Sanfilippo Syndrome Type B, a Lysosomal Storage Disease, Is Also a Tauopathy
Total Page：16
File Type：pdf, Size：1020Kb
Sanfilippo syndrome type B, a lysosomal storage disease, is also a tauopathy Kazuhiro Ohmia, Lili C. Kudob, Sergey Ryazantseva,c, Hui-Zhi Zhaoa, Stanislav L. Karstenb,d,e,1,2, and Elizabeth F. Neufelda,1,2 Departments of aBiological Chemistry, bNeurology and eObstetrics and Gynecology, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095; cElectron Imaging Center for Nanomachines at California NanoSystems Institute, University of California at Los Angeles, Los Angeles, CA 90095; dDivision of Neuroscience, Los Angeles Biomedical Research Institute at Harbor-University of California at Los Angeles Medical Center, Torrance, CA 90502 Contributed by Elizabeth F. Neufeld, March 24, 2009 (sent for review February 15, 2009) Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) behavioral anomalies, which have been variously reported as is an autosomal recessive, neurodegenerative disease of children, hypoactivity (3) and hyperactivity (4); the difference is probably characterized by profound mental retardation and dementia. The because of different ages of the animals studied and different primary cause is mutation in the NAGLU gene, resulting in defi- methods of testing. Other data show altered circadian rhythm, ciency of ␣-N-acetylglucosaminidase and lysosomal accumulation hearing and vision deficits and, in older mice, a loss of Purkinje of heparan sulfate. In the mouse model of MPS III B, neurons and cells and impaired motor coordination (5). microglia display the characteristic vacuolation of lysosomal stor- Gene therapy has been tested in the mice with some success age of undegraded substrate, but neurons in the medial entorhinal (4, 6–8) but is unlikely to be available for human patients in the cortex (MEC) display accumulation of several additional sub- near future. The disease is recessive and heterozygous mice have stances. We used whole genome microarray analysis to examine a normal phenotype. differential gene expression in MEC neurons isolated by laser Pathological abnormalities in the brain of NagluϪ/Ϫ mice have capture microdissection from Naglu؊/؊ and Naglu؉/؊ mice. Neu- been a major focus of research because they have the greatest rons from the lateral entorhinal cortex (LEC) were used as tissue potential for explaining the neurodegeneration in human pa- controls. The highest increase in gene expression (6- to 7-fold tients. Astrocytes and microglia are activated (9–11), as is the between mutant and control) in MEC and LEC neurons was that of entire immune system in the brain (12). Accumulation of the Lyzs, which encodes lysozyme, but accumulation of lysozyme primary storage product, heparan sulfate, is low (9), but can be protein was seen in MEC neurons only. Because of a report that detected histochemically by colloidal iron staining (13). Some lysozyme induced the formation of hyperphosphorylated tau (P- NagluϪ/Ϫ neurons also accumulate GM3 ganglioside and cho- tau) in cultured neurons, we searched for P-tau by immunohisto- lesterol (10, 14), ubiquitin, and a lysosomal form of subunit c of chemistry. P-tau was found in MEC of Naglu؊/؊ mice, in the same mitochondrial ATP synthase (SCMAS) (13). The accumulation neurons as lysozyme. In older mutant mice, it was also seen in the of these secondary products and of the primary product is not dentate gyrus, an area important for memory. Electron microscopy dispersed throughout the brain, but is limited to neurons in of dentate gyrus neurons showed cytoplasmic inclusions of paired certain areas, especially layer II of the medial entorhinal cortex helical filaments, P-tau aggregates characteristic of tauopathies—a and to a lesser extent, layer V of the somatosensory cortex. There group of age-related dementias that include Alzheimer disease. is no obvious explanation for the accumulation of these appar- Our findings indicate that the Sanfilippo syndrome type B should ently unrelated substances and for the focal nature of the also be considered a tauopathy. accumulation. On the hypothesis that something in the cellular environment hyperphosphorylated tau ͉ lysozyme ͉ mucopolysaccharidosis ͉ of neurons in the medial entorhinal cortex (MEC) of NagluϪ/Ϫ entorhinal cortex ͉ gene expression microarray mice made them prone to accumulate these materials, we undertook to examine gene expression in those cells and to he Sanfilippo syndrome type B (mucopolysaccharidosis III follow up candidate findings by examining protein expression. TB, MPS III B) is an autosomal recessive disorder caused by We used laser capture microdissection to obtain neuronal cell mutations in the NAGLU gene, which