Systemic inflammation and neurodegeneration in a mouse model of multiple sulfatase deficiency

Carmine Settembre*, Ida Annunziata*, Carmine Spampanato*, Daniela Zarcone†, Gilda Cobellis*‡, Edoardo Nusco*, Ester Zito*, Carlo Tacchetti†§, Maria Pia Cosma*, and Andrea Ballabio*¶ʈ

*Telethon Institute of Genetics and Medicine (TIGEM), 80131 Naples, Italy; †Department of Experimental Medicine and §MicroSCoBiO Research Center and IFOM Center of Cell Oncology and Ultrastructure, University of Genoa, 16126 Genoa, Italy; ‡Department of General Pathology, Second University of Naples, 80131 Naples, Italy; and ¶Medical Genetics, Department of Pediatrics, Federico II University, 80131 Naples, Italy

Communicated by C. Thomas Caskey, University of Texas Health Science Center, Houston, TX, January 18, 2007 (received for review November 17, 2006) Sulfatases are involved in several biological functions such as human diseases have been elucidated in detail, the function of degradation of macromolecules in the lysosomes. In patients with orphan sulfatases remains largely elusive (2). multiple sulfatase deficiency, mutations in the SUMF1 cause In a rare autosomal recessive disorder, known as multiple a reduction of sulfatase activities because of a posttranslational sulfatase deficiency (MSD), the activity of all sulfatases is modification defect. We have generated a mouse line carrying a profoundly reduced (1). The phenotype of MSD patients com- null mutation in the Sumf1 gene. Sulfatase activities are com- bines, with some phenotypic variability, all of the clinical symp- pletely absent in Sumf1؊/؊ mice, indicating that Sumf1 is indis- toms observed in each individual sulfatase deficiency (8). We pensable for sulfatase activation and that mammals, differently and others discovered that MSD is caused by mutations in the from bacteria, have a single sulfatase modification system. Simi- SUMF1 gene which is involved in the posttranslational modifi- larly to multiple sulfatase deficiency patients, Sumf1؊/؊ mice cation of sulfatases resulting in the conversion of a cysteine display frequent early mortality, congenital growth retardation, located in the catalytic site of sulfatases into ␣-formylglycine (9, skeletal abnormalities, and neurological defects. All examined 10). In mammals, SUMF1 is the only factor known to be involved tissues showed progressive cell vacuolization and significant lyso- in the sulfatase modification machinery and belongs to a gene somal storage of glycosaminoglycans. Sumf1؊/؊ mice showed a family that has been highly conserved from prokaryotes to generalized inflammatory process characterized by a massive pres- eukaryotes. Interestingly, sulfatases appear to be the only pro- ence of highly vacuolated macrophages, which are the main site of teins undergoing this unique biochemical modification (2). lysosomal storage. Activated microglia were detected in the cer- We generated Sumf1 KO mice in which, differently from MSD ebellum and brain cortex associated with remarkable astroglyosis patients, the activities of all sulfatases are completely absent. The and neuronal cell loss. Between 4 and 6 months of age, we detected phenotype of these mice is severe and progressive and resembles a strong increase in the expression levels of inflammatory cyto- the clinical features of patients with MSD. Massive GAG kines and of apoptotic markers in both the CNS and liver, demon- accumulation and cell vacuolization were observed in all tissues strating that inflammation and apoptosis occur at the late stage of and were associated with systemic inflammation, apoptosis, and disease and suggesting that they play an important role in both the neurodegeneration. systemic and CNS phenotypes observed in lysosomal disorders. Results This mouse model, in which the function of an entire protein family ؊/؊ has been silenced, offers a unique opportunity to study sulfatase Generation