Publisher Correction: Lysosomal storage diseases.

In the originally published version of Figure 3, APP was incorrectly linked to CMA. In addition, the label for NCP2 was omitted, and GlcSph was incorrectly labelled as GlcCer. This figure has now been corrected.

Author Correction: Lysosomal storage diseases.

In the version of the article originally published, in Figure 2 and the accompanying legend, LIMP 2 was incorrectly referred to as LIMP 1. The article has now been corrected.

Lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are a group of over 70 diseases that are characterized by lysosomal dysfunction, most of which are inherited as autosomal recessive traits. These disorders are individually rare but collectively affect 1 in 5,000 live births. LSDs typically present in infancy and childhood, although adult-onset forms also occur. Most LSDs have a progressive neurodegenerative clinical course, although symptoms in other organ systems are frequent. LSD-associated genes encode different lysosomal proteins, including lysosomal enzymes and lysosomal membrane proteins. The lysosome is the key cellular hub for macromolecule catabolism, recycling and signalling, and defects that impair any of these functions cause the accumulation of undigested or partially digested macromolecules in lysosomes (that is, 'storage') or impair the transport of molecules, which can result in cellular damage. Consequently, the cellular pathogenesis of these diseases is complex and is currently incompletely understood. Several LSDs can be treated with approved, disease-specific therapies that are mostly based on enzyme replacement. However, small-molecule therapies, including substrate reduction and chaperone therapies, have also been developed and are approved for some LSDs, whereas gene therapy and genome editing are at advanced preclinical stages and, for a few disorders, have already progressed to the clinic.

Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α-N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood-brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood-brain barrier, the fusion protein ("enzyme") in artificial cerebrospinal fluid ("vehicle") was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1-28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [ß-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, ß-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and ß-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

Defects in the medial entorhinal cortex and dentate gyrus in the mouse model of Sanfilippo syndrome type B.

Sanfilippo syndrome type B (MPS IIIB) is characterized by profound mental retardation in childhood, dementia and death in late adolescence; it is caused by deficiency of α-N-acetylglucosaminidase and resulting lysosomal storage of heparan sulfate. A mouse model, generated by homologous recombination of the Naglu gene, was used to study pathological changes in the brain. We found earlier that neurons in the medial entorhinal cortex (MEC) and the dentate gyrus showed a number of secondary defects, including the presence of hyperphosphorylated tau (Ptau) detected with antibodies raised against Ptau in Alzheimer disease brain. By further use of immunohistochemistry, we now show staining in neurons of the same area for beta amyloid, extending the resemblance to Alzheimer disease. Ptau inclusions in the dentate gyrus of MPS IIIB mice were reduced in number when the mice were administered LiCl, a specific inhibitor of Gsk3ß. Additional proteins found elevated in MEC include proteins involved in autophagy and the heparan sulfate proteoglycans, glypicans 1 and 5, the latter closely related to the primary defect. The level of secondary accumulations was associated with elevation of glypican, as seen by comparing brains of mice at different ages or with different mucopolysaccharide storage diseases. The MEC of an MPS IIIA mouse had the same intense immunostaining for glypican 1 and other markers as MPS IIIB, while MEC of MPS I and MPS II mice had weak staining, and MEC of an MPS VI mouse had no staining at all for the same proteins. A considerable amount of glypican was found in MEC of MPS IIIB mice outside of lysosomes. We propose that it is the extralysosomal glypican that would be harmful to neurons, because its heparan sulfate branches could potentiate the formation of Ptau and beta amyloid aggregates, which would be toxic as well as difficult to degrade.

From serendipity to therapy.

My postdoctoral training in the biosynthesis of plant polysaccharides at the University of California, Berkeley, led me, rather improbably, to study mucopolysaccharide storage disorders in the intramural program of the National Institutes of Health (NIH). I have traced the path from studies of mucopolysaccharide turnover in cultured cells to the development of therapy for patients. The key experiment started as an accident, i.e., the mixing of cells of different genotypes, resulting in correction of their biochemical defect. This serendipitous experiment led to identification of the enzyme deficiencies in the Hurler and Hunter syndromes, to an understanding of the biochemistry of lysosomal enzymes in general, and to the cell biology of receptor-mediated endocytosis and targeting to lysosomes. It paved the way for the development of enzyme replacement therapy with recombinant enzymes. I have also included studies performed after I moved to the University of California, Los Angeles (UCLA), including a recent unexpected finding in a neurodegenerative mucopolysaccharide storage disease, the Sanfilippo syndrome, with implications for therapy.

