Search

T Andrew Burrow, Sonya Barnes, Gregory A GrabowskiThe Division of Human Genetics, Cincinnati Children's Hospital Medical Center, and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USAAbstract: Gaucher disease is a phenotypically heterogeneous autosomal recessively inherited lysosomal storage disease, resulting from deficient activity of the enzyme glucocerebrosidase (GCase, acid ß-glucosidase) due to mutations in GBA1. Gaucher disease is the prototype for which disease-specific pharmacological therapy was successfully employed. The objective of this review is to provide a comprehensive review and critical examination of the prevalence, pathophysiology, natural history, and management of Gaucher disease.Keywords: lysosomal storage disease, pathophysiology, treatment, inborn errors of metabolism

OBJECTIVE: Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of [alpha]-synuclein (SNCA). A loss in lysosomal acid-[beta]-glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk. METHODS: We analyzed the effects of wild-type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols. RESULTS: We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293-SNCA cells increased GCase activity, as expected (ie, to 167% in MES-SNCA, 128% in PC12-SNCA, and 233% in HEK293-SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293-SNCA cells high GCase activity was associated with SNCA reduction by <=32% (p = 0.009). Inhibition of cellular GCase activity (to 8-20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA-induced SNCA accumulation could be pharmacologically reversed in D409V-expressing PC12-SNCA cells by rapamycin, an autophagy-inducer (<=40%; 10[mu]M; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, >=20%), we recorded an age-dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p = 0.019). In young Gaucher disease mice (V394Lgba+/+//prosaposin[ps]-null//ps-transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, <=10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week-old cathepsin D-deficient mice. INTERPRETATION: Our results demonstrate that GBA mutants promote SNCA accumulation in a dose- and time-dependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders. ANN NEUROL 2011; [ABSTRACT FROM AUTHOR]

Gaucher disease (GD) is caused by biallelic GBA1/Gba1 mutations that encode defective glucocerebrosidase (GCase). Progranulin (PGRN, encoded by GRN/Grn) is a modifier of GCase, but the interplay between PGRN and GCase, specifically GBA1/Gba1 mutations, contributing to GD severity is unclear. Mouse models were developed with various dosages of Gba1 D409V mutation against the PGRN deficiency (Grn-/-) [Grn-/-;Gba1D409V/WT (PG9Vwt), Grn-/-;Gba1D409V/D409V (PG9V), Grn-/-;Gba1D409V/Null (PG9VN)]. Disease progression in those mouse models was characterized by biochemical, pathological, transcriptomic, and neurobehavioral analyses. Compared to PG9Vwt, Grn-/-;Gba1WT/Null and Grn-/- mice that had a higher level of GCase activity and undetectable pathologies, homozygous or hemizygous D409V in PG9V or PG9VN, respectively, resulted in profound inflammation and neurodegeneration. PG9VN mice exhibited much earlier onset, shorter life span, tissue fibrosis, and more severe phenotypes than PG9V mice. Glycosphingolipid accumulation, inflammatory responses, lysosomal-autophagy dysfunction, microgliosis, retinal gliosis, as well as α-Synuclein increases were much more pronounced in PG9VN mice. Neurodegeneration in PG9VN was characterized by activated microglial phagocytosis of impaired neurons and programmed cell death due to necrosis and, possibly, pyroptosis. Brain transcriptomic analyses revealed the intrinsic relationship between D409V dosage, and the degree of altered gene expression related to lysosome dysfunction, microgliosis, and neurodegeneration in GD, suggesting the disease severity is dependent on a GCase activity threshold related to Gba1 D409V dosage and loss of PGRN. These findings contribute to a deeper understanding of GD pathogenesis by elucidating additional underlying mechanisms of interplay between PGRN and Gba1 mutation dosage in modulating GCase function and disease severity in GD and GBA1-associated neurodegenerative diseases., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mutations in GBA1 , encoding glucocerebrosidase (GCase), cause Gaucher disease (GD) and are also genetic risks in developing Parkinson's disease (PD). Currently, the approved therapies are only effective for directly treating visceral symptoms, but not for primary neuronopathic involvement in GD (nGD). Progranulin (PGRN), encoded by GRN , is a novel modifier of GCase, but the impact of PGRN in GBA1 mutation-associated pathologies in vivo remains unknown. Herein, Grn -/- mice crossed into Gba 9v/9v mice, a Gba1 mutant line homozygous for the Gba1 D409V mutation, generating Grn -/- Gba 9v/9v (PG9V) mice. PG9V mice exhibited neurobehavioral deficits, early onset, and more severe GD phenotypes compared to Grn -/- and Gba 9v/9v mice. Moreover, PG9V mice also displayed PD-like phenotype. Mechanistic analysis revealed that PGRN deficiency caused severe neuroinflammation with microgliosis and astrogliosis, along with impaired autophagy associated with the Gba1 mutation. A PGRN-derived peptide, termed ND7, ameliorated the disease phenotype in GD patient fibroblasts ex vivo . Unexpectedly, ND7 penetrated the blood-brain barrier (BBB) and effectively ameliorated the nGD manifestations and PD pathology in Gba 9v/null and PG9V mice. Collectively, this study not only provides the first line of in vivo but also ex vivo evidence demonstrating the crucial role of PGRN in GBA1/Gba1 mutation-related pathologies, as well as a clinically relevant mouse model for mechanistic and potential therapeutics studies for nGD and PD. Importantly, a BBB penetrant PGRN-derived biologic was developed that may provide treatment for rare lysosomal storage diseases and common neurodegenerative disorders, particularly nGD and PD.