of a Sumf1 Mouse Strain. By searching the Bay- function and the mechanisms underlying lysosomal storage genomics gene-trapping database, we identified an ES cell diseases. clone that was supposed to contain an insertion of the trapping vector within the Sumf1 gene. We carefully characterized both apoptosis ͉ macrophages ͉ sulfatase modifying factor 1 the insertion site and the Sumf1 fusion transcript in this ES cell clone. Genomic PCR [see supporting information (SI) Fig. 8 a and b] and Southern blotting (data not shown) experiments ulfatases catalyze the hydrolysis of sulfate ester bonds from demonstrated that the trapping vector was inserted in intron 3 Sa wide variety of substrates, ranging from complex mole- of the Sumf1 gene, and RT-PCR revealed that the fusion cules, such as glycosaminoglycans (GAGs), to sulfolipids and transcript was composed by the first three exons of Sumf1 steroid sulfates (1). Seventeen sulfatase are present in the fused by ␤-Geo mRNA (see SI Fig. 8c). This ES clone was (2). Based on current knowledge, two main injected into mouse blastocysts, and several chimeric animals functional categories can be identified. The first category in- were obtained. Germ-line transmission was obtained, and cludes six sulfatases that are localized into the lysosomes and three heterozygous animals were born. Mating of these het- exert their enzymatic activity at an acidic pH. These are primarily erozygous mice generated homozygous Sumf1Ϫ/Ϫ individuals, involved in the catabolism of GAGs and . The second category includes sulfatases acting at neutral pH and localized in other subcellular compartments such as the ER and the Golgi Author contributions: A.B. designed research; C. Settembre, I.A., C. Spampanato, G.C., and apparatus. These sulfatases are more likely to be involved in E.N. performed research; D.Z., E.Z., C.T., and M.P.C. contributed new reagents/analytic tools; C. Settembre, I.A., and A.B. analyzed data; and C. Settembre, C.T., and A.B. wrote the biosynthetic, rather than catabolic, pathways (2–4). paper. Eight known metabolic disorders are caused by the deficiency The authors declare no conflict of interest. of individual sulfatase activities. These disorders are all inherited Freely available online through the PNAS open access option. as monogenic traits and are associated with impaired desulfation Abbreviations: Sumf1, sulfatase-modifying factor 1; MPS, mucopolysaccharidoses; LSD, of specific substrate metabolites. Six of them are lysosomal lysosomal storage disease; GAG, glycosaminoglycan; MEF, mouse embryonic fibroblast. storage diseases (LSDs), including five different types of muco- ʈTo whom correspondence should be addressed at: Telethon Institute of Genetics and polysaccharidoses (MPSs) and metachromatic leukodystrophy Medicine (TIGEM), Via P. Castellino 111, 80131 Naples, Italy. E-mail: [email protected]. (5, 6), whereas the remaining two disorders are due to deficien- This article contains supporting information online at www.pnas.org/cgi/content/full/ cies of nonlysosomal sulfatases (4, 7). Although the natural 0700382104/DC1. substrates and metabolic pathways of sulfatases involved in © 2007 by The National Academy of Sciences of the USA

4506–4511 ͉ PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700382104 Downloaded by guest on September 25, 2021 Fig. 2. Growth, survival rates in Sumf1Ϫ/Ϫ mice. (a) Kaplan–Meyer survival curve of Sumf1Ϫ/Ϫ mice (n ϭ 39) over 180 days. (b) Mean weight values of WT (purple) and individual weight values of Sumf1Ϫ/Ϫ mice (blue). Crosses indicate the age (days) of death of Sumf1Ϫ/Ϫ mice. WT are the mean values of four (two females and two males) mice. Significant correlation between growth and survival rates is evident. (c) Sumf1Ϫ/Ϫ mice (KO) are smaller than their WT littermates at P40. (d) Sumf1Ϫ/Ϫ mice (on the left) display the typical flat facial profile of patients affected by MPSs.

