A Stable Micellar Formulation of RAD001 for Intracerebroventricular Delivery and the Treatment of Alzheimer's Disease and Other Neurological Disorders.

A large body of evidence, replicated in many mouse models of Alzheimer's disease (AD), supports the therapeutic efficacy of the oral mammalian target of rapamycin inhibitors (mTOR-Is). Our preliminary data show that intracerebroventricular (ICV) administration of everolimus (RAD001) soon after clinical onset greatly diminished cognitive impairment and the intracellular beta amyloid and neurofibrillary tangle load. However, RAD001 shows >90% degradation after 7 days in solution at body temperature, thus hampering the development of proper therapeutic regimens for patients. To overcome such a drawback, we developed a stable, liquid formulation of mTOR-Is by loading RAD001 into distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) micelles using the thin layer evaporation method. The formulation showed efficient encapsulation of RAD001 and a homogeneous colloidal size and stabilised RAD001, with over 95% of activity preserved after 14 days at 37 °C with a total decay only occurring after 98 days. RAD001-loaded DSPE-PEG2000 micelles were unchanged when stored at 4 and 25 °C over the time period investigated. The obtained formulation may represent a suitable platform for expedited clinical translation and effective therapeutic regimens in AD and other neurological diseases.

Proteomic analysis of murine Tsc1-deficient neural stem progenitor cells.

Tuberous sclerosis complex (TSC) is a rare, multisystem genetic disorder that leads to the development of benign tumors in multiple organs and neurological symptoms. TSC clinical manifestations show a great heterogenicity, with most patients presenting severe neuropsychiatric and neurological disorders. TSC is caused by loss-of-function mutations in either TSC1 or TSC2 genes, leading to overexpression of the mechanistic target of rapamycin (mTOR) and, consequently, abnormal cellular growth, proliferation and differentiation as well as to cell migration defects. Beside the growing interest, TSC remains a disorder poorly understood, with limited perspectives in the field of therapeutic strategies. Here we used murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene as a TSC model to unravel novel molecular aspects of the pathophysiology of this disease. 2D-DIGE-based proteomic analysis detected 55 differently represented spots in Tsc1-deficient cells, compared to wild-type counterparts, which were associated with 36 protein entries after corresponding trypsinolysis and nanoLC-ESI-Q-Orbitrap-MS/MS analysis. Proteomic results were validated using various experimental approaches. Bioinformatics associated differently represented proteins with oxidative stress and redox pathways, methylglyoxal biosynthesis, myelin sheath, protein S-nitrosylation and carbohydrate metabolism. Because most of these cellular pathways have already been linked to TSC features, these results were useful to clarify some molecular aspects of TSC etiopathogenesis and suggested novel promising therapeutic protein targets. SIGNIFICANCE: Tuberous Sclerosis Complex (TSC) is a multisystemic disorder caused by inactivating mutations of TSC1 or TSC2 genes, which induce overactivation of the mTOR component. The molecular mechanisms underlying the pathogenesis of TSC remain unclear, probably due to complexity of mTOR signaling network. To have a picture of protein abundance changes occurring in TSC disorder, murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene were used as a model of disease. Thus, Tsc1-deficient SVZ NSPCs and wild-type cells were comparatively evaluated by proteomics. This analysis evidenced changes in the abundance of proteins involved in oxidative/nitrosative stress, cytoskeleton remodelling, neurotransmission, neurogenesis and carbohydrate metabolism. These proteins might clarify novel molecular aspects of TSC etiopathogenesis and constitute putative molecular targets for novel therapeutic management of TSC-related disorders.

Biologically driven cut-off definition of lymphocyte ratios in metastatic breast cancer and association with exosomal subpopulations and prognosis.

High neutrophil to lymphocyte ratio (NLR) and monocyte to lymphocyte ratio (MLR) are respectively associated with systemic inflammation and immune suppression and have been associated with a poor outcome. Plasmatic exosomes are extracellular vesicles involved in the intercellular communication system that can exert an immunosuppressive function. Aim of this study was to investigate the interplay between the immune system and circulating exosomes in metastatic breast cancer (MBC). A threshold capable to classify patients according to MLR, NLR and PLR, was computed through a receiving operator curve analysis after propensity score matching with a series of female blood donors. Exosomes were isolated from plasma by ExoQuick solution and characterized by flow-cytometry. NLR, MLR, PLR and exosomal subpopulations potentially involved in the pre-metastatic niche were significantly different in MBC patients with respect to controls. MLR was significantly associated with number of sites at the onset of metastatic disease, while high levels of MLR and NLR were found to be associated with poor prognosis. Furthermore, exosomal subpopulations varied according to NLR, MLR, PLR and both were associated with different breast cancer subtypes and sites of distant involvement. This study highlights the nuanced role of immunity in MBC spread, progression and outcome. Moreover, they suggest potential interaction mechanisms between immunity, MBC and the metastatic niche.

