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EP-4735884-A1 - METHOD OF SCREENING COMPOUNDS FOR ASSESSING ENDOCYTIC ACTIVITY IN THE CONTEXT OF NEURODEGENERATIVE DISORDERS

EP4735884A1EP 4735884 A1EP4735884 A1EP 4735884A1EP-4735884-A1

Abstract

The present invention is drawn to an in vitro assay for screening the compounds. The compounds are assessed for their ability to induce endocytic activity in cells containing aggregates commonly found in the neurodegenerative disorders. It is proposed that the compounds that can induce endocytic activity would be capable of positively intervening in the neurodegenerative disorders.

Inventors

  • SUBRAMANYAM, DEEPA
  • SINGH, Surya Bansi
  • Majumdar, Amitabha

Assignees

  • National Centre For Cell Science

Dates

Publication Date
20260506
Application Date
20240626

Claims (20)

  1. 1. An in vitro method for screening compounds to assess endocytic activity comprising the steps of: i. contacting cell with a test compound and with a control; ii. measuring the endocytic activity in contacted cell obtained in step (i), by analysing movement of clathrin coated structures through live cell imaging and comparing with the control; iii. determining the test compounds that have endocytic activity obtained in step (ii).
  2. 2. The in vitro method for screening compounds as claimed in Claim 1, wherein the cell is selected from the group comprising mammalian neurons expressing pathogenic form of Huntington protein with CAG repeats in the range of 36-138 and Drosophila hemocytes.
  3. 3. The in vitro method for screening compounds as claimed in Claim 2, wherein the Drosophila hemocytes express pathogenic proteins comprising Huntington protein, HTTQ138, TDP43, A(beta)42, Alpha synuclein.
  4. 4. The in vitro method for screening compounds as claimed in Claim 2, wherein the cell has clathrin light chain tagged with a fluorescent protein at the N-terminal of the protein.
  5. 5. The in vitro method for screening compounds as claimed in Claim 4, wherein the fluorescent protein is selected from the group consisting of GFP, RFP, dsRed, tdTomato, YFP, Neon.
  6. 6. The in vitro method for screening compounds as claimed in claim 1, wherein the test compound is selected from the group comprising pharmacological modulators of actin polymerization comprising Latrulculin A, Cytochalasin D, mimics of Hipl or Arp2/3 complex components.
  7. 7. The in vitro method for screening compounds as claimed in claim 1, wherein the test compound exhibits endocytic activity in neurodegenerative disease.
  8. 8. The in vitro method for screening compounds as claimed in claim 7, wherein the neurodegenerative disease is selected from the group comprising Huntington’s disease, amyotrophic lateral sclerosis, Alzheimer’s disease and frontotemporal dementia.
  9. 9. The in vitro method for screening compounds as claimed in step (i) of claim 1, wherein the cells are contacted with the test compound and control comprising i. adding media in range of lOOpl to lOOOpl to the cells, ii. centrifugation of cells obtained in step (i) at 200 rpm to 1000 rpm, at 25-37 °C iii. plating the cells obtained in step (ii) in a separate cell culture dish using the media, iv. incubating the plate obtained in step (iii) for 15 to 25 minutes in the CO2 incubator at 25 °C, v. allowing cells obtained in step (iv) to adhere to the cell culture dish.
  10. 10. The in vitro method for screening compounds as claimed in claim 9, wherein the media is selected from the group comprising Schneider’s media or DMEM media supplemented with 10% fetal bovine serum, preferably Schneider’s media.
  11. 11. The in vitro method for screening compounds as claimed in step (i) of claim 1, wherein the cells in contact with the control are resuspended in solvent.
  12. 12. The in vitro method for screening compounds as claimed in claim 11, wherein the control comprises a wild type cell of Drosophila hemocyte origin, mammalian neurons or cells expressing non-pathogenic protein as a positive control.
  13. 13. The in vitro method for screening compounds as claimed in claim 12, wherein the non-pathogenic protein is HTTQ15.
  14. 14. The in vitro method for screening compounds as claimed in claim 11, wherein the solvent is a polar solvent selected from the group comprising DMSO, distilled water or ethanol.
  15. 15. The in vitro method for screening compounds as claimed in step (ii) of claim 1, wherein the endocytic activity in the cell obtained in claim 9 is observed and compared with the control obtained in claim 11 through light microscopy.
  16. 16. The in vitro method for screening compounds as claimed in step (iii) of claim 1, wherein the endocytic activity in the cell obtained in claim 9 is imaged and compared with the control as obtained in claim 11 through light microscopy to determine movement of clathrin light chain.
  17. 17. The in vitro method for screening compounds as claimed in claims 15-16, wherein the light microscopy is selected from the group comprising bright field microscopy, confocal microscopy, total internal reflection fluorescence microscopy or high content imaging platform.
  18. 18. A kit for screening compound for assessing endocytic activity comprising • pipette and pipette tips of appropriate volume, • microcentrifuge tubes or any container to hold the cells, • 1 vial containing Wild type cells expressing clathrin light chain tagged with fluorescent protein, • 1 vial containing non-pathogenic cells expressing clathrin light chain tagged with fluorescent protein to serve as control, • 1 vial containing pathogenic protein expressing clathrin light chain tagged with fluorescent protein, • 1 vial containing control drug or/and compound dissolved in solvent, • 1 vial of solvent control, • Cell culture media, • Cell culture glass-bottomed dishes or plates for plating cells and imaging.
  19. 19. The kit as claimed in claim 18, wherein wild type cell is selected from the group comprising Drosophila hemocyte origin, mammalian neurons.
  20. 20. The kit as claimed in claim 18, wherein non-pathogenic protein HTTQ15.

