Method of screening compounds for assessing endocytic activity in the context of neurodegenerative disorders

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.

Description

[0001] METHOD OF SCREENING COMPOUNDS FOR ASSESSING ENDOCYTIC ACTIVITY IN THE CONTEXT OF NEURODEGENERATIVE DISORDERS

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to the field of Biotechnology. In particular, it relates to a method of screening compounds for assessing endocytic activity.

[0004] BACKGROUND OF THE INVENTION

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] OBJECT OF THE INVENTION

[0011] An object of the invention is to provide a method of screening compounds for assessing endocytic activity in the context of neurodegenerative disorders.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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.

[0014] 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.

[0015] 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 aggregates. X - axis represents distance and Y - axis represents time in kymograph. Figure 2B depicts Graph showing radial movement of CCSs. 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 the vectors aligned to 180°, showing the centripetal movement of CCSs upon overexpression of Mrj in HTTQ138 background. Figure 2C depicts Kymograph representation of movement of CCSs in presence of HTTQ138 aggregates and HTTQ138+Arp2 / 3 condition. Kymograph, representing the spatial position of CCSs over time, shows the movement of CCSs upon Arp2 / 3 overexpression, whereas movement is stalled in presence of pathogenic Huntingtin aggregates. X - axis represents distance and Y - axis represents time in kymograph.

[0016] Figure 3 depicts treatment of Drosophila hemocytes with Etoposide and Camptothesin rescues clathrin-coated vesicle movement.

[0017] Figure 4 depicts overexpression of actin binding proteins rescues pathogenic Huntingtin- driven neurodegeneration in the Drosophila eye

[0018] SUMMARY OF THE INVENTION

[0019] 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. DESCRIPTION OF THE INVENTION

[0020] The present invention is drawn to an in vitro method of screening for compounds for assessing endocytic activity in the presence of protein aggregates involved in neurodegenerative conditions.

[0021] The in vitro method for screening compounds to assess endocytic activity comprising the steps of: i. contacting cells with a test compound and with a control; ii. measuring the endocytic activity in contacted cells 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).

[0022] The cells of the present invention may be selected from the group comprising Drosophila hemocytes, mammalian neurons, mammalian neuronal cell lines expressing any pathogenic form of the Huntington protein with CAG repeats in the range of 36-138.

[0023] The Drosophila hemocytes that may be used in the present invention as cells may express either the pathogenic form of Huntington protein, HTTQ138 which forms aggregates, or proteins from other neurogenerative disorders such as TDP43, A(beta)42, Alpha synuclein.

[0024] The cells of the present invention also contain a fluorescent protein tagged clathrin light chain at the N-terminal of the protein . The fluorescent protein tag may be selected from the group comprising but not restricted to GFP, RFP, dsRed, tdTomato, YFP, Neon.

[0025] The test compound can be selected from the group comprising pharmacological modulators of actin polymerization comprising Latrulculin A, Cytochalasin D, mimics of Hip 1 or Arp2 / 3 complex components. The test compound can regulate endocytic activity in neurodegenerative diseases.

[0026] The neurodegenerative diseases may be selected from the group comprising Huntington’s disease, amyotrophic lateral sclerosis, Alzheimer’s disease and frontotemporal dementia. The in vitro method for screening compounds 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 minutes to 25 minutes in the incubator at 25 °C, v. allowing cells obtained in step (iv) to adhere to the culture dish.

[0027] The media can be selected from the group comprising Schneider’s media or DMEM media supplemented with 10% fetal bovine serum, preferably Schneider’s media.

[0028] The cells in contact with the control are resuspended in the solvent.

[0029] The control comprises a wild type (WT) cell of Drosophila hemocyte origin, mammalian neurons or cells expressing non-pathogenic protein as a positive control.

[0030] The non-pathogenic protein is HTTQ15.

[0031] The solvent is a polar solvent selected from the group comprising DMSO, distilled water or ethanol.

[0032] The endocytic activity in the cell contacted with test compound and control is observed and compared with control through light microscopy.