not complete because of the presence of several (Ϸ70%) non- transduced cells. Sulfatase activities were also tested in Sumf1ϩ/Ϫ Fig. 1. Sulfatase activities in Sumf1Ϫ/Ϫ MEFs. ARSA (a), ARSB (b), ARSC (c), heterozygous mice and found within WT ranges (data not ARSE (d), SGSH (e), iduronate-2-sulfatase (IDS) (f), G6S (g), GAL6S (h), and shown). B-Gal (i) activities were measured in MEFs derived from two separate Sumf1Ϫ/Ϫ mice (KO1 and KO2) and WT littermates. Sulfatase activities are undetectable ؊/؊ Ϫ/Ϫ A Severe and Progressive Phenotype in Sumf1 Mice. In principle, in the Sumf1 MEFs and are partially rescued when the cells are transduced Ϫ/Ϫ by a lentiviral vector containing the human SUMF1 cDNA (KOϩSUMF1). WT the phenotype of Sumf1 mice should combine those observed Ϫ/Ϫ indicates average values and standard deviations of four WT mice. in each individual sulfatase deficiency. Sumf1 mice display KOϩSUMF1 indicate average values and standard deviations of two Sumf1Ϫ/Ϫ congenital growth retardation and frequent mortality in the first MEF lines from two different mice, each transduced in duplicate. All enzymatic weeks of life, with only 10% reaching 3 months of age (Fig. 2a). activities are indicated as nmol/h/mg. We observed that smaller Sumf1Ϫ/Ϫ mice live significantly less compared with those with higher weights (P Ͻ 0.0002, see Methods) (Fig. 2b). Growth and survival rates of Sumf1ϩ/Ϫ as demonstrated by genomic PCR (SI Fig. 8b). Nineteen litters heterozygous mice were indistinguishable from those of WT have been generated so far by crossing Sumf1ϩ/Ϫ heterozygous littermates. Fig. 2 c and d shows pictures of a 40-day-old GENETICS mice, yielding a total of 138 mice born. Among them, 38 were Sumf1Ϫ/Ϫ mouse and a WT littermate. Growth retardation and ϩ/ϩ ϩ/Ϫ Ϫ/Ϫ Sumf1 (27.5%), 62 Sumf1 (45%), and 38 Sumf1 a flat facial profile are evident in the Sumf1Ϫ/Ϫ mouse. Starting (27.5%), as expected by Mendelian laws, indicating that the at approximately 2 weeks of life, Sumf1Ϫ/Ϫ mice show a severe absence of all sulfatase activities is not embryonic-lethal in kyphosis, short limbs (Fig. 3a) and skull (Fig. 3b), loss of the mice, at least in the mixed C57B6/S129J background. RT-PCR spinal process of dorsal vertebrae, and joint deformities (Fig. 3 from tail mRNA demonstrated the presence of the fusion c and d). Hind limb clasping, head tremor, and seizures were also Ϫ Ϫ transcript in both the homozygous / mice and in the detected starting at 1 month of age, indicating neurological heterozygotes. Homozygous Ϫ/Ϫ mice totally lacked the WT involvement (data not shown). All these features are important Sumf1 transcript, demonstrating that they carry a null muta- components of the phenotype observed in patients with MSD (8) tion (SI Fig. 8c). Identical results were obtained by using liver and in human patients and murine models of MPSs (5–7, 11–16). and brain mRNA (data not shown). GAG Storage in Macrophages. Extraordinarily high levels of GAG ؊ ؊ Sumf1 / Mice Completely Lack All Sulfatase Activities. We tested staining, increasing with age, were detected in liver, kidney, and the activities of eight different sulfatases, , B, C, heart (Fig. 4a) as well as in brain, lung, synovium, heart valves, and E (ARSA, ARSB, ARSC, and ARSE), sulfamidase (SGSH), aorta, and trachea (data not shown) from Sumf1Ϫ/Ϫ mice, iduronate-2-sulfatase (IDS), N-acetylglucosamine-6-sulfatase indicating significant storage that is likely due to the simulta- (G6S), and N-acetylgalactosamine-6-sulfatase (GAL6S), each neous deficiency of several lysosomal sulfatases. Immunohisto- involved in a human disease (4–7), and of a control enzyme chemical analysis using MOMA-2 [monoclonal antibody against (␤-galactosidase), in total homogenates of mouse embryonic macrophages (17)] antibody revealed a massive presence of fibroblasts (MEFs) as well as in liver, kidney, and brain samples macrophages in all tissues examined from Sumf1Ϫ/Ϫ mice as early (data not shown) from Sumf1Ϫ/Ϫ mice and found them to be as at postnatal day (P)5, indicating the presence of a systemic completely absent (Fig. 1). inflammatory process (Fig. 4b). Interestingly, the sites of GAG Transduction of Sumf1Ϫ/Ϫ MEFs by using a lentiviral vector storage appeared to have a similar distribution to the presence containing the human SUMF1 cDNA resulted in significant of macrophages. Immunofluorescence analysis performed in rescue of the activities of all sulfatases (Fig. 1). The rescue was liver by using antibodies against macrophages (MOMA-2) and

Settembre et al. PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4507 Downloaded by guest on September 25, 2021 Fig. 3. Skeletal abnormalities in Sumf1Ϫ/Ϫ mice. (a) Alizarin red- and Alcian blue-stained skeletons from a P30 WT and a Sumf1Ϫ/Ϫ mouse. Growth retar- dation, spinal kyphosis, reduced length of long bones, and skull deformities are evident in the Sumf1Ϫ/Ϫmouse. (b) Facitron radiography of a P30 control mouse (Left) and a Sumf1Ϫ/Ϫ littermate (Right) shows that the Sumf1Ϫ/Ϫ skull is significantly shorter. (c) Loss of spinous process of dorsal vertebrae in P30 Sumf1Ϫ/Ϫ mice (Right) compared with control (Left). (d) Analysis of distal tibia shows enlarged epiphysis and metaphysis in Sumf1Ϫ/Ϫ mice (Right) compared with control (Left).

