epHero - a tandem-fluorescent probe to track the fate of apoptotic cells during efferocytosis.

The efficient removal of apoptotic cells via efferocytosis is critical for maintaining optimal tissue function. This involves the binding and engulfment of apoptotic cells by phagocytes and the subsequent maturation of the phagosome, culminating in lysosomal fusion and cargo destruction. However, current approaches to measure efferocytosis rely on labelling apoptotic targets with fluorescent dyes, which do not sufficiently distinguish between changes to the engulfment and acidification of apoptotic material. To address this limitation, we have developed a genetically coded ratiometric probe epHero which when expressed in the cytoplasm of target cells, bypasses the need for additional labelling steps. We demonstrate that epHero is a pH-sensitive reporter for efferocytosis and can be used to simultaneously track changes to apoptotic cell uptake and acidification, both in vitro and in mice. As proof-of-principle, we modify extracellular nutrition to show how epHero can distinguish between changes to cargo engulfment and acidification. Thus, tracking efferocytosis with epHero is a simple, cost-effective improvement on conventional techniques.

Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling.

Retromer prevents the destruction of numerous receptors by recycling them from endosomes to the trans-Golgi network or plasma membrane. This enables retromer to fine-tune the activity of many signaling pathways in parallel. However, the mechanism(s) by which retromer function adapts to environmental fluctuations such as nutrient withdrawal and how this affects the fate of its cargoes remains incompletely understood. Here, we reveal that macroautophagy/autophagy inhibition by MTORC1 controls the abundance of retromer+ endosomes under nutrient-replete conditions. Autophagy activation by chemical inhibition of MTOR or nutrient withdrawal does not affect retromer assembly or its interaction with the RAB7 GAP protein TBC1D5, but rather targets these endosomes for bulk destruction following their capture by phagophores. This process appears to be distinct from amphisome formation. TBC1D5 and its ability to bind to retromer, but not its C-terminal LC3-interacting region (LIR) or nutrient-regulated dephosphorylation, is critical for retromer to be captured by autophagosomes following MTOR inhibition. Consequently, endosomal recycling of its cargoes to the plasma membrane and trans-Golgi network is impaired, leading to their lysosomal turnover. These findings demonstrate a mechanistic link connecting nutrient abundance to receptor homeostasis.Abbreviations: AMPK, 5'-AMP-activated protein kinase; APP, amyloid beta precursor protein; ATG, autophagy related; BafA, bafilomycin A1; CQ, chloroquine; DMEM, Dulbecco's minimum essential medium; DPBS, Dulbecco's phosphate-buffered saline; EBSS, Earle's balanced salt solution; FBS, fetal bovine serum; GAP, GTPase-activating protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; LANDO, LC3-associated endocytosis; LP, leupeptin and pepstatin; MTOR, mechanistic target of rapamycin kinase; MTORC1, MTOR complex 1; nutrient stress, withdrawal of amino acids and serum; PDZ, DLG4/PSD95, DLG1, and TJP1/zo-1; RPS6, ribosomal protein S6; RPS6KB1/S6K1, ribosomal protein S6 kinase B1; SLC2A1/GLUT1, solute carrier family 2 member 1; SORL1, sortillin related receptor 1; SORT1, sortillin 1; SNX, sorting nexin; TBC1D5, TBC1 domain family member 5; ULK1, unc-51 like autophagy activating kinase 1; WASH, WASH complex subunit.

Basal autophagic flux measured in blood correlates positively with age in adults at increased risk of type 2 diabetes.

Preclinical data show that autophagy delays age-related disease. It has been postulated that age-related disease is-at least in part-caused by an age-related decline in autophagy. However, autophagic flux has never been measured in humans across a spectrum of aging in a physiologically relevant context. To address this critical gap in knowledge, the objective of this cross-sectional observational study was to measure basal autophagic flux in whole blood taken from people at elevated risk of developing type 2 diabetes and correlate it with chronological age. During this study, 119 people were recruited and five people were excluded during sample analysis such that 114 people were included in the final analysis. Basal autophagic flux measured in blood and correlations with parameters such as age, body weight, fat mass, AUSDRISK score, blood pressure, glycated hemoglobin HbA1c, blood glucose and insulin, blood lipids, high-sensitivity C-reactive protein, plasma protein carbonylation, and plasma ß-hexosaminidase activity were analysed. Despite general consensus in the literature that autophagy decreases with age, we found that basal autophagic flux increased with age in this human cohort. This is the first study to report measurement of basal autophagic flux in a human cohort and its correlation with age. This increase in basal autophagy could represent a stress response to age-related damage. These data are significant not only for their novelty but also because they will inform future clinical studies and show that measurement of basal autophagic flux in a human cohort is feasible.