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1 / Gba1 that encodes the enzyme termed as lysosomal acid β-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model ( Gba1 9V/- ) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4 + T and CD8 + T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba1 9V/- mice showed elevated production of CXCL9. Purified CD4 + T cells and the CD8 + T cells from Gba1 9V/- mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba1 9V/- T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba1 9V/- mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.

Substrate reduction therapy (SRT) in clinic adequately manages the visceral manifestations in Gaucher disease (GD) but has no direct effect on brain disease. To understand the molecular basis of SRT in GD treatment, we evaluated the efficacy and underlying mechanism of SRT in an immortalized neuronal cell line derived from a Gba knockout ( Gba -/- ) mouse model. Gba -/- neurons accumulated substrates, glucosylceramide, and glucosylsphingosine. Reduced cell proliferation was associated with altered lysosomes and autophagy, decreased mitochondrial function, and activation of the mTORC1 pathway. Treatment of the Gba -/- neurons with venglustat analogue GZ452, a central nervous system-accessible SRT, normalized glucosylceramide levels in these neurons and their isolated mitochondria. Enlarged lysosomes were reduced in the treated Gba -/- neurons, accompanied by decreased autophagic vacuoles. GZ452 treatment improved mitochondrial membrane potential and oxygen consumption rate. Furthermore, GZ452 diminished hyperactivity of selected proteins in the mTORC1 pathway and improved cell proliferation of Gba -/- neurons. These findings reinforce the detrimental effects of substrate accumulation on mitochondria, autophagy, and mTOR in neurons. A novel rescuing mechanism of SRT was revealed on the function of mitochondrial and autophagy-lysosomal pathways in GD. These results point to mitochondria and the mTORC1 complex as potential therapeutic targets for treatment of GD.

Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest.

Background: Enzyme replacement therapy (ERT) can positively affect the visceral manifestations of lysosomal storage diseases (LSDs). However, the exclusion of the intravenous ERT agents from the central nervous system (CNS) prevents direct therapeutic effects., Methods: Using a neuronopathic Gaucher disease (nGD) mouse model, CNS-ERT was created using a systemic, non-invasive, and CNS-selective delivery system based on nanovesicles of saposin C (SapC) and dioleoylphosphatidylserine (DOPS) to deliver to CNS cells and tissues the corrective, functional acid β-glucosidase (GCase)., Findings: Compared to free GCase, human GCase formulated with SapC-DOPS nanovesicles (SapC-DOPS-GCase) was more stable in serum, taken up into cells, mostly by a mannose receptor-independent pathway, and resulted in higher activity in GCase-deficient cells. In contrast to free GCase, SapC-DOPS-GCase nanovesicles penetrated through the blood-brain barrier into the CNS. The CNS targeting was mediated by surface phosphatidylserine (PS) of blood vessel and brain cells. Increased GCase activity and reduced GCase substrate levels were found in the CNS of SapC-DOPS-GCase-treated nGD mice, which showed profound improvement in brain inflammation and neurological phenotypes., Interpretation: This first-in-class CNS-ERT approach provides considerable promise of therapeutic benefits for neurodegenerative diseases., Funding: This study was supported by the National Institutes of Health grants R21NS 095047 to XQ and YS, R01NS 086134 and UH2NS092981 in part to YS; Cincinnati Children's Hospital Medical Center Research Innovation/Pilot award to YS and XQ; Gardner Neuroscience Institute/Neurobiology Research Center Pilot award to XQ and YS, Hematology-Oncology Programmatic Support from University of Cincinnati and New Drug State Key Project grant 009ZX09102-205 to XQ., Competing Interests: Declaration of Competing Interest The authors claim a provisional patent application has been filed on March 10, 2020. Consistent with current Cincinnati Children's Hospital Medical Center policies, the development and commercialization of this technology has been licensed to Bexion Pharmaceuticals, LLC, in which X. Qi, holds (< 5%) equity interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Gaucher disease (GD) is caused by GBA1 mutations leading to functional deficiency of acid-β-glucosidase (GCase). No effective treatment is available for neuronopathic GD (nGD). A subclass of neural stem and precursor cells (NPCs) expresses VLA4 (integrin α4β1, very late antigen-4) that facilitates NPC entry into the brain following intravenous (IV) infusion. Here, the therapeutic potential of IV VLA4+NPCs was assessed for nGD using wild-type mouse green fluorescent protein (GFP)-positive multipotent induced pluripotent stem cell (iPSC)-derived VLA4+NPCs. VLA4+NPCs successfully engrafted in the nGD (4L;C*) mouse brain. GFP-positive cells differentiated into neurons, astrocytes and oligodendrocytes in the brainstem, midbrain and thalamus of the transplanted mice and significantly improved sensorimotor function and prolonged life span compared to vehicle-treated 4L;C* mice. VLA4+NPC transplantation significantly decreased levels of CD68 and glial fibrillary acidic protein, as well as TNFα mRNA levels in the brain, indicating reduced neuroinflammation. Furthermore, decreased Fluoro-Jade C and NeuroSilver staining suggested inhibition of neurodegeneration. VLA4+NPC-engrafted 4L;C* midbrains showed 35% increased GCase activity, reduced substrate [glucosylceramide (GC, -34%) and glucosylsphingosine (GS, -11%)] levels and improved mitochondrial oxygen consumption rates in comparison to vehicle-4L;C* mice. VLA4+NPC engraftment in 4L;C* brain also led to enhanced expression of neurotrophic factors that have roles in neuronal survival and the promotion of neurogenesis. This study provides evidence that iPSC-derived NPC transplantation has efficacy in an nGD mouse model and provides proof of concept for autologous NPC therapy in nGD., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Gaucher disease is caused by mutations in GBA1 encoding acid β-glucosidase (GCase). Saposin C enhances GCase activity and protects GCase from intracellular proteolysis. Structure simulations indicated that the mutant GCases, N370S (0 S), V394L (4L) and D409V(9V)/H(9H), had altered function. To investigate the in vivo function of Gba1 mutants, mouse models were generated by backcrossing the above homozygous mutant GCase mice into Saposin C deficient (C*) mice. Without saposin C, the mutant GCase activities in the resultant mouse tissues were reduced by ~50% compared with those in the presence of Saposin C. In contrast to 9H and 4L mice that have normal histology and life span, the 9H;C* and 4L;C* mice had shorter life spans. 9H;C* mice developed significant visceral glucosylceramide (GC) and glucosylsphingosine (GS) accumulation (GC»GS) and storage macrophages, but lesser GC in the brain, compared to 4L;C* mice that presents with a severe neuronopathic phenotype and accumulated GC and GS primarily in the brain. Unlike 9V mice that developed normally for over a year, 9V;C* pups had a lethal skin defect as did 0S;C* mice resembled that of 0S mice. These variant Gaucher disease mouse models presented a mutation specific phenotype and underscored the in vivo role of Saposin C in the modulation of Gaucher disease.