encodes the lysosomal bodies, microarray-based technology to detect changes in gene enzyme ␣-N-acetylglucosaminidase (reviewed in ref. 1). The expression, and immunohistochemistry to examine proteins. Ϫ Ϫ resulting deficiency of the enzyme causes lysosomal accumula- Neurons of the lateral entorhinal cortex (LEC) of Naglu / mice, tion of its substrate, heparan sulfate. The disease is genetically which do not contain the secondary accumulations, were used as heterogeneous, with over a hundred mutations recorded to date controls for area-specific differences, while neurons of both (The Human Gene Mutation Database at the Institute of MEC and LEC of heterozygous mice were used as controls for Medical Genetics in Cardiff http://www.hgmd.cf.ac.uk/ac/ differences because of the disease. This approach yielded some index.php). Affected children have profound mental retarda- tion, intractable behavior problems, and eventual dementia, Author contributions: K.O., S.L.K., and E.F.N. designed research; K.O., L.C.K., S.R., H.-Z.Z., although somatic manifestations are relatively mild; death usu- and S.L.K. performed research; K.O., L.C.K., S.R., S.L.K., and E.F.N. analyzed data; and K.O., ally occurs in the second decade (2). The clinical manifestations L.C.K., S.L.K., and E.F.N. wrote the paper. of MPS III B are similar to those of MPS III A, MPS III C, and The authors declare no conflict of interest. MPS III D, which are the result of other enzymatic deficiencies Freely available online through the PNAS open access option. in the lysosomal degradation of heparan sulfate. There is no Data deposition: The microarray data have been deposited in the Gene Expression Omnibus effective therapy for any form of MPS III. database http://www.ncbi.nlm.nih.gov/geo/ (accession no GSE15758). A mouse model of MPS III B had been created by homologous 1S.L.K. and E.F.N. contributed equally to this work. recombination for the purpose of studying the pathophysiology 2To whom correspondence may be addressed. E-mail: [email protected] or of the disease and for developing treatment (3). The mutant [email protected]. Ϫ/Ϫ (Naglu ) mice are healthy when young but have limited fertility This article contains supporting information online at www.pnas.org/cgi/content/full/ and a shortened lifespan (about 8 months). They manifest some 0903223106/DCSupplemental. 8332–8337 ͉ PNAS ͉ May 19, 2009 ͉ vol. 106 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903223106 Downloaded by guest on October 2, 2021 Table 1. Differential gene expression in neurons of the MEC and LEC regions of the brain of Naglu؊/؊ mice Accession Gene symbol MEC p value LEC p value NM_017372 Lyzs 6.93 0.0002 6.16 0.002 NM_021281 Ctss 2.2 0.00 2.3 0.02 NM_147217 Gprc5c 2.5 0.05 2.0 - AK083103 Mobp 2.2 0.04 1.2 - NM_031254 Trem2 2.0 0.02 1.6 - NM_025745 4933407N01Rik 1.9 0.02 1.3 - NM_026380 Rgs8 1.9 0.00 1.4 - NM_022980 Dscr1l2 1.8 0.03 2.0 - NM_031998 Tsga14 1.8 0.05 1.1 - AK047894 Cit 1.8 0.02 1.2 - NM_010700 Ldlr 1.8 0.03 1.3 - NM_007694 Chgb (n ϭ 5) 1.8 Ͻ0.01 1.5 - AK038529 Cdh4 3.8 - 2.8 0.04 NM_001037727 Arhgap25 1.3 - 2.7 0.04 XM_885736 Fat1 3.8 - 2.6 0.01 NM_080728 Myh7 (n ϭ 2) 1.0 - 2.3 Ͻ0.05 AK017596 EST, unknown 2.1 - 1.9 0.03 NM_008528 Blnk 1.6 - 1.9 0.00 NM_027518 Gpr137c Ϫ1.1 - 1.9 0.00 NM_172443 Tbc1d16 1.3 - 1.9 0.04 NT_039170 Gls 1.3 - 1.9 0.04 NM_010271 Gpd1 1.3 - 1.9 0.02 NM_008380 Inhba 1.3 - 1.8 0.04 AK150790 H2-K1 1.5 - 1.8 0.00 AK020521 Gorasp2 1.0 - 1.8 0.04 AK078617 Trps1 1.0 - Ϫ1.8 0.02 NM_001045520 Clint1 1.1 - Ϫ1.8 0.01 Results are expressed as an average fold change of NagluϪ/Ϫ vs. Nagluϩ/Ϫ in 3 independent experiments (3 different pairs of animals) for MEC and LEC. Significance was calculated using paired t test (P Ͻ 0.05). The p value is not shown when significance was not reached (P Ͼ 0.05). The number of unique microarray probes for a given gene (n) is specified if greater than 1 unexpected findings of increased lysozyme and P-tau in the MEC istry also showed that even though lysozyme transcripts were and P-tau with paired helical filaments in the dentate gyrus of almost equally elevated in the MEC and LEC neurons of the the mutant mice, which may require reassessment of the patho- mutant mouse, the lysozyme protein was seen in the mutant genesis of the disease. mouse MEC (Fig. 1D) but not in LEC (Fig. 1E). As expected, it was not present in neurons of the control mouse MEC (Fig. 1F) Results or LEC (data not shown). While Fig. 1D shows staining in MEC Elevated Expression of the Gene Encoding Lysozyme Is Found in neurons of a mouse that was 6 months old, lysozyme was already Neurons of the Medial Entorhinal Cortex and Lateral Entorhinal present in MEC neurons of a 1-month-old NagluϪ/Ϫ mouse, Cortex.