Modifying Dietary Protein Impacts mTOR Signaling and Brain Deposition of Amyloid ß in a Knock-In Mouse Model of Alzheimer Disease.

BACKGROUND: Alzheimer disease (AD) is a neurodegenerative condition defined by the build-up of amyloid plaques in the brain and intraneuronal tangles of the protein tau. Autophagy is a cellular cleaning process involved in the degradation of proteins, including proteins directly responsible for amyloid plaques, but its activity is compromised in AD. The mechanistic target of rapamycin complex (mTORC) 1 inhibits autophagy when activated by amino acids. OBJECTIVES: We hypothesized that reducing amino acid intake by decreasing dietary protein could promote autophagy, which in turn could prevent amyloid plaque deposition in AD mice. METHODS: Homozygote (2-mo-old) and heterozygote (4-mo-old) amyloid precursor protein NL-G-F mice, a model of brain amyloid deposition, were used in this study to test this hypothesis. Male and female mice were fed with isocaloric low-protein, control, or high-protein diets for 4 mo and killed for analysis. Locomotor performance was measured using the inverted screen test, and body composition was measured using EchoMRI. Samples were analyzed using western blotting, enzyme-linked immunosorbent assay, mass spectrometry, and immunohistochemical staining. RESULTS: mTORC1 activity in the cerebral cortex was inversely covaried with protein consumption in both homozygote and heterozygote mice. Low-protein diet improved metabolic parameters and restored locomotor performance only in male homozygous mice. Dietary protein adjustment did not affect amyloid deposition in homozygous mice. However, in the heterozygous amyloid precursor protein NL-G-F mice, amyloid plaque was lower in male mice consuming the low protein compared with that in mice fed with the control diet. CONCLUSIONS: This study showed that reducing protein intake reduces mTORC1 activity and may prevent amyloid accumulation, at least in male mice. Moreover, dietary protein is a tool that can be used to change mTORC1 activity and amyloid deposition in the mouse brain, and the murine brain's response to dietary protein is sex specific.

Transcriptional targets of senataxin and E2 promoter binding factors are associated with neuro-degenerative pathways during increased autophagic flux.

Autophagy is an intracellular recycling process that degrades harmful molecules and enables survival during starvation, with implications for diseases including dementia, cancer and atherosclerosis. Previous studies demonstrate how a limited number of transcription factors (TFs) can increase autophagy. However, this knowledge has not resulted in translation into therapy, thus, to gain understanding of more suitable targets, we utilized a systems biology approach. We induced autophagy by amino acid starvation and mTOR inhibition in HeLa, HEK 293 and SH-SY5Y cells and measured temporal gene expression using RNA-seq. We observed 456 differentially expressed genes due to starvation and 285 genes due to mTOR inhibition (PFDR < 0.05 in every cell line). Pathway analyses implicated Alzheimer's and Parkinson's diseases (PFDR ≤ 0.024 in SH-SY5Y and HeLa) and amyotrophic lateral sclerosis (ALS, PFDR < 0.05 in mTOR inhibition experiments). Differential expression of the Senataxin (SETX) target gene set was predicted to activate multiple neurodegenerative pathways (PFDR ≤ 0.04). In the SH-SY5Y cells of neuronal origin, the E2F transcription family was predicted to activate Alzheimer's disease pathway (PFDR ≤ 0.0065). These exploratory analyses suggest that SETX and E2F may mediate transcriptional regulation of autophagy and further investigations into their possible role in neuro-degeneration are warranted.

Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.

Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer's disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of ß-hexosaminidase) result in accumulation of amyloid-ß (Aß) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for ß-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between ß-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of ß-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in App NL-G-F/NL-G-F (App KI/KI ) mice. App KI/KI and Hexb +/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in App KI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.

PICALM regulates cathepsin D processing and lysosomal function.

Degradation and clearance of cellular waste in the autophagic and endo-lysosomal systems is important for normal physiology and prevention of common late-onset diseases such as Alzheimer's disease (AD). Phosphatidylinostol-binding clathrin assembly protein (PICALM) is a robust AD risk factor gene and encodes an endosomal protein clathrin-binding cytosolic protein, reduction of which is known to exacerbate tauopathy. Although PICALM is known to regulate initiation of autophagy, its role in maturation of lysosomal enzymes required for proteolysis has not been studied. We sought to determine the importance of PICALM for cellular degradative function by disrupting exon 1 of PICALM using CRISPR/Cas9 in HeLa cells. PICALM disruption increased numbers of early endosomes. Proteomic analysis of endosome-enriched samples showed that disrupting exon 1 of PICALM increased the abundance of lysosomal enzymes in these organelles, and western blotting revealed disruption to processing and maturation of the lysosomal protease, cathepsin D, and a deficit in autophagy. This study shows PICALM is important for the correct maturation of lysosomal enzymes and efficient proteolytic function in the lysosome.