heparan sulfate (HS), demonstrated that macrophages are the primary site of GAG storage, thus establishing a link between storage and inflammation (Fig. 4c). Analysis of semithin sections of Sumf1Ϫ/Ϫ liver showed that, whereas vacuoles of macrophages are clear and very large in size, often filling the entire cytoplasm, hepatocytes display smaller and more closely packed vacuoles (Fig. 4 d and e). Fig. 5 shows that a significant number of macrophages was also detected in heart valves, synovium, and larynx.

Neuroinflammation and Neuronal Cell Loss. Particularly strong signs of inflammation were observed in the entire CNS of Sumf1Ϫ/Ϫ mice starting at 1 month of age. Activated microglia were detected in the cerebellum, where MOMA-2 labeling revealed that macrophages are located in the Purkinje cell layer (Fig. 6a). Comparison between MOMA-2 and calbindin immunofluores- Fig. 4. GAG staining and immunohistochemical detection of activated cence analyses showed that macrophages establish engulfing macrophages in the visceral organs of Sumf1Ϫ/Ϫ mice. (a) GAG staining. Alcian contacts with the few survived Purkinje cells (Fig. 6b). Fig. 6c blue-stained tissue sections of the liver, kidney, and heart from a 3-month-old shows electron microscopy performed in the cerebellum reveal- WT mouse (Upper) and a Sumf1Ϫ/Ϫ littermate (Lower). Tissues from the ing enlarged vacuoles in the cytoplasm of protoplasmic astro- Sumf1Ϫ/Ϫ mice show massive GAG accumulation (blue spots), which is absent cytes, which have a pale nucleus with a narrow rim of hetero- in the control. Sections were counterstained with nuclear-fast red reagent. (b) chromatin, surrounding Purkinje cells (Fig. 6c). Consistently, MOMA-2 immunohistochemistry on liver, kidney, and heart from a 3-month- Ϫ Ϫ significant and progressive loss of Purkinje cells was detected in old WT mouse (Upper) and a Sumf1 / littermate (Lower) shows the presence Ϫ/Ϫ Sumf1Ϫ/Ϫ mice (Fig. 6d). of macrophages in the Sumf1 tissues. (c) Immunofluorescence analyses of liver sections from 3-month-old Sumf1Ϫ/Ϫ mice by using MOMA-2 and heparan A similar situation was found in brain cortex, where macro- sulfate (HS) antibodies. Merge shows that macrophages are the primary site of phages completely encircle neurons (Fig. 6e). In addition, a GAG storage. (d) Semithin section of liver from 3-month-old Sumf1Ϫ/Ϫ mouse. remarkable astroglyosis was evident by the use of GFAP anti- Highly vacuolated macrophage (arrow) localize next to hepatocytes. Vacuoles body (Fig. 6f). Ultrastructural analysis of brain cortex shows the are significantly larger compared with those observed in hepatocytes (arrow- presence of numerous slightly electron dense granules, often head). (e) Semithin section of liver from 3-month-old Sumf1Ϫ/Ϫ mouse. Highly containing membrane remnants, filling the cytoplasm of proto- vacuolated macrophage (arrowheads) and endothelial cells (arrow) bound plasmic astrocytes (Fig. 6g), endothelial cells and pericytes liver sinusoids (sin). Vacuoles are significantly larger compared with those surrounding capillaries. observed in hepatocytes (Hep). [Scale bars: 20 ␮m(a–c); 7 ␮m(d); 20 ␮m(e).] Consistently, an increase in the expression levels of proapo- ptotic inflammatory cytokines TNF␣ and IL-12 and of MIP1␣ chemokine was detected at 6 months of age in RNA from total age and significantly increasing at 3 months when it becomes brain (Fig. 6h). A similar increase in the expression of inflam- massive and generalized (Fig. 7 a–c). Similar results were also matory cytokines was also detected in liver RNA (data not observed in the brain cortex (Fig. 7 d–f). shown). Discussion Massive Apoptosis at a Late Stage of Disease. In situ TUNEL Mutations in the SUMF1 gene in humans results in MSD, a staining of liver sections revealed the presence of apoptotic cells, recessively inherited Mendelian disorder in which the activity of morphologically identified as hepatocytes, starting at 1 month of all sulfatases is profoundly reduced because of a posttransla-