Proteome Alterations in Equine Osteochondrotic Chondrocytes.

Osteochondrosis is a failure of the endochondral ossification that affects developing joints in humans and several animal species. It is a localized idiopathic joint disorder characterized by focal chondronecrosis and growing cartilage retention, which can lead to the formation of fissures, subchondral bone cysts, or intra-articular fragments. Osteochondrosis is a complex multifactorial disease associated with extracellular matrix alterations and failure in chondrocyte differentiation, mainly due to genetic, biochemical, and nutritional factors, as well as traumas. This study describes the main proteomic alterations occurring in chondrocytes isolated from osteochondrotic cartilage fragments. A comparative analysis performed on equine osteochondrotic and healthy chondrocytes showed 26 protein species as differentially represented. In particular, quantitative changes in the extracellular matrix, cytoskeletal and chaperone proteins, and in cell adhesion and signaling molecules were observed in osteochondrotic cells, compared to healthy controls. Functional group analysis annotated most of these proteins in "growth plate and cartilage development", while others were included in "glycolysis and gluconeogenesis", "positive regulation of protein import", "cell-cell adhesion mediator activity", and "mitochondrion nucleoid". These results may help to clarify some chondrocyte functional alterations that may play a significant role in determining the onset and progression of equine osteochondrosis and, being related, of human juvenile osteochondrosis.

Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation.

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of ß-hexosaminidase (Hex, EC 3.2.1.52) and ß-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.

KRIT1 Loss-Of-Function Associated with Cerebral Cavernous Malformation Disease Leads to Enhanced S-Glutathionylation of Distinct Structural and Regulatory Proteins.

Loss-of-function mutations in the KRIT1 gene are associated with the pathogenesis of cerebral cavernous malformations (CCMs), a major cerebrovascular disease still awaiting therapies. Accumulating evidence demonstrates that KRIT1 plays an important role in major redox-sensitive mechanisms, including transcriptional pathways and autophagy, which play major roles in cellular homeostasis and defense against oxidative stress, raising the possibility that KRIT1 loss has pleiotropic effects on multiple redox-sensitive systems. Using previously established cellular models, we found that KRIT1 loss-of-function affects the glutathione (GSH) redox system, causing a significant decrease in total GSH levels and increase in oxidized glutathione disulfide (GSSG), with a consequent deficit in the GSH/GSSG redox ratio and GSH-mediated antioxidant capacity. Redox proteomic analyses showed that these effects are associated with increased S-glutathionylation of distinct proteins involved in adaptive responses to oxidative stress, including redox-sensitive chaperonins, metabolic enzymes, and cytoskeletal proteins, suggesting a novel molecular signature of KRIT1 loss-of-function. Besides providing further insights into the emerging pleiotropic functions of KRIT1, these findings point definitively to KRIT1 as a major player in redox biology, shedding new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis.

Early intrathecal infusion of everolimus restores cognitive function and mood in a murine model of Alzheimer's disease.