The complement system is well appreciated for its role as an important effector of innate immunity that is activated by the classical, lectin or alternative pathway. C5a is one important mediator of the system that is generated in response to canonical and non-canonical C5 cleavage by circulating or cell-derived proteases. In addition to its function as a chemoattractant for neutrophils and other myeloid effectors, C5a and its sister molecule C3a have concerted roles in cell homeostasis and surveillance. Through activation of their cognate G protein coupled receptors, C3a and C5a regulate multiple intracellular pathways within the mitochondria and the lysosomal compartments that harbor multiple enzymes critical for protein, carbohydrate and lipid metabolism. Genetic mutations of such lysosomal enzymes or their receptors can result in the compartmental accumulation of specific classes of substrates in this organelle summarized as lysosomal storage diseases (LSD). A frequent LSD is Gaucher disease (GD), caused by autosomal recessively inherited mutations in GBA1, resulting in functional defects of the encoded enzyme, acid β-glucosidase (glucocerebrosidase, GCase). Such mutations promote excessive accumulation of β-glucosylceramide (GC or GL1) in innate and adaptive immune cells frequently associated with chronic inflammation. Recently, we uncovered an unexpected link between the C5a and C5a receptor 1 (C5aR1) axis and the accumulation of GL1 in experimental and clinical GD. Here, we will review the pathways of complement activation in GD, its role as a mediator of the inflammatory response, and its impact on glucosphingolipid metabolism. Further, we will discuss the potential role of the C5a/C5aR1 axis in GL1-specific autoantibody formation and as a novel therapeutic target in GD., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To explore the role of LAL (lysosomal acid lipase) in macrophage cholesterol efflux and whole-body reverse cholesterol transport., Approach and Results: Immortalized peritoneal macrophages from lal -/- mice showed reduced expression of ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1), reduced production of the regulatory oxysterol 27-hydroxycholesterol, and impaired suppression of cholesterol synthesis on exposure to acetylated low-density lipoprotein when compared with lal +/+ macrophages. LAL-deficient mice also showed reduced hepatic ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8) expression compared with lal +/+ mice. LAL-deficient macrophages loaded with [ 3 H]-cholesteryl oleate-labeled acetylated low-density lipoprotein showed impaired efflux of released [ 3 H]-cholesterol to apoA-I (apolipoprotein A-I), with normalization of [ 3 H]-cholesteryl ester levels and partial correction of ABCA1 expression and cholesterol efflux to apoA-I when treated with exogenous rhLAL (recombinant human LAL protein). LAL-deficient mice injected intraperitoneally with lal -/- macrophages cholesterol loaded and labeled in the same way exhibited only 1.55±0.35% total injected [ 3 H]-cholesterol counts appearing in the feces for 48 h (n=30), compared with 5.38±0.92% in lal +/+ mice injected with labeled lal +/+ macrophages (n=27), P <0.001. To mimic the therapeutic condition of delivery of supplemental LAL in vivo, injection of labeled lal -/- macrophages into lal +/+ mice resulted in a significant increase in reverse cholesterol transport (2.60±0.46% of 3 H-cholesterol counts in feces at 48 hours [n=19]; P <0.001 when compared with injection into lal -/- mice)., Conclusions: These results indicate a critical role for LAL in promoting both macrophage and whole-body reverse cholesterol transport and the ability of supplemental LAL to be taken up and correct reverse cholesterol transport in vivo., (© 2018 American Heart Association, Inc.)