Description

METHOD OF SCREENING COMPOUNDS FOR ASSESSING ENDOCYTIC ACTIVITY IN THE CONTEXT OF NEURODEGENERATIVE DISORDERS FIELD OF THE INVENTION The present invention generally relates to the field of Biotechnology. In particular, it relates to a method of screening compounds for assessing endocytic activity. BACKGROUND OF THE INVENTION Neurodegenerative diseases are defined by a group of neurological disorders which adversely affect the structure and function of neurons. Neurodegenerative disorders include Huntington’s disease, amyotrophic lateral sclerosis, Alzheimer’s disease, frontotemporal dementia etc. In most of these neurodegenerative disorders, aggregates are observed in the nucleus and cytoplasm of affected neurons. It has been demonstrated that such aggregates can spread from one cell to another, with aggregates accumulating at synaptic terminals in the central brain that spread over time to other regions resulting in a loss of neuronal population and degeneration of neurons in basal ganglia and in cortical region. It has been noted that Drosophila brain, mimics similar protein aggregation resulting in neuronal toxicity and death. It has also been noted that endocytic activity is compromised in the presence of certain aggregates. It is supposed that drugs which can induce endocytic activity in the presence of such aggregates can positively intervene in such neurodegenerative disorders. There are certain in vitro assays to determine the presence of neurodegenerative disorders. Many such assays examine the miRNA mis-regulation, mitochondrial behaviour, or transcriptomic based analysis. US2013/0303562 utilises a Drosophila neuronal culture model expressing either HTTQ15 (non-aggregating) form, or HTTQ138 (aggregating form) to assess the effects of various compounds using a high throughput-based screening approach to assess number and size of aggregates. However, this assay does not examine the ability of the compounds to induce endocytic activity in the presence of the aggregates. In Cardoso et al., Methods in Molecular Biology, vol. 2233, Page 71-91, a screening technique utilizing uptake of specific ligands as a readout for endocytosis has been disclosed. The paper discloses a method to screen endocytosis inhibitors to identify drugs that can inhibit ligand uptake through endocytosis, using fluorescently-labeled transferrin or EGF (readout for clathrin mediated endocytosis), or Dextran (for macropinocytosis), or cholera toxin (for clathrin-independent carriers(CLIC)/ Glycosylphosphotidylinositol- anchored protein (GPI-AP) enriched compartments (GEEC) endocytosis) on U2OS cells. The method involves addition of ligand to the cultured cells and then measuring uptake of the ligands upon treatment with a control compound or an inhibitor of endocytosis. This uptake is measured after fixing the cells and using microscopy to assess uptake. However, the method does not study the movement of clathrin coated structures/vesicles in the context of pathogenic aggregating proteins involved in neurodegeneration and does not disclose a rescue of movement. It is considered that the ability of the compounds to induce endocytic activity may be an indicator for the positive intervention of the compounds in neurogenerative disorders. Accordingly, there is a need for a screening method to assess the endocytic activity. OBJECT OF THE INVENTION An object of the invention is to provide a method of screening compounds for assessing endocytic activity in the context of neurodegenerative disorders. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A depicts a Kymograph representation of movement of clathrin coated structures (CCSs). Kymograph, representing the spatial position of CCSs over time, shows the movement of CCSs in wild type cells, whereas movement is stalled in presence of pathogenic Huntingtin aggregates. X - axis represents distance and Y - axis represents time in kymograph. Figure IB depicts Graph showing radial movement of CCSs in wild type cells. X - axis represents distance covered by CCSs in pm and Y - axis represents radial speed of CCSs in pm/ sec. Polar histogram of distribution of vectors obtained from particle image velocimetry (PIV) analysis shows the vectors aligned to 180°, showing the centripetal movement of CCSs in wild type cells. Figure 1C depicts Graph showing stalled movement of CCSs in HTTQ138 cells. X - axis represents distance covered by CCSs in pm and Y - axis represents radial speed of CCSs in pm/ sec. Polar histogram of distribution of vectors obtained from PIV analysis shows broad distribution of the angles, indicating the absence of any directional centripetal movement of CCSs in presence of HTTQ138. Figure 2A depicts Kymograph representation of movement of CCSs in presence of HTTQ138 aggregates and HTTQ138+Mrj condition. Kymograph, representing the spatial position of CCSs over time, shows the movement of CCSs upon Mrj overexpression, whereas movement is stalled in presence of pathogenic Huntingtin agg