Inhibiting mTOR activity using AZD2014 increases autophagy in the mouse cerebral cortex.

Autophagy is a catabolic process that collects and degrades damaged or unwanted cellular materials such as protein aggregates. Defective brain autophagy has been linked to diseases such as Alzheimer's disease. Autophagy is regulated by the protein kinase mTOR (mechanistic target of rapamycin). Although already demonstrated in vitro, it remains contentious whether inhibiting mTOR can enhance autophagy in the brain. To address this, mice were intraperitoneally injected with the mTOR inhibitor AZD2014 for seven days. mTOR complex 1 (mTORC1) activity was decreased in liver and brain. Autophagic activity was increased by AZD2014 in both organs, as measured by immunoblotting for LC3 (microtubule-associated proteins-1A/1B light chain 3B) and measurement of autophagic flux in the cerebral cortex of transgenic mice expressing the EGFP-mRFP-LC3B transgene. mTOR activity was shown to correlate with changes in LC3. Thus, we show it is possible to promote autophagy in the brain using AZD2014, which will be valuable in tackling conditions associated with defective autophagy, especially neurodegeneration.

TSC-insensitive Rheb mutations induce oncogenic transformation through a combination of constitutively active mTORC1 signalling and proteome remodelling.

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5' AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

Comparison of chloroquine-like molecules for lysosomal inhibition and measurement of autophagic flux in the brain.

Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.

Measurement of autophagic flux in humans: an optimized method for blood samples.

Autophagic flux is a critical cellular process that is vastly under-appreciated in terms of its importance to human health. Preclinical studies have demonstrated that reductions in autophagic flux cause cancer and exacerbate chronic diseases, including heart disease and the pathological hallmarks of dementia. Autophagic flux can be increased by targeting nutrition-related biochemical signaling. To date, translation of this knowledge has been hampered because there has been no way to directly measure autophagic flux in humans. In this study we detail a method whereby human macroautophagic/autophagic flux can be directly measured from human blood samples. We show that whole blood samples can be treated with the lysosomal inhibitor chloroquine, and peripheral blood mononuclear cells isolated from these samples could be used to measure autophagic machinery protein LC3B-II. Blocking of autophagic flux in cells while still in whole blood represents an important advance because it preserves genetic, nutritional, and signaling parameters inherent to the individual. We show this method was reproducible and defined LC3B-II as the best protein to measure autophagic flux in these cells. Finally, we show that this method is relevant to assess intra-individual variation induced by an intervention by manipulating nutrition signaling with an ex vivo treatment of whole blood that comprised leucine and insulin. Significantly, this method will enable the identification of factors that alter autophagic flux in humans, and better aid their translation in the clinic. With further research, it could also be used as a novel biomarker for risk of age-related chronic disease.Abbreviations: AMPK: AMP-activated protein kinase; ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; CQ: chloroquine; DMSO: dimethyl sulfoxide; DPBS: Dulbecco's phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; KO: knockout; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; PBMCs: peripheral blood mononuclear cells; PMNs: polymorphonuclear cells; RPMI: Roswell Park Memorial Institute; SQSTM1: sequestosome 1; TBST: Tris-buffered saline containing 0.1% (v:v) Tween 20; TEM: transmission electron microscopy.

Retromer regulates the lysosomal clearance of MAPT/tau.

The macroautophagy/autophagy-lysosome axis enables the clearance and degradation of cytoplasmic components including protein aggregates, damaged organelles and invading pathogens. Protein aggregation and lysosomal system dysfunction in the brain are common features of several late-onset neurological disorders including Alzheimer disease. Spatial overlap between depletion of the endosomal-sorting complex retromer and MAPT/tau aggregation in the brain have been previously reported. However, whether retromer dysfunction plays a direct role in mediating MAPT aggregation remains unclear. Here, we demonstrate that the autophagy-lysosome axis is the primary mode for the clearance of aggregated species of MAPT using both chemical and genetic approaches in cell models of amyloid MAPT aggregation. We show that depletion of the central retromer component VPS35 causes a block in the resolution of autophagy. We establish that this defect underlies marked accumulation of cytoplasmic MAPT aggregates upon VPS35 depletion, and that VPS35 overexpression has the opposite effect. This work illustrates how retromer complex integrity regulates the autophagy-lysosome axis to suppress MAPT aggregation and spread.

