangliosidosis (β-gal−/−) mouse brains were spotted on HPTLC, separated with the appropriate solvent system, and incubated with filipin complex for 90 min. A: The primary filipin-positive bands in the neutral lipid microsomes are cholesterol (chol) and phosphatidylcholine (PC). GA1 is detectable in the β-gal−/− MS fraction. B: GM1 is the only detectable band among the gangliosides of β-gal−/− mice. No fluorescent signal is detected in control β-gal+/− microsomes. Cholesterol and GM1 relative fluorescence is displayed in Table 2.

TABLE 2.

Relative UV fluorescence of GM1 and cholesterol in microsomal fractions

	Fluoresencea
	area units/mg protein
Genotype	GM1	Cholesterol
β-gal+/−	NDb	954 ± 67
β-gal−/−	1,117 ± 75	899 ± 81

Values are expressed as the mean ± standard error of the mean (SEM). N = 3 mice per group.

^aRelative fluorescence at 365 nm was determined using a CAMAG TLC scanner on HPTLC plates as seen in Fig. 6.

^bND, no detectable fluorescence.

DISCUSSION

Using filipin as a cholesterol marker, Broekman et al. (13) showed that GM1 accumulation was accompanied by strong filipin staining in lysosomal membranes. These findings suggested that cholesterol accumulated in lysosomes along with GM1. In this study, we showed that GM1 accumulation was not associated with significant increases of cholesterol content in MS membranes. Our findings raise concerns in using filipin as an accurate marker for quantitative cholesterol content in GM1 gangliosidosis brain. The cholesterol-trapping hypothesis proposes that the accumulation of one type of lipid raft molecule in late endosomes will lead to an accumulation of other types of raft lipids, eventually impeding raft lipid turnover and cholesterol trafficking (34). If GM1 and cholesterol were tightly associated in raft lipids, it follows that the accumulation of GM1 should directly cause the accumulation of cholesterol. However, our biochemical lipid analysis showed that GM1 accumulation alone was capable of producing a filipin-positive signal in the brain of β-gal−/− mice. We did not quantify cholesterol in lysosomes specifically in this study, but the presumed colocalization of GM1 would overstate the presence of cholesterol in any lipid subdomain examined.

Although filipin binding is greater for cholesterol than for phosphatidylcholine on HPTLC, filipin was considered somewhat promiscuous in its lipid binding capabilities (16). Our data suggest that the promiscuity of the filipin complex can also extend to gangliosides. Our findings also suggest that accumulation of GM1 can produce misinformation on cholesterol content when filipin is used as a cholesterol marker. This is an obvious caveat for using filipin as a histochemical marker for known lysosomal storage disorders, because gangliosides can accumulate as either primary or secondary storage material in several storage diseases (26–29, 43, 44).

Our findings might help to resolve several inconsistencies associated with storage of cholesterol and gangliosides seen in GM1 gangliosidosis and Niemann-Pick Type C disease. For example, cyclodextrin treatment abrogates filipin signal and corrects cholesterol storage in NPC1 fibroblasts and mouse models (45–47). However, cyclodextrin has no effect on filipin signal in GM1 gangliosidosis fibroblasts or mouse tissue (45). Earlier studies in human GM1 gangliosidosis brain showed a decrease in cholesterol content, depending on the severity of the disease (48). Filipin also inhibits the internalization of the Vibrio cholorae cholera toxin, which avidly binds to GM1 (49, 50). This finding further suggests a cross-reactivity of filipin with GM1.

We did not attempt to evaluate membrane fractions based on their detergent-resistant properties. The analysis of GM1 and cholesterol in detergent-resistant membranes or lipid rafts holds the caveat that different raft preparations can produce disparate results (51, 52). The membrane fractionation performed here yielded results consistent with previous findings regarding ganglioside GM1 and asialo GA1 storage in late endosomes of β-gal^−/− mice (30). We also confirmed the previous findings of Lomnitski and coworkers (39) that phosphatidylethanolamine and phosphatidylcholine are enriched in PMs and microsomes, respectively. Although the P2 fractions examined here did contain mitochondria, we have previously demonstrated that purified mitochondria from normal mouse brain contain only trace amounts of ganglioside (40). Further, a report from Sano et al. (25) showed that the GM1 storage seen in mitochondrial preparations from GM1 gangliosidosis mouse brain is primarily due to its presence in mitochondria-associated ER membranes. We conclude that the appearance of filipin-positive staining in tissues from GM1 gangliosidosis results from the cross-reactivity of filipin with GM1 ganglioside in addition to binding of filipin with cholesterol. Although our findings should not decrease the utility of filipin as a histochemical marker for nonesterified cholesterol, they do question the specificity of filipin as a marker solely for cholesterol in certain disease conditions.