The discovery that mammalian target of rapamycin (mTOR) inhibition increases lifespan in mice and restores/delays many aging phenotypes has led to the identification of a novel potential therapeutic target for the treatment of Alzheimer's disease (AD). Among mTOR inhibitors, everolimus, which has been developed to improve the pharmacokinetic characteristics of rapamycin, has been extensively profiled in preclinical and clinical studies as anticancer and immunosuppressive agent, but no information is available about its potential effects on neurodegenerative disorders. Using a reliable mouse model of AD (3 × Tg-AD mice), we explored whether short-term treatment with everolimus injected directly into the brain by osmotic pumps was able to modify AD-like pathology with low impact on peripheral organs. We first established in non-transgenic mice the stability of everolimus at 37 °C in comparison with rapamycin and, then, evaluated its pharmacokinetics and pharmacodynamics profiles through either a single peripheral (i.p.) or central (i.c.v.) route of administration. Finally, 6-month-old (symptomatic phase) 3 × Tg-AD mice were treated with continuous infusion of either vehicle or everolimus (0.167 µg/µl/day, i.c.v.) using the osmotic pumps. Four weeks after the beginning of infusion, we tested our hypothesis following an integrated approach, including behavioral (tests for cognitive and depressive-like alterations), biochemical and immunohistochemical analyses. Everolimus (i) showed higher stability than rapamycin at 37 °C, (ii) poorly crossed the blood-brain barrier after i.p. injection, (iii) was slowly metabolized in the brain due to a longer t1/2 in the brain compared to blood, and (iv) was more effective in the CNS when administered centrally compared to a peripheral route. Moreover, the everolimus-induced mTOR inhibition reduced human APP/Aß and human tau levels and improved cognitive function and depressive-like phenotype in the 3 × Tg-AD mice. The intrathecal infusion of everolimus may be effective to treat early stages of AD-pathology through a short and cyclic administration regimen, with short-term outcomes and a low impact on peripheral organs.

mTOR Signaling and Neural Stem Cells: The Tuberous Sclerosis Complex Model.

The mechanistic target of rapamycin (mTOR), a serine-threonine kinase, plays a pivotal role in regulating cell growth and proliferation. Notably, a great deal of evidence indicates that mTOR signaling is also crucial in controlling proliferation and differentiation of several stem cell compartments. Consequently, dysregulation of the mTOR pathway is often associated with a variety of disease, such as cancer and metabolic and genetic disorders. For instance, hyperactivation of mTORC1 in neural stem cells (NSCs) is associated with the insurgence of neurological manifestation characterizing tuberous sclerosis complex (TSC). In this review, we survey the recent contributions of TSC physiopathology studies to understand the role of mTOR signaling in both neurogenesis and tumorigenesis and discuss how these new insights can contribute to developing new therapeutic strategies for neurological diseases and cancer.

TFEB activation restores migration ability to Tsc1-deficient adult neural stem/progenitor cells.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder caused by mutations in either of two genes, TSC1 or TSC2, resulting in the constitutive activation of the mammalian target of rapamycin complex 1 (mTORC1). mTOR inhibitors are now considered the treatment of choice for TSC disease. A major pathological feature of TSC is the development of subependymal giant cell astrocytomas (SEGAs) in the brain. Nowadays, it is thought that SEGAs could be a consequence of aberrant aggregation and migration of neural stem/progenitor cells (NSPCs). Therefore, reactivation of cell migration of NSPCs might be the crucial step for the treatment of patients. In order to identify potential in vitro targets activating migration, we generated Tsc1-deficient NSPCs. These cells summarize most of the biochemical and morphological characteristics of TSC neural cells, such as the mTORC1 activation, the formation of abnormally enlarged astrocytes-like cells, the reduction of autophagy flux and the impairment of cell migration. Moreover, nuclear translocation, namely activation of the transcription factor EB (TFEB) was markedly impaired. Herein, we show that compounds such as everolimus, ionomycin and curcumin, which directly or indirectly stimulate TFEB nuclear translocation, restore Tsc1-deficient NSPC migration. Our data suggest that reduction of TFEB activation, caused by mTORC1 hyperactivation, contributes to the migration deficit characterizing Tsc1-deficient NSPCs. The present work highlights TFEB as a druggable protein target for SEGAs therapy, which can be additionally or alternatively exploited for the mTORC1-directed inhibitory approach.

Rapamycin Loaded Solid Lipid Nanoparticles as a New Tool to Deliver mTOR Inhibitors: Formulation and in Vitro Characterization.