A novel fluorescent probe reveals starvation controls the commitment of amyloid precursor protein to the lysosome.

Alzheimer's disease is the most important cause of dementia but there is no therapy that has been demonstrated to stop or slow disease progression. Amyloid precursor protein (APP) is the source of amyloid-ß (Aß), which aggregates in Alzheimer's disease to form toxic oligomeric species. The endo-lysosomal system can clear APP and Aß from the cell if these molecular species are trafficked through to the lysosome. Currently, there are no easy methods available for the analysis of lysosomal APP trafficking. We therefore generated a fusion protein (tandem-fluorescent, or tf-APP) that allows detection of changes in APP trafficking using accessible techniques such as flow cytometry. This permits rapid analysis or screening of genes and compounds that alter APP processing in the cell. Using our novel molecular probe, we determined that starvation induces trafficking of APP and APP-carboxy-terminal fragments (APP-CTFs) to the degradative endo-lysosomal network. In line with this finding, suppression of mTOR signalling using AZD8055 also strongly induced trafficking of APP to the endo-lysosomal system. Remarkably, activation of mTOR signalling via RHEB over-expression inhibited the starvation-induced autophagy but did not affect trafficking of tf-APP. These results show tf-APP can be used to determine how APP is trafficked through the lysosomal system of the cell. This molecular probe is therefore useful for determining the molecular mechanism behind the commitment of APP to the degradative pathway or for screening compounds that can induce this effect. This is important as clearance of APP and APP-CTF provides an important potential therapeutic strategy for Alzheimer's disease.

Lipid composition of microdomains is altered in neuronopathic Gaucher disease sheep brain and spleen.

Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase activity that leads to accumulation of glucosylceramide and glucosylsphingosine. Membrane raft microdomains are discrete, highly organized microdomains with a unique lipid composition that provide the necessary environment for specific protein-lipid and protein-protein interactions to take place. In this study we purified detergent resistant membranes (DRM; membrane rafts) from the occipital cortex and spleen from sheep affected with acute neuronopathic Gaucher disease and wild-type controls. We observed significant increases in the concentrations of glucosylceramide, hexosylsphingosine, BMP and gangliosides and decreases in the percentage of cholesterol and phosphatidylcholine leading to an altered DRM composition. Altered sphingolipid/cholesterol homeostasis would dramatically disrupt DRM architecture making them less ordered and more fluid. In addition, significant changes in the length and degree of lipid saturation within the DRM microdomains in the Gaucher brain were also observed. As these DRM microdomains are involved in many cellular events, an imbalance or disruption of the cell membrane homeostasis may impair normal cell function. This disruption of membrane raft microdomains and imbalance within the environment of cellular membranes of neuronal cells may be a key factor in initiating a cascade process leading to neurodegeneration.

Glycosphingolipid analysis in a naturally occurring ovine model of acute neuronopathic Gaucher disease.

Gaucher disease arises from mutations in the ß-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the ß-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in ß-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process.

Absence of α-galactosidase cross-correction in Fabry heterozygote cultured skin fibroblasts.

Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from deficiency of α-galactosidase A (GLA). Traditionally, heterozygotes were considered asymptomatic carriers of FD, but it is now apparent that the asymptomatic female carrier is the exception and most heterozygotes suffer significant multisystemic disease. To determine why the process of cross-correction does not occur effectively in FD heterozygotes, we investigated GLA production and secretion in cultured skin fibroblasts as well as GLA levels in plasma. The maturation of GLA was similar in FD heterozygotes and control fibroblasts, confirming that both produce the 46kDa mature form; the same as that present in control plasma. However, the proportion of GLA secreted into the culture media was substantially less than eight other lysosomal proteins. Artificial generation of FD heterozygotes in cellulo, along with another lysosomal storage disorder, mucopolysaccharidosis type II, revealed no cross-correction in the FD system, whereas MPS II fibroblasts were able to cross-correct. In plasma, GLA was present as the 46kDa mature form, which lacks the mannose 6-phosphorylated moiety and is not able to be efficiently endocytosed by affected cells. Our evidence shows that fibroblasts secrete minimal amounts of GLA and consequently normal fibroblasts are unable to cross-correct FD fibroblasts. We suggest that symptomatic FD heterozygotes arise due to the secretion of primarily the mature form, with only small amounts of the mannose 6-phosphorylated form of GLA from unaffected cells. This limits capacity for enzyme cross correction of affected cells, despite uptake of exogenous recombinant GLA.

Liquid chromatography/electrospray ionisation-tandem mass spectrometry quantification of GM2 gangliosides in human peripheral cells and plasma.

