Engineered arylsulfatase A with increased activity, stability and brain delivery for therapy of metachromatic leukodystrophy.

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme's bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.

Proteomic investigation of neural stem cell to oligodendrocyte precursor cell differentiation reveals phosphorylation-dependent Dclk1 processing.

Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify doublecortin-like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. All data are available via ProteomeXchange with identifier PXD040652.

Brain cell type-specific endocytosis of arylsulfatase A identifies limitations of enzyme-based therapies for metachromatic leukodystrophy.

Enzyme replacement therapies, allogeneic bone marrow transplantation and gene therapies are treatment options for lysosomal storage diseases caused by inherited deficiencies of soluble lysosomal enzymes. Independent from the approach, the enzyme must be delivered to lysosomes of deficient patient cells. Little is known about the dissemination of enzyme within a tissue where cells compete for uptake via different receptor systems, binding affinities and endocytic rates. To evaluate dissemination and lysosomal targeting of a lysosomal enzyme in the CNS, we analysed receptor-mediated endocytosis of arylsulfatase A (ASA) by different types of brain-derived cell lines and primary murine brain cells. For ASA expressed by chinese hamster ovary cells for enzyme replacement therapy of metachromatic leukodystrophy, endocytic rates decline from microglia to neurons and astrocytes and to oligodendrocytes. Only immature oligodendrocytes endocytose significant amounts of enzyme. Uptake by non-microglial cells is due to mannose 6-phosphate receptors, whereas several receptor systems participate in endocytosis by microglial cells. Interestingly, ASA expressed by microglial cells cannot be taken up in a mannose 6-phosphate dependent manner. The resulting failure to correct non-microglial cells corroborates in vivo data and indicates that therapeutic effects of allogeneic bone marrow transplantation and hematopoietic stem cell gene therapy on metachromatic leukodystrophy are independent of metabolic cross-correction of neurons, astrocytes and oligodendrocytes by receptor-mediated endocytosis.

Decreased turnover of the CNS myelin protein Opalin in a mouse model of hereditary spastic paraplegia 35.

Spastic paraplegia 35 (SPG35) (OMIM: 612319) or fatty acid hydroxylase-associated neurodegeneration (FAHN) is caused by deficiency of fatty acid 2-hydroxylase (FA2H). This enzyme synthesizes sphingolipids containing 2-hydroxylated fatty acids, which are particularly abundant in myelin. Fa2h-deficient (Fa2h-/-) mice develop symptoms reminiscent of the human disease and therefore serve as animal model of SPG35. In order to understand further the pathogenesis of SPG35, we compared the proteome of purified CNS myelin isolated from wild type and Fa2h-/- mice at different time points of disease progression using tandem mass tag labeling. Data analysis with a focus on myelin membrane proteins revealed a significant increase of the oligodendrocytic myelin paranodal and inner loop protein (Opalin) in Fa2h-/- mice, whereas the concentration of other major myelin proteins was not significantly changed. Western blot analysis revealed an almost 6-fold increase of Opalin in myelin of Fa2h-/- mice aged 21-23 months. A concurrent unaltered Opalin gene expression suggested a decreased turnover of the Opalin protein in Fa2h-/- mice. Supporting this hypothesis, Opalin protein half-life was reduced significantly when expressed in CHO cells synthesizing 2-hydroxylated sulfatide, compared to cells synthesizing only non-hydroxylated sulfatide. Degradation of Opalin was inhibited by inhibitors of lysosomal degradation but unaffected by proteasome inhibitors. Taken together, these results reveal a new function of 2-hydroxylated sphingolipids namely affecting the turnover of a myelin membrane protein. This may play a role in the pathogenesis of SPG35.

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice.

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.

Proteaphagy in Mammalian Cells Can Function Independent of ATG5/ATG7.

The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), ß-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Because these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.

CLN6 deficiency causes selective changes in the lysosomal protein composition.

Neuronal ceroid lipofuscinoses (NCLs) collectively account for the highest prevalence of inherited neurodegenerative diseases in childhood. This disease group is classified by the deposition of similar autofluorescence storage material in lysosomes that is accompanied by seizures, blindness and premature mortality in later disease stages. Defects in several genes affecting various proteins lead to NCL, one of them being CLN6, a transmembrane protein resident in the endoplasmic reticulum. Dysfunctionality of CLN6 causes variant late infantile NCL (vLINCL). The function of CLN6 and how its deficiency affects lysosomal integrity remains unknown. In this work, we performed a comparative proteomic analysis of isolated lysosomal fractions from liver tissue of nclf mice, a natural mouse model displaying a similar disease course than its human counterpart. We could identify a drastic reduction in the protein amounts of selected lysosomal proteins, amongst them several members of the NCL protein family. Most of these proteins were N-glycosylated, soluble hydrolases and their reduction in protein levels was verified by western blotting and enzymatic assays. Hereby we could directly link Cln6 dysfunction to changes in the lysosomal compartment and to other NCL forms.