Recently, the use of mammalian target of rapamycin (mTOR) inhibitors, in particular rapamycin (Rp), has been suggested to improve the treatment of neurodegenerative diseases. However, as Rp is a strong immunosuppressant, specific delivery to the brain has been postulated to avoid systemic exposure. In this work, we fabricated new Rp loaded solid lipid nanoparticles (Rp-SLN) stabilized with polysorbate 80 (PS80), comparing two different methods and lipids. The formulations were characterized by differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), wide angle X-ray scattering (WAXS), cryo-transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS) and particle tracking. In vitro release and short-term stability were assessed. Biological behavior of Rp-SLN was tested in SH-SY5Y neuroblastoma cells. The inhibition of mTOR complex 1 (mTORC1) was evaluated over time by a pulse-chase study compared to free Rp and Rp nanocrystals. Compritol Rp-SLN resulted more stable and possessing proper size and surface properties with respect to cetyl palmitate Rp-SLN. Rapamycin was entrapped in an amorphous form in the solid lipid matrix that showed partial crystallinity with stable Lß, sub-Lα and Lß' arrangements. PS80 was stably anchored on particle surface. No drug release was observed over 24 h and Rp-SLN had a higher cell uptake and a more sustained effect over a week. The mTORC1 inhibition was higher with Rp-SLN. Overall, compritol Rp-SLN show suitable characteristics and stability to be considered for further investigation as Rp brain delivery system.

Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.

Use of Polylactide-Co-Glycolide-Nanoparticles for Lysosomal Delivery of a Therapeutic Enzyme in Glycogenosis Type II Fibroblasts.

Glycogenosis type II, or Pompe Disease, is a lysosomal storage disease caused by the deficiency of acid alpha-glucosidase (GAA), leading to glycogen accumulation in muscles. A recombinant human GAA (rhGAA, Myozyme®) is currently used for enzyme replacement therapy. Despite its efficacy in most of patients, some of them show a diminished response to the treatment with rapidly progressive clinical deterioration, due to immuno-mediated enzyme inactivation. To demonstrate that Nanoparticles (NPs) could be profitably exploited to carry macromolecules, PLGA NPs loaded with rhGAA (GAA-NPs) were prepared by double emulsion solvent evaporation. Their surface morphology, particle size, zeta-potential and biochemical activity were assessed. "Pulse and chase" experiments were made by administrating GAA-NPs on patients' fibroblasts. Biochemical activity tests showed a more efficient cellular uptake of rhGAA loaded to NPs and a more significant stability of the enzyme (up to 7 days) in vitro, if compared to the same amount of rhGAA free enzyme. This data allows to envision in vivo experiments, in significant animal models, to further characterize lysosomal enzyme loaded-NPs' efficacy and toxicity.

Expression of the glycolytic enzymes enolase and lactate dehydrogenase during the early phase of Toxoplasma differentiation is regulated by an intron retention mechanism.

The intracellular parasite Toxoplasma gondii converts from a rapidly replicating tachyzoite form during acute infection to a quiescent encysted bradyzoite stage that persists inside long-lived cells during chronic infection. Bradyzoites adopt reduced metabolism and slow replication while waiting for an opportunity to recrudesce the infection within the host. Interconversion between these two developmental stages is characterized by expression of glycolytic isoenzymes that play key roles in parasite metabolism. The parasite genome encodes two isoforms of lactate dehydrogenase (LDH1 and LDH2) and enolase (ENO1 and ENO2) that are expressed in a stage-specific manner. Expression of different isoforms of these enzymes allows T. gondii to rapidly adapt to diverse metabolic requirements necessary for either a rapid replication of the tachyzoite stage or a quiescent lifestyle typical of the bradyzoites. Herein we identified unspliced forms of LDH and ENO transcripts produced during transition between these two parasite stages suggestive of an intron retention mechanism to promptly exchange glycolytic isoforms for rapid adaptation to environmental changes. We also identified key regulatory elements in the ENO transcription units, revealing cooperation between the ENO2 5'-untranslated region and the ENO2 intron, along with identifying a role for the ENO1 3'-untranslated region in stage-specific expression.

Evaluation of a LC-MS method for everolimus preclinical determination in brain by using [(13)C2D4]RAD001 internal standard.

Isotopic internal standards are increasingly frequent in LC-MS analysis to control biological matrix effects in the quantitation of immunosuppressant drugs, such as everolimus (RAD001). Here we present the evaluation of a LC-MS method, exploiting [(13)C2D4]RAD001 as internal standard, for preclinical determination of RAD001 in mice brain tissue. Samples were purified by solid phase extraction. Brain and blood were collected from vehicle-treated and RAD001-treated mice. The QTOF MS detector was set to select RAD001 ammonium adducts (m/z 975.6152) and [(13)C2D4]RAD001 (m/z 981.6481). Two different UHPLC columns were preliminarily tested. The method showed linear behavior between 4 and 100ng/mL (r(2)=0.99943) and linearity was preserved in the presence of blood (r(2)=0.99107) and brain (r(2)=0.99098) matrix components. Intra-day and inter-day precision (3-19%) and accuracy (82-109%) were comparable between standards and spiked blood and brain samples. As resulting from recovery comparison (82-98%), [(13)C2D4]RAD001 compensated ion suppression phenomena maintaining method performance over a wide range of consecutive analytical runs. The comparison with a HPLC-UV method showed reliability of the method with good correlation between blood (r(2)=0.94319) and brain (r(2)=0.97773) samples and acceptable biases (<15%). This validation suggests that the investigated method could be useful for the preclinical monitoring of RAD001 brain therapeutic concentrations in animal models.