4508 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700382104 Settembre et al. Downloaded by guest on September 25, 2021 Fig. 5. Inflammation in heart valves, synovium, and larynx. Immunofluores- cence analyses of atrioventricular valve (a and b), synovium (c and d), and larynx (e and f) sections from 1-month-old Sumf1Ϫ/Ϫ mice and control litter- mates by using MOMA-2 antibodies revealed massive presence of macro- phages in the tissues from Sumf1Ϫ/Ϫ mice. (Scale bars: 20 ␮m.)

tional modification defect. However, a variable but significant residual sulfatase activity is detected in MSD cases (1), thus raising the possibility that another factor, in addition to SUMF1, participates to the posttranslational modification of sulfatases in mammals, similarly to what was described in bacteria (18). This putative factor may be responsible for the residual activity Fig. 6. Inflammation in the CNS of Sumf1Ϫ/Ϫ mice. (a) Immunofluorescence detected in MSD patients. We have now demonstrated that with MOMA-2 antibody reveals massive presence of activated microglia in the Ϫ/Ϫ GENETICS Sumf1Ϫ/Ϫ mice have a complete deficiency of sulfatase activities, cerebellum of Sumf1 mice, particularly along the Purkinje cell layer. (b) Purkinje cells immunostained with calbindin marker (green) are surrounded thus excluding this possibility and indicating that mammals have Ϫ Ϫ by microglia. (c) Cerebellum from 3-month-old Sumf1 / mice. Electron mi- a single sulfatase modification system. The residual sulfatase crograph shows large vacuoles present in the cytoplasm of protoplasmic activities detected in MSD patients may be due to the hypomor- astrocytes (A) surrounding a Purkinje cell (P). (d) Immunostaining with calbi- phic nature of the mutations. ndin shows loss of Purkinje cells in 2- and 6-month-old Sumf1Ϫ/Ϫ mice. Quan- Animal models of individual sulfatase deficiencies are found tification of progressive loss of Purkinje cells in Sumf1Ϫ/Ϫ mice. Histogram in several species. Some of them represent spontaneous mutants, shows the percentage of Purkinje cells lost in cerebellar sections of Sumf1Ϫ/Ϫ whereas others were generated as KO mice. The latter are mice compared with control sections (values represent means of n ϭ 3 mice for Ͻ available for five diseases due to individual sulfatase deficiencies, each group. *, Student’s t test P 0.05). (e) Double staining using MOMA-2 (red) and Neun (green) as macrophage and neuronal markers, respectively, namely metachromatic leukodystrophy, and MPSs (i.e., MPSII, shows that activated microglia are juxtaposed to neurons in the cortex of MPSIIIA, MPSIVA, and MPSVI) (11–16). In all instances the Sumf1Ϫ/Ϫ mice. Microglia staining is not detected in control mice. (f) Double biochemical abnormalities in the mouse models replicated those staining with GFAP (green) and Neun (red) shows massive astroglyosis (GFAP- observed in the corresponding human disease. However, in some positive cells) in the cortex of Sumf1Ϫ/Ϫ mice. (g) Cortex from 3-month-old cases there were differences between the clinical phenotypes of Sumf1Ϫ/Ϫ mouse. Electron micrograph of endothelial cells surrounding a human diseases and those observed in the corresponding animal blood capillary vessel (BV) contain cytoplasmic vacuoles (arrowheads). Larger models. In principle, a Sumf1 KO mouse should recapitulate all vacuoles are present in the cytoplasm of a neighboring protoplasmic astrocyte (arrows). (h) Measurement of expression levels of TNF␣ and IL-12 cytokines of the features found in individual sulfatase deficiencies and, in and MIP1␣ chemokine at 2 and 6 months of age in RNA from total brain (values addition, should display the potential consequences of the represent means value of three independent experiment for each group). *, deficiency of all of the other