Evolutionary redesign of the lysosomal enzyme arylsulfatase A increases efficacy of enzyme replacement therapy for metachromatic leukodystrophy.

Protein engineering is a means to optimize protein therapeutics developed for the treatment of so far incurable diseases including cancers and genetic disorders. Here we report on an engineering approach in which we successfully increased the catalytic rate constant of an enzyme that is presently evaluated in enzyme replacement therapies (ERT) of a lysosomal storage disease (LSD). Although ERT is a treatment option for many LSDs, outcomes are lagging far behind expectations for most of them. This has been ascribed to insufficient enzyme activities accumulating in tissues difficult to target such as brain and peripheral nerves. We show for human arylsulfatase A (hARSA) that the activity of a therapeutic enzyme can be substantially increased by reversing activity-diminishing and by inserting activity-promoting amino acid substitutions that had occurred in the evolution of hominids and non-human mammals, respectively. The potential of this approach, here designated as evolutionary redesign, was highlighted by the observation that murinization of only 1 or 3 amino acid positions increased the hARSA activity 3- and 5-fold, with little impact on stability, respectively. The two kinetically optimized hARSA variants showed no immunogenic potential in ERT of a humanized ARSA knockout mouse model of metachromatic leukodystrophy (MLD) and reduced lysosomal storage of kidney, peripheral and central nervous system up to 3-fold more efficiently than wild-type hARSA. Due to their safety profile and higher therapeutic potential the engineered hARSA variants might represent major advances for future enzyme-based therapies of MLD and stimulate analogous approaches for other enzyme therapeutics.

Generation of Antibodies Targeting Cleavable Cross-Linkers.

Chemical cross-linking has become a powerful tool for the analysis of protein structures and interactions by mass spectrometry. A particular strength of this approach is the ability to investigate native states in vivo, investigating intact organelles, cells, or tissues. For such applications, the cleavable cross-linkers disuccinimidyl sulfoxide (DSSO) and disuccinimidyl dibutyric urea (DSBU) are gaining increasing popularity, as they allow for the analysis of complex mixtures. It is inherently difficult to follow the reaction of cross-linkers with proteins in intact biological structures, stalling the optimization of in vivo cross-linking experiments. We generated polyclonal antibodies targeting DSSO- and DSBU-modified proteins, by injection of cross-linked bovine serum albumin (BSA) in rabbits. We show that the cross-linker-modified BSA successfully triggered an immune response, and that DSSO- and DSBU-specific antibodies were generated by the animals. Using affinity-purified antibodies specific for the individual cross-linkers, we demonstrate their application to the detection of cross-linker-modified proteins in Western blot and immunocytochemistry experiments of intact and permeabilized cells. Furthermore, we show their ability to immunoprecipitate DSSO/DSBU-modified proteins and provide evidence for their affinity toward water-quenched dead-links. These antibodies provide a valuable tool for the investigation of proteins modified with the cross-linkers DSSO and DSBU.

Lysosomal proteome analysis reveals that CLN3-defective cells have multiple enzyme deficiencies associated with changes in intracellular trafficking.

Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.

Quantitative proteomics of synaptosome S-nitrosylation in Alzheimer's disease.

Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2-/- mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies.

BDNF and NGF signals originating from sensory ganglia promote cranial motor axon growth.

After exiting the hindbrain, branchial motor axons reach their targets in association with sensory ganglia. The trigeminal ganglion has been shown to promote motor axon growth from rhombomeres 2/3 and 4/5, but it is unknown whether this effect is ganglion specific and through which signals it is mediated. Here, we addressed these questions by co-cultures of ventral rhombomere 8 explants with cranial and spinal sensory ganglia in a collagen gel matrix. Our results show that all cranial sensory ganglia and even a trunk dorsal root ganglion can promote motor axon growth and that ganglia isolated from older embryos had a stronger effect on the axonal growth than younger ones. We found that brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are necessary and sufficient for this effect. Altogether, our results demonstrate that the promoting effect of sensory ganglia on cranial motor axon growth is stage dependent, but not ganglion specific and is mediated by BDNF and NGF signals.

Synthesis and structure-activity relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and discovery of a competitive inhibitor.

Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) causing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase (CST) catalyses the transfer of a sulphate group from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) to cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised and investigated analogues of the substrate galactosylceramide with variations at the anomeric position, the acyl substituent and the carbohydrate moiety, and investigated their structure-activity relationships. While most of the compounds behaved as substrates, α-galactosylceramide 16 was identified as the first competitive CST inhibitor. Compound 16 can serve as a new lead structure for the development of drugs for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently not available.

