Manual Lysosomes (Medical Intelligence Unit)

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History and Morphology of the Lysosome

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Used book in very good condition. Thus, a genetic program controls lysosomal biogenesis and function, providing a potential therapeutic target to enhance cellular clearing in lysosomal storage disorders and neurodegenerative diseases. Prior art reports the description of a system to increase the activity of some cathepsins following the inhibition of the lysosomal system; however, these results are rather partial, controversial, and the molecular mechanism has not been analyzed into details.

In the published literature there are no papers that reveal the presence of a lysosomal gene network or that identify TFEB as a possible modulator of the lysosomal activity. Description of the invention. The authors of the invention identified a gene network that comprises the genes encoding lysosomal proteins of critical importance for the degradation of toxic compounds.

These proteins are involved, directly or indirectly, in a high number of human diseases.


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The regulatory element responsible for the modulation of these genes has been identified in their promoter sequences. Such regulatory element, which authors called CLEAR, represents itself a target for the modulation - and therefore the enhancement - of the production of the lysosomal proteins responsible for the degradation of toxic compounds. Authors demonstrated that the lysosomal activity can be modulated by increasing or decreasing the amount of TFEB.

In particular, the lysosomal enhancement resulting from the increase in TFEB levels is able to clear the cell from the toxic protein responsible for the neurodegenerative Huntington's disease. The enhancement of the cellular degradative pathways by the activation of the lysosomal system may be advantageously used for the therapy of lysosomal storage disorders and of neurodegenerative diseases.

Such treatment may be performed by using: 1 TFEB or synthetic or biotechnological derivatives thereof, as peptide fragments, chimeric peptides etc. CA A1 describes the use of TFEB for cancer treatment and for modulating cell proliferation or differentiation. Esumi Noriko et al. The document discloses experiments with yeast cells, that identified several modificators of the clearance of neurotoxic peptides, suggesting that some putative human orthologs of yeast genes should act in the same way.

A possible link between TFEB expression and clearance of neurotoxic peptides, in a diagnostic perspective, is suggested, with no data. Finally, the CLEAR regulatory element - allowing the lysosomal system modulation - is not disclosed in any prior art documents. Technologies able to enhance the lysosomal activity have not been described so far. In the instant invention, lysosomal storage disorders are intended as inherited diseases in which a defect in one of many proteins participating in lysosomal biogenesis or metabolism leads to the intralysosomal storage of undegraded molecules, as described in "Lysosomes", author: Paul Saftig, Landes Bioscience, It is an object of the invention a molecule being able to enhance the cellular degradative pathways to prevent or antagonize the accumulation of toxic compounds in a cell, characterized by: a acting either direcly or indirectly on a CLEAR element to enhance the expression of at least a gene involved in cellular degradative pathways, said CLEAR element comprising at least one repeat of a nucleotide sequence having Seq Id No.

Id No. In a particular aspect of the invention the chimeric molecule comprises the TFEB protein and a nuclear localization signal NLS , more preferably the chimeric molecule has the sequence of Seq Id No. In a preferred aspect, the molecule of the invention acts either direcly or indirectly on a. CLEAR element to enhance the expression of at least a gene expressing a lysosomal protein, involved in cellular degradative pathways. In a preferred aspect, the molecule of the invention is for neurodegenerative disorders' treatments. Neurodegenerative diseases comprise but are not limited to the following: Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease,.


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Preferably the neurodegenerative disorder belongs to the group of Alzheimer, Parkinson and Huntington diseases. In an alternative preferred aspect, the molecule of the invention is for for lysosomal storage disorders' treatments. It is another aspect of the invention a nucleic acid containing a sequence encoding for the molecule according as above disclosed,.

It is another aspect of the invention a vector comprising under appropriate regulative sequence the above nucleic acid, preferably for gene therapy. The invention shall be described with reference to experimental non limitating evidences. A regulatory gene network controlling the expression of lysosomal genes. Dashed box contains all the elements corresponding to the genes that were used for Gene Ontology analysis see text. B Luciferase assay using constructs carrying four tandem copies of either intact upper or mutated middle, mutations in red CLEAR elements.

Red bars show the fold change of mRNA levels in mimic-miRtransfected cells vs. Randomly chosen non-lysosomal genes were used as controls. D Chromatin immunoprecipitation ChIP analysis. TFEB overexpression induces lysosomal biogenesis. Blue bars indicate the proportion of cells with fluorescence intensity greater than the indicated threshold P4 gate. C Electron microscopy analysis.

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Thin sections exhibit more lysosome profiles arrows with typical ultrastructure see details in inset corresponding to dash boxed area in TFEB overexpressing transfectants over the control. Scale bar, nm. A ChIP analysis following lysosomal storage of sucrose. The histogram shows the ratio expressed as fold change between the amounts of FLAG-immunoprecipitated chromatin in sucrose-treated versus non-treated cells.

Lysosomal genes show an average two- to three-fold increase of immunoprecipitated chromatin, whereas no significant changes are observed for control genes. B Expression analysis of lysosomal genes following sucrose supplementation. C Immunofluorescence microscopy analysis of TFEB subcellular localization following sucrose supplementation. TFEB enhances cellular clearance. The graph shows relative amounts of 3H-glucosamine incorporated into GAGs over time.

The diagram reports a visual representation of the expression correlation of 40 lysosomal disease genes with all known lysosomal genes. Each column represents the , gene probes of the Affymetrix HG-U A platform ranked by their correlation of expression with the gene indicated at the top. Blue bars represent the position of lysosomal genes within the ranked lists. The analysis shows that there is an enrichment of lysosomal genes within the first 5th percentile of ranked lists of expression correlation.

The columns include the first gene probes of the expression correlation lists for selected lysosomal genes. Lysosomal genes are highlighted in orange. Other genes associated to the lysosomal function are highlighted in yellow. The conservation of each residue within columns is visualized as the relative height of symbols. The legend to colour code is reported as a schematic diagram in the figure. Luciferase activities were measured in the presence or absence of a plasmid construct containing the precursor sequence of hsa-miR Blue bars show the fold change of monitored genes in mimic-miR transfected cells vs.

No significant changes were observed for any of the genes tested. The diagram shows that 20 genes, all containing CLEAR sites in their promoters, are represented in both categories.

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Lysosomes [electronic resource] in SearchWorks catalog

This is likely to be an underestimate as it is based on highly stringent statistical criteria and on a single cell type. The graph shows the enrichment plots generated by GSEA analysis of ranked gene expression data left: upregulated, red; right: downregulated, blue. The enrichment score is shown as a blue line, and the vertical blue bars below the plot indicate the position of lysosomal genes carrying CLEAR sites in their promoters. The arrows indicate the storage of glycosaminoglycans in untreated MSD cells.

The experiment shows that cells treated with TFEB no longer display accumulation of undigested glycosaminoglycans. Untreated cells show an extensive vacuolization due to the storage of undigested glycosaminoglycans. Cells treated with TFEB show that the cellular vacuolization is largely reversed.