sulfatases which have not been Student’s t test P Ͻ 0.05. [Scale bars: 20 ␮m(a, b, d–f); 2.8 ␮m(c); 1.8 ␮m(g).] associated to a human disease yet. The majority of Sumf1Ϫ/Ϫ mice die within the first month of age, indicating that this is a very severe condition, more than any Heart insufficiency, respiratory problems and neurological im- previously described murine model of lysosomal storage dis- pairment may represent either alternative or concomitant eases. Among the possible causes of death are feeding difficulties causes. Because of the high complexity of MSD at this point we due to the severe growth deficiency and skeletal deformities. do not know which of the many metabolic disturbances occurring

Settembre et al. PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 ͉ 4509 Downloaded by guest on September 25, 2021 for a more global lysosomal defect affecting intracellular turnover. It is conceivable that, in LSDs, damaged cells stimulate the infiltration of macrophages. In our mice, we cannot distinguish between infiltrating and resident macrophages. However, a study performed in a murine model of Sandhoff disease demonstrated that macrophages in the brain derive from the recruitment of circulating blood monocytes, which is mediated by the MIP1␣ chemokine (21). Consistent with this observation we detected a significant increase of MIP1␣ Sumf1Ϫ/Ϫ mice. Overall, these data also demonstrate that the inflammatory response in LSDs is not linked to the presence of a specific type of lysosomal storage, because the storage material in Sandhoff disease (i.e., ganglio- sides) is very different from that accumulating in Sumf1Ϫ/Ϫ mice. Fig. 7. Massive apoptosis in Sumf1Ϫ/Ϫ. (a–c) In situ TUNEL analysis of liver The precise role of inflammation in the phenotype of LSDs is sections of control (a), 1-month-old (b), and 3-month-old (c) Sumf1Ϫ/Ϫ mice still a matter of debate. MPS patients suffer from degenerative shows a very high number of apoptotic cells in liver. (d and e) In situ TUNEL joint disease, breathing abnormalities, and heart valves insuffi- analysis of brain sections of Sumf1Ϫ/Ϫ mice shows few apoptotic cells in ciency, which is one of the main causes of death in MPS VI (4). Ϫ Ϫ cerebral cortex in 2-month-old Sumf1 / brain (d), whereas apoptosis is Interestingly, in Sumf1Ϫ/Ϫ mice, we found massive macrophage generalized in cerebral cortex at 6 month of age (e). (f) Apoptotic cell count Ϫ/Ϫ infiltration in all of these tissues, as observed in inflammatory in the brain cortex from 2- and 6-month-old Sumf1 mice and in 6-month-old diseases. For example, it is known that expansion of monocytes/ control mice (values represent mean of n ϭ 3 mice for each group; *, Student’s t test P Ͻ 0.05). macrophages in synovium is a key pathological feature of rheumatoid arthritis (22). This suggests that some features of LSDs result from inflammatory conditions similar to those in this disease play a major role in the growth deficiency and observed in autoimmune diseases. mortality observed in Sumf1Ϫ/Ϫ mice. However, it is likely that Many LSDs are associated with neurodegeneration (23), even several different factors act synergistically. For example, though the pathways leading from lysosomal storage to neuronal Sumf1Ϫ/Ϫ mice are smaller than their WT littermates at birth, cell damage or loss are not well understood. Previous studies suggesting a role of factors acting during development. In described an inflammatory response in the CNS of patients and addition, their postnatal growth curve is clearly abnormal and murine models of MPSs and other LSDs (24–27). Crossing a this maybe related to metabolic dysfunction and feeding diffi- mouse model of LSD, i.e., Sandhoff disease, with a KO mouse ␣ culties. More detailed studies are needed to clarify these issues. lacking the MIP1 leukocyte chemokine, caused a reduction of Predominant aspects of the phenotype observed in