Sanfilippo syndrome type B, a lysosomal storage disease, is also a tauopathy.

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of alpha-N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu(-/-) and Naglu(+/-) mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu(-/-) mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies-a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.

Aptamer-based endocytosis of a lysosomal enzyme.

Enzyme replacement therapy for lysosomal storage diseases is currently based on endocytosis of lysosomal enzymes via the mannose or mannose 6-phosphate receptors. We are developing a technology for endocytosis of lysosomal enzymes that depends on generic, chemically conjugated reagents. These reagents are aptamers (single-stranded nucleic acid molecules) selected to bind to the extracellular domain of the mouse transferrin receptor. After selection, an RNA aptamer and a DNA aptamer were modified with biotin and linked to dye-labeled streptavidin for detection by confocal microscopy. Aptamer-streptavidin conjugates showed saturable uptake into mouse fibroblasts (Ltk(-) cells), which could be inhibited by an excess of free aptamer but not by tRNA, calf thymus DNA, or transferrin. The RNA aptamer-streptavidin conjugate was mouse-specific, as human cells (293T) did not take it up unless first transfected with the mouse transferrin receptor. Some streptavidin separated from the recycling pathway of transferrin and colocalized with lysosomes. After characterization in the model system, the DNA aptamer was conjugated to a lysosomal enzyme, alpha-l-iduronidase, from which mannose 6-phosphate had been removed. The aptamer had been modified by attachment of terminal glycerol for oxidation by periodate and reaction of the resulting aldehyde with amino groups on the protein. Dephospho-alpha-L-iduronidase-aptamer conjugate was taken up in saturable manner by alpha-L-iduronidase-deficient mouse fibroblasts, with half-maximal uptake estimated as 1.6 nM. Endocytosed enzyme-aptamer conjugate corrected glycosaminoglycan accumulation, indicating that it reached lysosomes and was functional in those organelles. Both uptake and correction were inhibited by unconjugated aptamer, confirming the role of the aptamer in receptor-mediated endocytosis.

Lysosomal accumulation of SCMAS (subunit c of mitochondrial ATP synthase) in neurons of the mouse model of mucopolysaccharidosis III B.

The neurodegenerative disease MPS III B (Sanfilippo syndrome type B) is caused by mutations in the gene encoding the lysosomal enzyme alpha-N-acetylglucosaminidase, with a resulting block in heparan sulfate degradation. A mouse model with disruption of the Naglu gene allows detailed study of brain pathology. In contrast to somatic cells, which accumulate primarily heparan sulfate, neurons accumulate a number of apparently unrelated metabolites, including subunit c of mitochondrial ATP synthase (SCMAS). SCMAS accumulated from 1 month of age, primarily in the medial entorhinal cortex and layer V of the somatosensory cortex. Its accumulation was not due to the absence of specific proteases. Light microscopy of brain sections of 6-months-old mice showed SCMAS to accumulate in the same areas as glycosaminoglycan and unesterified cholesterol, in the same cells as ubiquitin and GM3 ganglioside, and in the same organelles as Lamp 1 and Lamp 2. Cryo-immuno electron microscopy showed SCMAS to be present in Lamp positive vesicles bounded by a single membrane (lysosomes), in fingerprint-like layered arrays. GM3 ganglioside was found in the same lysosomes, but was not associated with the SCMAS arrays. GM3 ganglioside was also seen in lysosomes of microglia, suggesting phagocytosis of neuronal membranes. Samples used for cryo-EM and further processed by standard EM procedures (osmium tetroxide fixation and plastic embedding) showed the disappearance of the SCMAS fingerprint arrays and appearance in the same location of "zebra bodies", well known but little understood inclusions in the brain of patients with mucopolysaccharidoses.