We recently reported that progranulin (PGRN) is a novel regulator of glucocerebrosidase and its deficiency associates with Gaucher Diseases (GD) (Jian et al., 2016a; Jian et al., 2018). To isolate the relevant downstream molecules, we performed a whole genome microarray and mass spectrometry analysis, which led to the isolation of Chitinase-3-like-1 (CHI3L1) as one of the up-regulated genes in PGRN null mice. Elevated levels of CHI3L1 were confirmed by immunoblotting and immunohistochemistry. In contrast, treatment with recombinant Pcgin, a derivative of PGRN, as well as imigluerase, significantly reduced the expressions of CHI3L1 in both PGRN null GD model and the fibroblasts from GD patients. Serum levels of CHIT1, a clinical biomarker for GD, were significantly higher in GD patients than healthy controls (51.16±2.824ng/ml vs 35.07±2.099ng/ml, p<0.001). Similar to CHIT1, serum CHI3L1 was also significantly increased in GD patients compared with healthy controls (1736±152.1pg/ml vs 684.7±68.20pg/ml, p<0.001). Whereas the PGRN level is significantly reduced in GD patients as compared to the healthy control (91.56±3.986ng/ml vs 150.6±4.501, p<0.001). Collectively, these results indicate that CHI3L1 may be a previously unrecognized biomarker for diagnosing GD and for evaluating the therapeutic effects of new GD drug(s)., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

To better understand regional brain glycosphingolipid (GSL) accumulation in Gaucher disease (GD) and its relationship to neuropathology, a feasibility study using mass spectrometry and immunohistochemistry was conducted using brains derived from a GD mouse model (4L/PS/NA) homozygous for a mutant GCase (V394L [4L]) and expressing a prosaposin hypomorphic (PS-NA) transgene. Whole brains from GD and control animals were collected using one hemisphere for MALDI FTICR IMS analysis and the other for quantitation by LC-ESI-MS/MS. MALDI IMS detected several HexCers across the brains. Comparison with the brain hematoxylin and eosin (H&E) revealed differential signal distributions in the midbrain, brain stem, and CB of the GD brain versus the control. Quantitation of serial brain sections with LC-ESI-MS/MS supported the imaging results, finding the overall HexCer levels in the 4L/PS-NA brains to be four times higher than the control. LC-ESI-MS/MS also confirmed that the elevated hexosyl isomers were glucosylceramides rather than galactosylceramides. MALDI imaging also detected differential analyte distributions of lactosylceramide species and gangliosides in the 4L/PS-NA brain, which was validated by LC-ESI-MS/MS. Immunohistochemistry revealed regional inflammation, altered autophagy, and defective protein degradation correlating with regions of GSL accumulation, suggesting that specific GSLs may have distinct neuropathological effects.

Neuroinflammation is an intrinsic component of the neurodegeneration of inborn errors of neurometabolic diseases. Diseases resulting in lysosomal, peroxisomal, and autophagocytic disruption lead to neuroinflammation by different mechanisms relating to accumulated substrates and/or downstream deficiencies that cause presymptomatic microglial activation, axonal instabilities and/or direct hyperactivation of intrinsic inflammatory mechanisms. Only in selected diseases is the blood-brain barrier (BBB) breached, thereby permitting peripheral adaptive immune mechanisms to amplify intrinsic immune reactions in the central nervous system. These result in evoking several different programmed cell death pathways, including apoptosis, necroptosis, and pyroptosis, with the subsequent neuronal death of specific types and in selected regions of the brain or spinal cord. In addition to correction of the primary genetic or metabolic defects, successful therapeutic interventions require greater molecular understanding of the specific neuroinflammatory components of neurometabolic diseases to permit identification of significant targets for intervention., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