Sumf1Ϫ/Ϫ macrophage infiltration, thus decreasing apoptosis and improv- mice were growth retardation and skeletal abnormalities. Most ing neurological phenotype. In addition, the treatment of this mouse model with antiinflammatory drugs resulted in pheno- mice displayed spinal deformities resulting in a severe kyphosis Ϫ/Ϫ and a short and coarse skull which becomes particularly evident typic improvement (28). In Sumf1 mice, the elevation of after the first month of life. Joint deformities were also evident proapoptotic inflammatory cytokines, as well as the presence of in most mice. All these features are important components of the massive apoptosis, were observed only at late stages suggesting phenotype of LSDs and are observed in patients with MSD (8) that inflammation-mediated apoptosis represents a final step in and of both human patients and murine models of MPSs (5, LSD pathogenesis. Ϫ Ϫ In conclusion, silencing of the activity of the entire sulfatase 12–16). In addition, bone histological analysis of Sumf1 / mice protein family in Sumf1Ϫ/Ϫ mice offers the opportunity to study showed clear signs of defective osteogenesis (C.S., unpublished sulfatase function and the cascade of events leading from data). Growth deficiency and defective osteogenesis may also be lysosomal dysfunction to cell damage and death in LSDs. related to arylsulfatase E (ARSE) deficiency, causing X-linked recessive chondrodysplasia punctata (CDPX) in humans (7). Methods Further studies of Sumf1Ϫ/Ϫ mice may help identifying the Generation of MEFs. MEFs were isolated by trypsinization of metabolic pathway involved in this condition and the natural littermate embryos dissected at 14 days of gestation from a cross substrate of ARSE which is still unknown. of heterozygous SUMF1 mutant mice. Homogeneous cell sus- The hallmark of LSDs is the accumulation of compounds pensions were plated in six-well plates in DMEM supplemented because of a block of a specific catabolic pathway causing with 20% FBS and penicillin/streptomycin. significant cell vacuolization (19). A detailed analysis of Sumf1 KO tissue sections showed massive and generalized cell vacuol- Sulfatase Enzymatic Assays. Experiments with MEFs were per- ization, particularly in macrophages. However, although macro- formed in early passages (p Ͻ 6). For ARSA, ARSB, ARSC, and phage vacuoles appear clear and very large, often filling the ARSE, the enzymatic activity was tested as described (10). entire cytoplasm, hepatocytes and neurons display smaller and Iduronate 2-sulfatase, sulfamidase, N-acetylglucosamine more closely packed vacuoles, suggesting that there may be 6-sulfatase and N-acetylgalactosamine-6-sulfatase were assayed differences in the origin and nature of vacuoles in macrophages with fluorogenic substrates. ␣-D-galactosidase enzymatic activity compared with other cell types. The activation of macrophages was measured by using 4-MU-␣-D-galactose as a substrate. The in LSDs may represent an attempt of the organism to remove activity was determined by incubating cell homogenates with 2 undigested material or damaged cells (20). Macrophages were mM 4 MU-␣-D-galactose in 0.5 M sodium acetate buffer (pH found to encircle other cell types, suggesting the presence of 5.0) in 300 ␮l of incubation mixture. active phagocytosis. A striking example is the Purkinje cell layer, where microglia become the predominant cell type, and are Infections with Lentiviral Vectors. The human SUMF1 cDNA was associated with significant neuronal cell loss. Therefore, it is cloned into the plasmid pHRcPPT.CMV. MEFs were infected by likely that GAG storage in macrophages derives not only from incubation for 16 h with 50 ng of p24 SUMF1 virus in 0.5 ml of endogenous sources but also from their phagocytic activity. fresh culture medium for 2 days before harvesting for sulfatase Furthermore, GAG storage in macrophages may be responsible tests.