Abnormal cortical lysosomal ß-hexosaminidase and ß-galactosidase activity at post-synaptic sites during Alzheimer's disease progression.

A critical role of endosomal-lysosomal system alteration in neurodegeneration is supported by several studies. Dysfunction of the lysosomal compartment is a common feature also in Alzheimer's disease. Altered expression of lysosomal glycohydrolases has been demonstrated not only in the brain and peripheral tissues of Alzheimer's disease patients, but also in presymptomatic subjects before degenerative phenomenon becomes evident. Moreover, the presence of glycohydrolases associated to the plasma membrane have been widely demonstrated and their alteration in pathological conditions has been documented. In particular, lipid microdomains-associated glycohydrolases can be functional to the maintenance of the proper glycosphingolipids pattern, especially at cell surface level, where they are crucial for the function of cell types such as neurons. In this study we investigated the localization of ß-hexosaminidase and ß-galactosidase glycohydrolases, both involved in step by step degradation of the GM1 to GM3 gangliosides, in lipid microdomains from the cortex of both an early and advanced TgCRND8 mouse model of Alzheimer's disease. Throughout immunoprecipitation experiments of purified cortical lipid microdomains, we demonstrated for the first time that ß-hexosaminidase and ß-galactosidase are associated with post-synaptic vesicles and that their activities are increased at both the early and the advanced stage of Alzheimer's disease. The early increase of lipid microdomain-associated ß-hexosaminidase and ß-galactosidase activities could have relevant implications for the pathophysiology of the disease since their possible pharmacological manipulation could shed light on new reliable targets and biological markers of Alzheimer's disease.

Oncogenic H-Ras up-regulates acid ß-hexosaminidase by a mechanism dependent on the autophagy regulator TFEB.

The expression of constitutively active H-RasV12 oncogene has been described to induce proliferative arrest and premature senescence in many cell models. There are a number of studies indicating an association between senescence and lysosomal enzyme alterations, e.g. lysosomal ß-galactosidase is the most widely used biomarker to detect senescence in cultured cells and we previously reported that H-RasV12 up-regulates lysosomal glycohydrolases enzymatic activity in human fibroblasts. Here we investigated the molecular mechanisms underlying lysosomal glycohydrolase ß-hexosaminidase up-regulation in human fibroblasts expressing the constitutively active H-RasV12. We demonstrated that H-Ras activation increases ß-hexosaminidase expression and secretion by a Raf/extracellular signal-regulated protein kinase dependent pathway, through a mechanism that relies on the activity of the transcription factor EB (TFEB). Because of the pivotal role of TFEB in the regulation of lysosomal system biogenesis and function, our results suggest that this could be a general mechanism to enhance lysosomal enzymes activity during oncogene-induced senescence.

Hypermethylation contributes to down-regulation of lysosomal ß-hexosaminidase α subunit in prostate cancer cells.

ß-Hexosaminidase, involved in degradation of glycoproteins and glycosphingolipids, is altered in several tumours leading to enhanced migration capacity. To date, the expression of the ß-hexosaminidase isoenzymes in prostate cancer cells has not been elucidated. By using PC3, LNCaP, DUCaP, MDAPCa 2b, and hyperplasic prostate (BPH-1) cell lines, we analysed the ß-hexosaminidase activity in each cell line and determined ß-hexosaminidase α subunit gene expression in PC3, LNCaP, and BPH-1. We then investigated the methylation status of the gene promoter and determined the cellular responses of PC3 and LNCaP after transfection with ß-hexosaminidase α subunit. We found that each prostate cancer cell line had a decrease in total hexosaminidase activity and that the lack of hexosaminidase A activity, observed in PC3 and LNCaP cells, was associated with mRNA disappearance. The HEXA promoter region in LNCaP and PC3 cell lines had methylated CpG islands, as confirmed by 5'-Aza-2'-deoxycitidine treatment, in PC3 cells, used as cell cancer model. We also tested, the involvement of hexosaminidase A in the migration capacity by migration assay using Hex α subunit-transfected PC3. Finally, we found that, after Hex α subunit transfection, both PC3 and LNCaP were less susceptible to exogenous ceramide treatment. Results indicate a likely contribution of the lysosomal enzyme to the acquisition of cancerous features.