The advent of the first effective specific therapy for a lysosomal storage disease (LSDs), Gaucher disease type 1, by Roscoe O. Brady was foundational for development of additional treatments for this group of rare diseases. The past 26years, since the approval of enzyme therapy for Gaucher disease type 1, have witnessed a burgeoning understanding of LSDs at genetic, molecular, biochemical, cell biologic, and clinical levels. Simultaneously, this expansion of knowledge has exposed our incomplete understanding of the individual pathophysiologies of LSDs as well as difficult challenges for improvement in therapy and therapeutic outcomes for afflicted individuals. Here, 10 such challenges/problems representing major impediments, which need to be overcome, to move forward toward the goals of more effective and complete therapies for these devastating diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In Gaucher disease (GD), deficiency of lysosomal acid β-glucosidase results in a broad phenotypic spectrum that is classified into three types based on the absence (type 1 [GD1]) or presence and severity of primary central nervous system involvement (type 2 [GD2], the fulminant neuronopathic form, and type 3 [GD3], the milder chronic neuronopathic form). Enzyme replacement therapy (ERT) with imiglucerase ameliorates and prevents hematological and visceral manifestations in GD1, but data in GD3 are limited to small, single-center series. The effects of imiglucerase ERT on hematological, visceral and growth outcomes (note: ERT is not expected to directly impact neurologic outcomes) were evaluated during the first 5years of treatment in 253 children and adolescents (<18years of age) with GD3 enrolled in the International Collaborative Gaucher Group (ICGG) Gaucher Registry. The vast majority of GBA mutations in this diverse global population consisted of only 2 mutations: L444P (77%) and D409H (7%). At baseline, GD3 patients exhibited early onset of severe hematological and visceral disease and growth failure. During the first year of imiglucerase treatment, hemoglobin levels and platelet counts increased and liver and spleen volumes decreased, leading to marked decreases in the number of patients with moderate or severe anemia, thrombocytopenia, and hepatosplenomegaly. These improvements were maintained through Year 5. There was also acceleration in linear growth as evidenced by increasing height Z-scores. Despite devastating disease at baseline, the probability of surviving for at least 5years after starting imiglucerase was 92%. In this large, multinational cohort of pediatric GD3 patients, imiglucerase ERT provided a life-saving and life-prolonging benefit for patients with GD3, suggesting that, with proper treatment, many such severely affected patients can lead productive lives and contribute to society., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuronopathic Gaucher disease (nGD) manifests as severe neurological symptoms in patients with no effective treatment available. Ryanodine receptors (Ryrs) are a family of calcium release channels on intracellular stores. The goal of this study is to determine if Ryrs are potential targets for nGD treatment. A nGD cell model (CBE-N2a) was created by inhibiting acid β-glucosidase (GCase) in N2a cells with conduritol B epoxide (CBE). Enhanced cytosolic calcium in CBE-N2a cells was blocked by either ryanodine or dantrolene, antagonists of Ryrs and by Genz-161, a glucosylceramide synthase inhibitor, suggesting substrate-mediated ER-calcium efflux occurs through ryanodine receptors. In the brain of a nGD (4L;C*) mouse model, expression of Ryrs was normal at 13 days of age, but significantly decreased below the wild type level in end-stage 4L;C* brains at 40 days. Treatment with dantrolene in 4L;C* mice starting at postnatal day 5 delayed neurological pathology and prolonged survival. Compared to untreated 4L;C* mice, dantrolene treatment significantly improved gait, reduced LC3-II levels, improved mitochondrial ATP production and reduced inflammation in the brain. Dantrolene treatment partially normalized Ryr expression and its potential regulators, CAMK IV and calmodulin. Furthermore, dantrolene treatment increased residual mutant GCase activity in 4L;C* brains. These data demonstrate that modulating Ryrs has neuroprotective effects in nGD through mechanisms that protect the mitochondria, autophagy, Ryr expression and enhance GCase activity. This study suggests that calcium signalling stabilization, e.g. with dantrolene, could be a potential disease modifying therapy for nGD., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

To study the neuronal deficits in neuronopathic Gaucher Disease (nGD), the chronological behavioral profiles and the age of onset of brain abnormalities were characterized in a chronic nGD mouse model (9V/null). Progressive accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) in the brain of 9V/null mice were observed at as early as 6 and 3 months of age for GC and GS, respectively. Abnormal accumulation of α-synuclein was present in the 9V/null brain as detected by immunofluorescence and Western blot analysis. In a repeated open-field test, the 9V/null mice (9 months and older) displayed significantly less environmental habituation and spent more time exploring the open-field than age-matched WT group, indicating the onset of short-term spatial memory deficits. In the marble burying test, the 9V/null group had a shorter latency to initiate burying activity at 3 months of age, whereas the latency increased significantly at ≥12 months of age; 9V/null females buried significantly more marbles to completion than the WT group, suggesting an abnormal response to the instinctive behavior and an abnormal activity in non-associative anxiety-like behavior. In the conditional fear test, only the 9V/null males exhibited a significant decrease in response to contextual fear, but both genders showed less response to auditory-cued fear compared to age- and gender-matched WT at 12 months of age. These results indicate hippocampus-related emotional memory defects. Abnormal gait emerged in 9V/null mice with wider front-paw and hind-paw widths, as well as longer stride in a gender-dependent manner with different ages of onset. Significantly higher liver- and spleen-to-body weight ratios were detected in 9V/null mice with different ages of onsets. These data provide temporal evaluation of neurobehavioral dysfunctions and brain pathology in 9V/null mice that can be used for experimental designs to evaluate novel therapies for nGD., Competing Interests: The authors have declared that no competing interests exist.

Background: Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of β-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD., Methods: Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed., Findings: Serum PGRN levels were significantly lower in GD patients (96.65±53.45ng/ml) than those in healthy controls of the general population (164.99±43.16ng/ml, p<0.0001) and of Ashkenazi Jews (150.64±33.99ng/ml, p<0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, "aged" and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients., Interpretation: Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCase's lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD., (Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.)