Cardiac manifestations in the mouse model of mucopolysaccharidosis I.

Mucopolysaccharidosis I (MPS I, alpha-l-iduronidase deficiency disease) is a heritable lysosomal storage disorder involving multiple organs, including the heart. Malfunction of the heart is also a major manifestation in the mouse model of MPS I, progressing in severity from 6 to 10 months (of a 1 year life span). In comparisons of MPS I with wild-type mice, the heart was found enlarged, with thickened septal and posterior walls, primarily because of infiltration of the muscle by storage-laden cells. Heart valves were enlarged and misshapen, and contained large numbers of highly vacuolated interstitial cells. The thickened aortic wall contained vacuolated smooth muscle cells and interrupted elastic fibers. Hemodynamic measurements and echocardiography revealed reduced left ventricular function as well as mitral and aortic regurgitation. But despite these abnormalities, free-roaming MPS I mice implanted with radio telemetry devices showed surprisingly normal heart rate and blood pressure, though their electrocardiograms were abnormal. An incidental finding of the telemetry studies was a disturbed circadian rhythm in the MPS I mice. Restoration of enzyme activity in the heart of one mouse, by transplantation of retrovirally modified bone marrow, resulted in normalization of left ventricular function as well as loss of storage vacuoles in myocytes and endothelial cells, though not in valvular interstitial cells. This study demonstrates the usefulness of the mouse model for in-depth studies of the cardiovascular component of MPS I.

Retrovirally transduced bone marrow has a therapeutic effect on brain in the mouse model of mucopolysaccharidosis IIIB.

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder caused by mutations in NAGLU, the gene encoding alpha-N-acetylglucosaminidase. The disease is characterized by profound mental retardation and eventual neurodegeneration, but relatively mild somatic manifestations. There is no available therapy. We have used a mouse knockout model of the disease to test therapy by genetically modified bone marrow. Bone marrow from Naglu -/- male mice was transduced with human NAGLU cDNA in an MND-MFG vector, and transplanted into 6- to 8-week-old lethally irradiated female -/- mice. Sham-treated mice received bone marrow transduced with eGFP cDNA in an MND vector. alpha-N-Acetylglucosaminidase activity in plasma and leukocytes, measured 3 and 6 months after transplantation, varied from marginal to nearly 30 times wild-type. A low level of alpha-N-acetylglucosaminidase activity, as little as provided by transplantation of unmodified Naglu +/+ bone marrow, could normalize biochemical defects (glycosaminoglycan storage and beta-hexosaminidase elevation) in liver and spleen, but a very high level was required for an effect on kidney. Effects on the brain were best seen by examination of cellular morphology using light and electron microcopy. Mice that expressed very high levels of alpha-N-acetylglucosaminidase in blood had an increased number of normal-appearing neurons in the cortex and other parts of the brain, while microglia with engorged lysosomes had almost completely disappeared. Immunohistochemistry showed a marked decrease of staining for subunit c of mitochondrial ATP synthase and for Lamp1, markers of neuronal and microglial pathology, respectively, as well as a decrease in staining for glial fibrillary acid protein, a marker of activated astrocytes. These results show that genetically modified cells of hematopoietic origin can reduce the pathologic manifestations of MPS IIIB in the Naglu -/- mouse brain.

Treatment of the mouse model of mucopolysaccharidosis I with retrovirally transduced bone marrow.