4510 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700382104 Settembre et al. Downloaded by guest on September 25, 2021 Genotyping of Sumf1 KO Mice. We genotyped the mice by PCR on performed by using Vectastain ABC kit (Vector Laboratories, tail DNAs using ␤-gal-specific primers coupled with Sumf1- Burlingame, CA) according to the manufacturer’s instructions. specific primers. RT-PCR was performed by using the RNeasy kit (Qiagen, Valencia, CA), followed by cDNA synthesis per- In Situ Detection of Apoptotic Cells (TUNEL). TUNEL staining was formed with SuperScript First Strand kit (Invitrogen, Carlsbad, performed by using the ApopTag Peroxidase In Situ Apoptosis CA). Animal use and analyses were conducted in accordance Detection kit (Oncor, Gaithersburg, MD) according to the man- with the guidelines of the Animal Care and Use Committee of ufacturer’s instructions. Cardarelli Hospital in Naples and authorized by the Italian GAG Staining. GAG staining was performed on organs fixed in Ministry of Health. All mice to be killed were deeply anesthe- methacarn (methanol 60%, chloroform 30%, and acetic acid tized with 100 mg/kg phenobarbital and subsequently perfused 10%) and stained with Alcian blue. through the left ventricle with PBS. Real-Time PCR. Real-time PCR was performed by using the iQ Skeletal Staining. Carcasses were fixed overnight in ethanol l00% SYBR green supermix kit (Bio-Rad, Hercules, CA) following and stained with 30 mg of Alcian blue (Sigma, St. Louis, MO) the manufacturer instructions and by using an iCycler iQ system in 1:5 ethanol/acetic acid (final volume 100 ml) for 3 days. Bones (Bio-Rad). were stained by using 1% KOH and 75 ␮g/ml Alizarin red S (Sigma). Electron Microscopy and Semithin Sections. For semithin sections, 0.5- to 0.7-␮m sections were stained with toluidine blue. For Antibodies. The antibodies used were MOMA-2 (rat anti mouse electron microscopy, gray-silver sections were observed with a 1:250; Serotech, Ontario, Canada), GFAP (G-A-5 1:200; Sigma), FEI CM10 or Tecnai 12G2. NeuN (MAB377 1:100; Chemicon, Temecula, CA), anti-Calbindin- D Statistical Analysis. The experiment was carried out on 16 -28K (EG-20 1:200; Sigma) anti-heparan sulfate (F58–10E4 1:200; Ϫ/Ϫ Seigagaku, Tokyo, Japan). The secondary antibodies were from Sumf1 mice. We divided the observed samples into two groups based on the survival time (Ts). Group 1 (n1 ϭ 9) Ts was Molecular Probes (Eugene, OR) (Invitrogen). Ͻ40 days, and group 2 (n2 ϭ 7) Ts was Ͼ40 days. For the statistical analysis, we measured the weight of each mouse at Immunofluorescence. Organs were fixed in 4% paraformaldehyde postnatal day (P)25, and then we computed an unpaired t test. in PBS, pH 7.4, overnight and embedded in optimal cutting The corresponding P value was 0.0002. temperature compound (Tissue Tek). Cryostat sections were cut Student’s t tests were used to compare the mean levels of three at 10 ␮m. The immunofluorescence was performed by using a independent experiments in Figs. 6 and 7. P Ͻ 0.05 was standard protocol. Photographs were taken by using a fluores- considered statistically significant. cence microscope Zeiss (Thornwood, NY) Axioplan 2 integrated with the AxioCam MR camera. We thank Consuelo Venturi for electron microscopy and Pietro De Camilli, Graciana Diez-Roux, Alessandro Fraldi, Gerard Karsenty, Histology and Immunohistochemistry. Histological analyses were Alberto Mantovani, Giancarlo Parenti, Marco Sardiello, Antonio Sica, and Miche`leStuder for helpful suggestions. C. Settembre is the recipient performed by using standard operating procedures generated by of a predoctoral fellowship of the European School of Molecular the EMPReSS platform (European Mouse Phenotyping Re- Medicine (SEMM). This work was supported by the Italian Telethon source of Standardised Screens). Immunohistochemistry was Foundation and by the Italian Ministry of Agriculture (MiPAF).

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