Gaucher disease (GD) is caused by a deficiency of glucocerebrosidase and the consequent lysosomal accumulation of unmetabolized glycolipid substrates. Enzyme-replacement therapy adequately manages the visceral manifestations of nonneuronopathic type-1 Gaucher patients, but not the brain disease in neuronopathic types 2 and 3 GD. Substrate reduction therapy through inhibition of glucosylceramide synthase (GCS) has also been shown to effectively treat the visceral disease. Here, we evaluated the efficacy of a novel small molecule inhibitor of GCS with central nervous system (CNS) access (Genz-682452) to treat the brain disease. Treatment of the conduritol β epoxide-induced mouse model of neuronopathic GD with Genz-682452 reduced the accumulation of liver and brain glycolipids (>70% and >20% respectively), extent of gliosis, and severity of ataxia. In the genetic 4L;C* mouse model, Genz-682452 reduced the levels of substrate in the brain by >40%, the extent of gliosis, and paresis. Importantly, Genz-682452-treated 4L;C* mice also exhibited an ~30% increase in lifespan. Together, these data indicate that an orally available antagonist of GCS that has CNS access is effective at attenuating several of the neuropathologic and behavioral manifestations associated with mouse models of neuronopathic GD. Therefore, Genz-682452 holds promise as a potential therapeutic approach for patients with type-3 GD.

Defective lysosomal acid β-glucosidase (GCase) in Gaucher disease causes accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) that distress cellular functions. To study novel pathological mechanisms in neuronopathic Gaucher disease (nGD), a mouse model (4L;C*), an analogue to subacute human nGD, was investigated for global profiles of differentially expressed brain mRNAs (DEGs) and miRNAs (DEmiRs). 4L;C* mice displayed accumulation of GC and GS, activated microglial cells, reduced number of neurons and aberrant mitochondrial function in the brain followed by deterioration in motor function. DEGs and DEmiRs were characterized from sequencing of mRNA and miRNA from cerebral cortex, brain stem, midbrain and cerebellum of 4L;C* mice. Gene ontology enrichment and pathway analysis showed preferential mitochondrial dysfunction in midbrain and uniform inflammatory response and identified novel pathways, axonal guidance signaling, synaptic transmission, eIF2 and mammalian target of rapamycin (mTOR) signaling potentially involved in nGD. Similar analyses were performed with mice treated with isofagomine (IFG), a pharmacologic chaperone for GCase. IFG treatment did not alter the GS and GC accumulation significantly but attenuated the progression of the disease and altered numerous DEmiRs and target DEGs to their respective normal levels in inflammation, mitochondrial function and axonal guidance pathways, suggesting its regulation on miRNA and the associated mRNA that underlie the neurodegeneration in nGD. These analyses demonstrate that the neurodegenerative phenotype in 4L;C* mice was associated with dysregulation of brain mRNAs and miRNAs in axonal guidance, synaptic plasticity, mitochondria function, eIF2 and mTOR signaling and inflammation and provides new insights for the nGD pathological mechanism., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Gaucher's disease is caused by defects in acid β-glucosidase 1 (GBA1) and has been also proposed as an inflammatory disease. GBA1 cleaves glucosylceramide to form ceramide, an established bioactive lipid, and defects in GBA1 lead to aberrant accumulation in glucosylceramide and insufficient formation of ceramide. We investigated if the pro-inflammatory kinase p38 is activated in Gaucher's disease, since ceramide has been proposed to suppress p38 activation. Three Gaucher's disease mouse models were employed, and p38 was found to be activated in lung and liver tissues of all Gaucher's disease mice. Most interestingly, neuronopathic Gaucher's disease type mice, but not non-neuronopathic ones, displayed significant activation of p38 and up-regulation of p38-inducible proinflammatory cytokines in brain tissues. In addition, all type of Gaucher's disease mice also showed increases in serum IL-6. As cellular signalling is believed to represent an in vivo inflammatory phenotype in Gaucher's disease, activation of p38 and possibly its-associated formation of proinflammatory cytokines were assessed in fibroblasts established from neuronopathic Gaucher's disease mice. In mouse Gaucher's disease cells, p38 activation and IL-6 formation by TNF-α treatment were enhanced as compared to those of wild type. Furthermore, human fibroblasts from Gaucher's disease patients also displayed increases in p38 activation and IL-6 formation as comparison to healthy counterpart. These results raise the potential that proinflammatory responses such as p38 activation and IL-6 formation are augmented in Gaucher's disease.

Study of the natural history of Gaucher disease has revealed marked phenotypic variation. Correlations to genotypes could provide insight into individual susceptibility to varying disease severity, which may impact whole-life medical care, reproductive decisions, and therapeutic choices for affected families. Importantly, pre-symptomatic or prospective interventions or the use of therapies with significant risk require accurate risk-benefit analyses based on the prognosis for individual patients. The body of international data held within the International Collaborative Gaucher Group (ICGG) Gaucher Registry provides an unprecedented opportunity to characterize the phenotypes of Gaucher disease types 1 and 3 and to appreciate demographic and ethnic factors that may influence phenotypes. The diversity of GBA gene mutations from patients with Gaucher disease represented in the ICGG Gaucher Registry database and in the literature provides the basis for initial genotype/phenotype correlations, the outcomes of which are summarized here., (© 2015 Wiley Periodicals, Inc.)

Gaucher disease (GD) is caused by insufficient activity of acid β-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD, induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs, NPCs, and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition, GD2 neurons showed increased α-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons, but intriguingly, those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes, sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings, providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge, this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease.