Investigation of Oligodendrocyte Precursor Cell Differentiation by Quantitative Proteomics.

Oligodendrocytes, the myelinating cells of the central nervous system, are essential for correct brain function. They originate from oligodendrocyte precursor cells through a differentiation process which is only incompletely understood and impaired in a variety of demyelinating diseases. Better knowledge of this differentiation holds the promise to develop novel therapies for these disorders. The differentiation of rat oligodendrocyte precursor cells to oligodendrocytes in vitro is investigated. After confirmation of differentiation by immunohistochemical analysis using cell type-specific marker proteins, a quantitative proteomics study using tandem mass tags (TMT) is conducted. Four time points of differentiation covering early, intermediate, and late stages are investigated. Data analysis by Mascot and MaxQuant identified 5259 protein groups of which 471 are not described in the context of cells of the oligodendroglial lineage before. Quantitative analysis of the dataset revealed distinct regulation patterns for proteins of different functional categories including metabolic processes, regulation of the cell cycle, and transcriptional control of protein expression. The present data confirm a significant number of proteins known to play a role in oligodendrocytes and myelination. Furthermore, novel candidate proteins are identified which may play an important role in this differentiation process providing a valuable resource for oligodendrocyte research.

Identification of progesterone receptor membrane component-1 as an interaction partner and possible regulator of fatty acid 2-hydroxylase.

The fatty acid 2-hydroxylase (FA2H) is essential for synthesis of 2-hydroxylated fatty acids in myelinating and other cells, and deficiency of this enzyme causes a complicated form of hereditary spastic paraplegia also known as fatty acid hydroxylase-associated neurodegeneration. Despite its important role in sphingolipid metabolism, regulation of FA2H and its interaction with other proteins involved in the same or other metabolic pathways is poorly understood. To identify potential interaction partners of the enzyme, quantitative mass spectrometry using stable isotope labeling of cells was combined with formaldehyde cross-linking and proximity biotinylation, respectively. Besides other enzymes involved in sphingolipid synthesis and intermembrane transfer of ceramide, and putative redox partners of FA2H, progesterone receptor membrane component-1 (PGRMC1) and PGRMC2 were identified as putative interaction partners. These two related heme-binding proteins are known to regulate several cytochrome P450 enzymes. Bimolecular fluorescence complementation experiments confirmed the interaction of FA2H with PGRMC1. Moreover, the PGRMC1 inhibitor AG-205 significantly reduced synthesis of hydroxylated ceramide and glucosylceramide in FA2H-expressing cells. This suggests that PGRMC1 may regulate FA2H activity, possibly through its heme chaperone activity.

Lrp1/LDL Receptor Play Critical Roles in Mannose 6-Phosphate-Independent Lysosomal Enzyme Targeting.

Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P-independent transport routes. Here, we quantify the lysosomal proteome in M6P-deficient mouse fibroblasts (PT(ki)) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P-independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non-phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT(ki) cells. These findings establish Lrp1 and LDL receptors in M6P-independent secretion-recapture targeting mechanism for lysosomal enzymes.

N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time.

Canavan disease (CD) is a severe, lethal leukodystrophy caused by deficiency in aspartoacylase (ASPA), which hydrolyzes N-acetylaspartate (NAA). In the brains of CD patients, NAA accumulates to high millimolar concentrations. The pathology of the disease is characterized by loss of oligodendrocytes and spongy myelin degeneration in the CNS. Whether accumulating NAA, absence of NAA-derived acetate, or absence of any unknown functions of the ASPA enzyme is responsible for the pathology of the disease is not fully understood. We generated ASPA-deficient (Aspa(nur7/nur7)) mice that are also deficient for NAA synthase Nat8L (Nat8L(-/-)/Aspa(nur7/nur7)). These mice have no detectable NAA. Nevertheless, they exhibited normal myelin content, myelin sphingolipid composition, and full reversal of spongy myelin and axonal degeneration. Surprisingly, although pathology was fully reversed, the survival time of the mice was not prolonged. In contrast, Aspa(nur7/nur7) mice with only one intact Nat8L allele accumulated less NAA, developed a less severe pathology, phenotypic improvements, and, importantly, an almost normal survival time. Therefore, inhibition of NAA synthase is a promising therapeutic option for CD. The reduced survival rate of Nat8L(-/-)/Aspa(nur7/nur7) mice, however, indicates that complete inhibition of NAA synthase may bear unforeseeable risks for the patient. Furthermore, we demonstrate that acetate derived from NAA is not essential for myelin lipid synthesis and that loss of NAA-derived acetate does not cause the myelin phenotype of Aspa(nur7/nur7) mice. Our data clearly support the hypothesis that NAA accumulation is the major factor in the development of CD.