GM2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of GM2 gangliosides (GM2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify GM2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of GM2 was achieved using LC/ESI-MS/MS with d35GM1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all GM2 gangliosidosis patients were demarcated from controls by elevated GM2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in GM2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted ß-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for GM2 gangliosidosis by allowing quantification of a reduction in the primary storage burden.

Lipid composition of membrane rafts, isolated with and without detergent, from the spleen of a mouse model of Gaucher disease.

Biological membranes are composed of functionally relevant liquid-ordered and liquid-disordered domains that coexist. Within the liquid-ordered domains are low-density microdomains known as rafts with a unique lipid composition that is crucial for their structure and function. Lipid raft composition is altered in sphingolipid storage disorders, and here we determined the lipid composition using a detergent and detergent-free method in spleen tissue, the primary site of pathology, in a mouse model of the sphingolipid storage disorder, Gaucher disease. The accumulating lipid, glucosylceramide, was 30- and 50-fold elevated in the rafts with the detergent and detergent-free method, respectively. Secondary accumulation of di- and trihexosylceramide resided primarily in the rafts with both methods. The phospholipids distributed differently with more than half residing in the rafts with the detergent-free method and less than 10% with the detergent method, with the exception of the fully saturated species that were primarily in the rafts. Individual isoforms of sphingomyelin correlated with detergent-free extraction and more than half resided in the raft fractions. However, this correlation was not seen with the detergent extraction method as sphingomyelin species were spread across both the raft and non-raft domains. Therefore caution must be exercised when interpreting phospholipid distribution in raft domains as it differs considerably depending on the method of isolation. Importantly, both methods revealed the same lipid alterations in the raft domains in the spleen of the Gaucher disease mouse model highlighting that either method is appropriate to determine membrane lipid changes in the diseased state.

Transgenic mice expressing human glucocerebrosidase variants: utility for the study of Gaucher disease.

Gaucher disease is an autosomal recessively inherited storage disorder caused by deficiency of the lysosomal hydrolase, acid ß-glucosidase. The disease manifestations seen in Gaucher patients are highly heterogeneous as is the responsiveness to therapy. The elucidation of the precise factors responsible for this heterogeneity has been challenging as the development of clinically relevant animal models of Gaucher disease has been problematic. Although numerous murine models for Gaucher disease have been described each has limitations in their specific utility. We describe here, transgenic murine models of Gaucher disease that will be particularly useful for the study of pharmacological chaperones. We have produced stable transgenic mouse strains that individually express wild type, N370S and L444P containing human acid ß-glucosidase and show that each of these transgenic lines rescues the lethal phenotype characteristic of acid ß-glucosidase null mice. Both the N370S and L444P transgenic models show early and progressive elevations of tissue sphingolipids with L444P mice developing progressive splenic Gaucher cell infiltration. We demonstrate the potential utility of these new transgenic models for the study of Gaucher disease pathogenesis. In addition, since these mice produce only human enzyme, they are particularly relevant for the study of pharmacological chaperones that are specifically targeted to human acid ß-glucosidase and the common mutations underlying Gaucher disease.

Selective reduction of bis(monoacylglycero)phosphate ameliorates the storage burden in a THP-1 macrophage model of Gaucher disease.

Bis(monoacylglycero)phosphate (BMP) assists lysosomal function by facilitating interaction of hydrolases and activator proteins with sphingolipid substrates. Impaired lysosomal degradation of the sphingolipid glucosylceramide (GC) occurs in Gaucher disease due to an inherited deficiency of acid ß-glucosidase, with secondary BMP alterations. We investigated the nature of BMP accumulation and whether its correction reduced the storage burden in a THP-1 macrophage model of Gaucher disease. Using sucrose gradients and detergent solubility, 98% of BMP resided in the detergent-soluble membranes (DSM) rather than in the detergent-resistant membranes (DRM) where 73% of GC predominated. There was a 2-fold widespread elevation in BMP, including the saturated, mono- and polyunsaturated species. Linoleic acid in the culture media selectively reduced BMP from 4.2 nmol/mg to 0.49 nmol/mg (except 18:1/18:2) and prevented up to one third of GC, dihexosylceramide (DHC), and trihexosylceramide (THC) from accumulating. The 2-fold reduction in these sphingolipids occurred only in the DRM and did not reduce 18:1/16:0. However, once GC had accumulated, linoleic acid could not reverse it, DHC, or THC, despite effectively reducing BMP. These results imply a causative link for BMP in the pathobiology of Gaucher disease and demonstrate that linoleic acid can shield the cell from excessive substrate accumulation.