Saposin B (Sap B) is an essential activator protein for arylsulfatase A in the hydrolysis of sulfatide, a lipid component of myelin. To study Sap B's role in hearing and balance, a Sap B-deficient (B(-/-)) mouse was evaluated. At both light and electron microscopy (EM) levels, inclusion body accumulation was seen in satellite cells surrounding spiral ganglion (SG) neurons from postnatal month 1 onward, progressing into large vacuoles preceding satellite cell degeneration, and followed by SG degeneration. EM also revealed reduced or absent myelin sheaths in SG neurons from postnatal month 8 onwards. Hearing loss was initially seen at postnatal month 6 and progressed thereafter for frequency-specific stimuli, whereas click responses became abnormal from postnatal month 13 onward. The progressive hearing loss correlated with the accumulation of inclusion bodies in the satellite cells and their subsequent degeneration. Outer hair cell numbers and efferent function measures (distortion product otoacoustic emissions and contralateral suppression) were normal in the B(-/-) mice throughout this period. Alcian blue staining of SGs demonstrated that these inclusion bodies corresponded to sulfatide accumulation. In contrast, changes in the vestibular system were much milder, but caused severe physiologic deficits. These results demonstrate that loss of Sap B function leads to progressive sulfatide accumulation in satellite cells surrounding the SG neurons, leading to satellite cell degeneration and subsequent SG degeneration with a resultant loss of hearing. Relative sparing of the efferent auditory and vestibular neurons suggests that alternate glycosphingolipid metabolic pathways predominate in these other systems., (Copyright © 2015 the authors 0270-6474/15/353263-13$15.00/0.)

Background: Oliver-McFarlane syndrome is characterised by trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Laurence-Moon syndrome presents similarly, though with progressive spinocerebellar ataxia and spastic paraplegia and without trichomegaly. Both recessively inherited disorders have no known genetic cause., Methods: Whole-exome sequencing was performed to identify the genetic causes of these disorders. Mutations were functionally validated in zebrafish pnpla6 morphants. Embryonic expression was evaluated via in situ hybridisation in human embryonic sections. Human neurohistopathology was performed to characterise cerebellar degeneration. Enzymatic activities were measured in patient-derived fibroblast cell lines., Results: Eight mutations in six families with Oliver-McFarlane or Laurence-Moon syndrome were identified in the PNPLA6 gene, which encodes neuropathy target esterase (NTE). PNPLA6 expression was found in the developing human eye, pituitary and brain. In zebrafish, the pnpla6 curly-tailed morphant phenotype was fully rescued by wild-type human PNPLA6 mRNA and not by mutation-harbouring mRNAs. NTE enzymatic activity was significantly reduced in fibroblast cells derived from individuals with Oliver-McFarlane syndrome. Intriguingly, adult brain histology from a patient with highly overlapping features of Oliver-McFarlane and Laurence-Moon syndromes revealed extensive cerebellar degeneration and atrophy., Conclusions: Previously, PNPLA6 mutations have been associated with spastic paraplegia type 39, Gordon-Holmes syndrome and Boucher-Neuhäuser syndromes. Discovery of these additional PNPLA6-opathies further elucidates a spectrum of neurodevelopmental and neurodegenerative disorders associated with NTE impairment and suggests a unifying mechanism with diagnostic and prognostic importance., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

A Caucasian male with Gaucher disease type 3, treated with continuous enzyme therapy (ET) for 11 years, experienced progressive mesenteric and retroperitoneal lymphadenopathy, lung disease, and neurological involvement leading to death at an age of 12.5 years. Autopsy showed significant pathology of the brain, lymph nodes, and lungs. Liver and spleen glucosylceramide (GluCer) and glucosylsphingosine (GluS) levels were nearly normal and storage cells were cleared. Clusters of macrophages and very elevated GluCer and GluS levels were in the lungs, and brain parenchymal and perivascular regions. Compared to normal brain GluCer (GC 18:0), GluCer species with long fatty acid acyl chains were increased in the patient's brain. This profile was similar to that in the patient's lungs, suggesting that these lipids were present in brain perivascular macrophages. In the patient's brain, generalized astrogliosis, and enhanced LC3, ubiquitin, and Tau signals were identified in the regions surrounding macrophage clusters, indicating proinflammation, altered autophagy, and neurodegeneration. These findings highlight the altered phenotypes resulting from increased longevity due to ET, as well as those in poorly accessible compartments of brain and lung, which manifested progressive disease involvement despite ET., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Gaucher disease is a lysosomal storage disease caused by defective activity of acid β-glucosidase (GCase), which leads to the accumulation of its major substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in many cells. To modulate cellular substrate concentration in viable mouse models of Gaucher disease (Gba1 mutants), a novel mouse model was created with enhanced glycosphingolipid biosynthesis. This was accomplished by cross-breeding Gba1 mutant mice with mice expressing a transgene (GCStg) containing the mouse glucosylceramide synthase (GCS, Ugcg) cDNA driven by the ROSA promoter, yielding GCStg/Gba1 mice. The GCStg rescued Ugcg null mice from embryonic lethality. GCStg/Gba1 mice showed 2-3 fold increases in tissue GCS activity as well as accelerated GlcCer accumulation and the appearance of lipid-laden CD68 positive macrophages in visceral organs. Although GlcCer/GlcSph concentrations were elevated in the brain, there was no neurodegenerative phenotype up to 1 yr of age conceivably due to the greater residual GCase hydrolytic activity in the brains than in the visceral tissues of 9V/null mice. These studies provide 'proof of principle' for threshold substrate flux that modifies phenotypic development in Gaucher disease and other lysosomal storage diseases.