Mucopolysaccharidosis I is a lysosomal storage disorder caused by mutations in the IDUA gene, resulting in deficiency of alpha-L-iduronidase and accumulation of glycosaminoglycans. Bone marrow transplantation has been the only available therapy, soon to be joined by enzyme replacement. We have tested retroviral gene therapy in a knockout mouse model of the disease. Bone marrow from Idua-/- male donor mice was transduced with human IDUA cDNA in an MND vector and transplanted into 6-8-week-old, lethally irradiated female Idua-/- mice. Sham-treated mice received Idua-/- bone marrow that was either unmodified or transduced with eGFP. Unmodified Idua+/+ (wild type) bone marrow was transplanted for comparison. Recipient mice were sacrificed 2-6 months after transplantation. Three biochemical parameters were used to gauge therapeutic success: appearance of alpha-L-iduronidase activity, reduction of beta-hexosaminidase activity and reduction of soluble glycosaminoglycan accumulation. Transplantation of unmodified +/+ bone marrow was effective in reducing storage in liver and spleen, but not in kidney or brain. The level of alpha-L-iduronidase activity achieved by transplantation of IDUA-transduced bone marrow varied greatly between experiments. But even modest activity resulted in correction of pathology of kidney, bladder epithelium, fibrocartilage, choroid plexus, and thalamus, as seen by light microscopy, while electron microscopy showed the presence of some normal neurons in the cortex. The partial correction of brain pathology is attributed to migration of donor hematopoietic cells, demonstrated by the presence of the Y chromosome and of normal microglia in the brain of mice receiving IDUA cDNA.

Activated microglia in cortex of mouse models of mucopolysaccharidoses I and IIIB.

Alpha-N-acetylglucosaminidase deficiency (mucopolysaccharidosis IIIB, MPS IIIB) and alpha-l-iduronidase deficiency (MPS I) are heritable lysosomal storage diseases; neurodegeneration is prominent in MPS IIIB and in severe cases of MPS I. We have obtained morphologic and molecular evidence for the involvement of microglia in brain pathology of mouse models of the two diseases. In the cortex, a subset of microglia (sometimes perineuronal) consists of cells that are probably phagocytic; they have large storage vacuoles, react with MOMA-2 (monoclonal antibody against macrophages) and Griffonia simplicifolia isolectin IB(4), and stain intensely for the lysosomal proteins Lamp-1, Lamp-2, and cathepsin D as well as for G(M3) ganglioside. MOMA-2-positive cells appear at 1 and 6 months in MPS IIIB and MPS I mice, respectively, but though their number increases with age, they remain sparse. However, a profusion of cells carrying the macrophage CD68/macrosialin antigen appear in the cortex of both mouse models at 1 month. mRNA encoding CD68/macrosialin also increases at that time, as shown by microarray and Northern blot analyses. Ten other transcripts elevated in both mouse models are associated with macrophage functions, including complement C4, the three subunits of complement C1q, lysozyme M, cathepsins S and Z, cytochrome b558 small subunit, macrophage-specific protein 1, and DAP12. An increase in IFN-gamma and IFN-gamma receptor was observed by immunohistochemistry. These functional increases may represent activation of resident microglia, an influx and activation of blood monocytes, or both. They show an inflammatory component of brain disease in the two MPS, as is known for many neurodegenerative disorders.

Attenuated plasticity in neurons and astrocytes in the mouse model of Sanfilippo syndrome type B.

Sanfilippo syndrome type B (MPS III B) is a neurodegenerative disorder characterized by profound mental retardation and early death. It is caused by deficiency of a lysosomal enzyme involved in heparan sulfate (HS) degradation. Because HS accumulation can be a major feature of this disease, we have examined crucial molecular systems associated with HS function. Using a knockout mouse with disruption of the gene responsible for HS degradation, we evaluated the effects of possible HS accumulation on neuroplasticity that are within the spectrum of action of fibroblast growth factors (FGFs) and their receptor (FGFR). We found that levels of mRNA for the FGFR-1 were attenuated in the mutant mice by the age of 6 months, whereas the mRNAs for FGF-1 and FGF-2 were reduced or unchanged in the brain regions tested. Neurogenesis, in which FGF-2 is involved, was inhibited in the MPS III B mouse brain at both young and adult ages. We also examined the expression of the glial fibrillary acidic protein (GFAP) gene and GFAP-positive cell density in both normal and injured conditions to study the functional response of astrocytes to insult. We found that, although the mutation alone caused drastic induction of reactive astrocytes, acute injury to the mutant brains failed to induce additional reactive astrocytes. Our results showed important alterations in the expression of several genes involved in the maintenance of neuroplasticity in the MPS III B. This in turn may result in reduction of neuronal health and brain function.