Anti-inflammatory Therapy With Simvastatin Improves Neuroinflammation and CNS Function in a Mouse Model of Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency of the lysosomal enzyme arylsulfatase A. The prevailing late-infantile variant of MLD is characterized by widespread and progressive demyelination of the central nervous system (CNS) causing death during childhood. In order to gain insight into the pathomechanism of the disease and to identify novel therapeutic targets, we analyzed neuroinflammation in two mouse models reproducing a mild, nondemyelinating, and a more severe, demyelinating, variant of MLD, respectively. Microgliosis and upregulation of cytokine/chemokine levels were clearly more pronounced in the demyelinating model. The analysis of the temporal cytokine/chemokine profiles revealed that the onset of demyelination is preceded by a sustained elevation of the macrophage inflammatory protein (MIP)-1α followed by an upregulation of MIP-1ß, monocyte chemotactic protein (MCP)-1, and several interleukins. The tumor necrosis factor (TNF)-α remains unchanged. Treatment of the demyelinating mouse model with the nonsteroidal anti-inflammatory drug simvastatin reduced neuroinflammation, improved the swimming performance and ataxic gait, and retarded demyelination of the spinal cord. Our data suggest that neuroinflammation is causative for demyelination in MLD mice and that anti-inflammatory treatment might be a novel therapeutic option to improve the CNS function of MLD patients.

Arylsulfatase A Overexpressing Human iPSC-derived Neural Cells Reduce CNS Sulfatide Storage in a Mouse Model of Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder resulting from a functional deficiency of arylsulfatase A (ARSA), an enzyme that catalyzes desulfation of 3-O-sulfogalactosylceramide (sulfatide). Lack of active ARSA leads to the accumulation of sulfatide in oligodendrocytes, Schwann cells and some neurons and triggers progressive demyelination, the neuropathological hallmark of MLD. Several therapeutic approaches have been explored, including enzyme replacement, autologous hematopoietic stem cell-based gene therapy, intracerebral gene therapy or cell-based gene delivery into the central nervous system (CNS). However, long-term treatment of the blood-brain-barrier protected CNS remains challenging. Here we used MLD patient-derived induced pluripotent stem cells (iPSCs) to generate long-term self-renewing neuroepithelial stem cells and astroglial progenitors for cell-based ARSA replacement. Following transplantation of ARSA-overexpressing precursors into ARSA-deficient mice we observed a significant reduction of sulfatide storage up to a distance of 300 µm from grafted cells. Our data indicate that neural precursors generated via reprogramming from MLD patients can be engineered to ameliorate sulfatide accumulation and may thus serve as autologous cell-based vehicle for continuous ARSA supply in MLD-affected brain tissue.

Comparison of five peptide vectors for improved brain delivery of the lysosomal enzyme arylsulfatase A.

Enzyme replacement therapy (ERT) is a treatment option for lysosomal storage disorders (LSDs) caused by deficiencies of soluble lysosomal enzymes. ERT depends on receptor-mediated transport of intravenously injected recombinant enzyme to lysosomes of patient cells. The blood-brain barrier (BBB) prevents efficient transfer of therapeutic polypeptides from the blood to the brain parenchyma and thus hinders effective treatment of LSDs with CNS involvement. We compared the potential of five brain-targeting peptides to promote brain delivery of the lysosomal enzyme arylsulfatase A (ASA). Fusion proteins between ASA and the protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep peptide (Ang-2), and the receptor-binding domains of human apolipoprotein B (ApoB) and ApoE (two versions, ApoE-I and ApoE-II) were generated. All ASA fusion proteins were enzymatically active and targeted to lysosomes when added to cultured cells. In contrast to wild-type ASA, which is taken up by mannose-6-phosphate receptors, all chimeric proteins were additionally endocytosed via mannose-6-phosphate-independent routes. For ASA-Ang-2, ASA-ApoE-I, and ASA-ApoE-II, uptake was partially due to the low-density lipoprotein receptor-related protein 1. Transendothelial transfer in a BBB cell culture model was elevated for ASA-ApoB, ASA-ApoE-I, and ASA-ApoE-II. Brain delivery was, however, increased only for ASA-ApoE-II. ApoE-II was also superior to wild-type ASA in reducing lysosomal storage in the CNS of ASA-knock-out mice treated by ERT. Therefore, the ApoE-derived peptide appears useful to treat metachromatic leukodystrophy and possibly other neurological disorders more efficiently.