Methods to discriminate the distribution of acidic glycohydrolases between the endosomal-lysosomal systems and the plasma membrane.

The endosomal-lysosomal system plays important roles in cellular physiology. Beyond the well-known function as terminal degradative compartment, necessary to maintain the health of the cell, lysosomes are critical for many other cellular processes, such as termination of signaling mediated by cell surface receptors and processing of internalized peptides in antigen-presenting cells. Moreover, the intracellular membrane trafficking related to the endosomal-lysosomal system plays a pivotal role in diverse physiological and pathological processes, such as exocytosis, plasma membrane repair, and endocytosis. Increasing evidences suggest that several lysosomal glycohydrolases, together with nonlysosomal glycohydrolases, are associated with cell membranes in their active form, and they are localized into lipid microdomains. The role of these forms in physiological and pathological conditions, such as differentiation and aging, neurodegenerative diseases, and cancer spreading, is under investigation. Here we provide general methods to purify lipid microdomain proteins and to discriminate cell surface lipid microdomains-associated glycohydrolases from those not exposed on cell surface. The methods reported here have been developed to characterize the membrane-associated forms of the acidic glycohydrolases ß-hexosaminidase and ß-galactosidase, but they may be applied to any other protein of interest.

Assessment of safety and efficiency of nitrogen organic fertilizers from animal-based protein hydrolysates--a laboratory multidisciplinary approach.

BACKGROUND: Protein hydrolysates or hydrolysed proteins (HPs) are high-N organic fertilizers allowing the recovery of by-products (leather meal and fluid hydrolysed proteins) otherwise disposed of as polluting wastes, thus enhancing matter and energy conservation in agricultural systems while decreasing potential pollution. Chemical and biological characteristics of HPs of animal origin were analysed in this work to assess their safety, environmental sustainability and agricultural efficacy as fertilizers. Different HPs obtained by thermal, chemical and enzymatic hydrolytic processes were characterized by Fourier transform infrared spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their safety and efficacy were assessed through bioassays, ecotoxicological tests and soil biochemistry analyses. RESULTS: HPs can be discriminated according to their origin and hydrolysis system by proteomic and metabolomic methods. Three experimental systems, soil microbiota, yeast and plants, were employed to detect possible negative effects exerted by HPs. The results showed that these compounds do not significantly interfere with metabolomic activity or the reproductive system. CONCLUSION: The absence of toxic and genotoxic effects of the hydrolysates prepared by the three hydrolytic processes suggests that they do not negatively affect eukaryotic cells and soil ecosystems and that they can be used in conventional and organic farming as an important nitrogen source derived from otherwise highly polluting by-products.

Chaperone therapy for GM2 gangliosidosis: effects of pyrimethamine on ß-hexosaminidase activity in Sandhoff fibroblasts.

Sphingolipidoses are inherited genetic diseases due to mutations in genes encoding proteins involved in the lysosomal catabolism of sphingolipids. Despite a low incidence of each individual disease, altogether, the number of patients involved is relatively high and resolutive approaches for treatment are still lacking. The chaperone therapy is one of the latest pharmacological approaches to these storage diseases. This therapy allows the mutated protein to escape its natural removal and to increase its quantity in lysosomes, thus partially restoring the metabolic functions. Sandhoff disease is an autosomal recessive inherited disorder resulting from ß-hexosaminidase deficiency and characterized by large accumulation of GM2 ganglioside in brain. No enzymatic replacement therapy is currently available, and the use of inhibitors of glycosphingolipid biosynthesis for substrate reduction therapy, although very promising, is associated with serious side effects. The chaperone pyrimethamine has been proposed as a very promising drug in those cases characterized by a residual enzyme activity. In this review, we report the effect of pyrimethamine on the recovery of ß-hexosaminidase activity in cultured fibroblasts from Sandhoff patients.