Key Message: Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent. Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein's N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.

The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp(399) and the di-isoleucines, Ile(402) and Ile(403). A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

There are several inherited disorders that involve abnormal storage of lipids in tissues leading to severe compromise of organs. Sadly, these are often accompanied by lifelong morbidity and early mortality. Disorders such as Gaucher, Fabry, and lysosomal acid lipase deficiencies (Wolman and cholesteryl ester storage diseases) have been known for many years, and provide a difficult and frustrating set of problems for patients, their families, and their physicians. With recombinant methods of protein synthesis, it is now possible to literally replace the defective enzymes that underlie the basic pathophysiology of many such disorders. The delivery of these enzymes into the affected cells is possible because of their location in the lysosomes where the natural degradation of their lipid substrates occurs. I have asked 2 well-known investigators to join us for this Roundtable. These are professors who have been involved with the research that has made this type of therapy possible and who have participated in the clinical trials that demonstrated the value of enzyme replacement therapy. They are Dr. Robert Desnick, dean of Genetic and Genomic Medicine and professor and chairman emeritus of the Department of Genetics and Genomic Sciences at the Icahn School of Medicine at Mount Sinai in New York City, and Dr. Gregory Grabowski, professor of Microbiology, Biochemistry, and Pediatrics, at the University of Cincinnati College of Medicine. Dr. Grabowski recently retired from that school to become the chief science officer of Synageva, a company involved in producing enzymes for this type of therapy., (Copyright © 2014 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Gaucher disease, a prevalent lysosomal storage disease (LSD), is caused by insufficient activity of acid β-glucosidase (GCase) and the resultant glucosylceramide (GC)/glucosylsphingosine (GS) accumulation in visceral organs (Type 1) and the central nervous system (Types 2 and 3). Recent clinical and genetic studies implicate a pathogenic link between Gaucher and neurodegenerative diseases. The aggregation and inclusion bodies of α-synuclein with ubiquitin are present in the brains of Gaucher disease patients and mouse models. Indirect evidence of β-amyloid pathology promoting α-synuclein fibrillation supports these pathogenic proteins as a common feature in neurodegenerative diseases. Here, multiple proteins are implicated in the pathogenesis of chronic neuronopathic Gaucher disease (nGD). Immunohistochemical and biochemical analyses showed significant amounts of β-amyloid and amyloid precursor protein (APP) aggregates in the cortex, hippocampus, stratum and substantia nigra of the nGD mice. APP aggregates were in neuronal cells and colocalized with α-synuclein signals. A majority of APP co-localized with the mitochondrial markers TOM40 and Cox IV; a small portion co-localized with the autophagy proteins, P62/LC3, and the lysosomal marker, LAMP1. In cultured wild-type brain cortical neural cells, the GCase-irreversible inhibitor, conduritol B epoxide (CBE), reproduced the APP/α-synuclein aggregation and the accumulation of GC/GS. Ultrastructural studies showed numerous larger-sized and electron-dense mitochondria in nGD cerebral cortical neural cells. Significant reductions of mitochondrial adenosine triphosphate production and oxygen consumption (28-40%) were detected in nGD brains and in CBE-treated neural cells. These studies implicate defective GCase function and GC/GS accumulation as risk factors for mitochondrial dysfunction and the multi-proteinopathies (α-synuclein-, APP- and Aβ-aggregates) in nGD., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TG) and cholesteryl esters (CE) in lysosomes. Mutations of the LIPA gene lead to Wolman disease (WD) and cholesterol ester storage disease (CESD). The disease hallmarks include hepatosplenomegaly and extensive storage of CE and/or TG. The effects of intravenous investigational enzyme therapy (ET) on survival and efficacy were evaluated in Lipa knock out, lal-/- mice with advanced disease using recombinant human LAL (rhLAL). Comparative ET was conducted with lower doses (weekly, 0.8 and 3.2mg/kg) beginning at 16 weeks (study 1), and with higher dose (10mg/kg) in early (8-weeks), middle (16-weeks) and late (24-weeks) disease stages (study 2). In study 1, rhLAL extended the life span of lal-/- mice in a dose dependent manner by 52 (0.8 mg/kg) or 94 (3.2mg/kg) days. This was accompanied by partial correction of cholesterol and TG levels in spleen and liver. In study 2, the high dose resulted in a significant improvement in organ size (liver, spleen and small intestine) and tissue histology as well as significant decreases in cholesterol and TG in all three groups. In the treated livers and spleens the cholesterol and TG levels were reduced to below treatment initiation levels indicating a reversal of disease manifestations, even in advanced disease. ET diminished liver fibrosis and macrophage proliferation. These results show that LAL deficiency can be improved biochemically and histopathologically by various dosages of ET, even in advanced disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)