[0033] The endocytic activity in the test compound is observed to determined movement of clathrin light chain through light microscopy.

[0034] The light microscopy is selected from the group comprising bright field microscopy, confocal microscopy, total internal reflection fluorescence microscopy or high content imaging platform.

[0035] Based on the test conducted above, it can be elucidated that Clathrin light chain tagged to fluorescent protein displays centripetal movement in WT cells and cells containing nonaggregating forms of the protein. However, in cells containing pathogenic aggregates of HTTQ138, or TDP-43, there is a complete loss of this movement. Addition of the positive control will restore this movement.

[0036] In an aspect, the present invention discloses a kit comprising the components,

[0037] • pipette and pipette tips of appropriate volume,

[0038] • microcentrifuge tubes or any container to hold the cells,

[0039] • 1 Vial containing WT cells expressing clathrin light chain tagged with fluorescent protein,

[0040] • 1 vial containing HTTQ15 cells expressing clathrin light chain tagged with fluorescent protein,

[0041] • 1 vial containing HTTQ138 (or other pathogenic aggregate) cells expressing clathrin light chain tagged with fluorescent protein,

[0042] • 1 vial containing control drug or / and compound dissolved in solvent,

[0043] • 1 vial of solvent control,

[0044] • Cell culture media,

[0045] • Cell culture glass-bottomed dishes or plates for plating cells and imaging.

[0046] The wild type cells in the kit can be selected from the group comprising wild type cell of Drosophila hemocyte origin, mammalian neurons.

[0047] The non-pathogenic protein in the kit is HTTQ15.

[0048] The pathogenic protein selected from the group comprising Huntington protein, HTTQ138, TDP43, A(beta)42, Alpha synuclein

[0049] The media in the kit can be selected from the group comprising Schneider’s media or DMEM media supplemented with 10% fetal bovine serum, preferably Schneider’s media.

[0050] The solvent in the kit is a polar solvent selected from the group comprising DMSO, distilled water or ethanol

[0051] The imaging of cells in the kit is through light microscopy, and wherein the light microscopy can be selected from the group comprising bright field microscopy, confocal microscopy, total internal reflection fluorescence microscopy or high content imaging platform. Without being limited by theory, it is considered that cells expressing HTTQ138 or TDP43 aggregates show radial speeds below O.OOlum / sec at the periphery of the cell. Any increase in speed will be considered as a positive effect. Additionally, the distribution of vectors obtained from PIV analysis reveal that in WT cells vectors are aligned to 180° showing the centripetal movement of CCSs. Whereas vectors display a broad distribution of angles, indicating the absence of any directional centripetal movement indicating the loss of directionality of CCS movement in the presence of HTTQ138. Any enhancement of vectors aligned to 180° post treatment with test compounds in the presence of pathogenic Htt or TDP43 will be considered as a positive effect.

[0052] The Application is now illustrated by way of examples. The examples are only meant to serve as illustrations and cannot be used to limit the scope of the invention.

[0053] Examples

[0054] Example 1: Screening process of the present invention.

[0055] Drosophila hemocytes expressing either the pathogenic form of Huntingtin protein, HTTQ138 which forms aggregates, or the non-pathogenic form, HTTQ15, which serves as a control. These cells also contain a GFP-tagged clathrin light chain protein, which serves to follow clathrin coated structure movement, an essential feature of clathrin mediated endocytosis. Live cell imaging on hemocytes were performed followed by analysis of movement of Clathrin coated structures which is briefly described below.

[0056] 1.1. Generation of recombinant fly lines:

[0057] Recombinant fly lines were generated by crossing collagen GAL4 (CgGAL4) with Clathrin light chain tagged to GFP (UAS-Clc GFP). Flies from this line were crossed with flies expressing Huntingtin containing 138 polyQ repeats tagged with red fluorescence protein (UAS mRFPHTT Q138) or Wild type or UAS-mRFP-HTTQ15 fly line. Flies from recombinant line CgGAL4 > UAS Clc GFP were crossed with virgin flies collected from lines expressing TDP43 to look at dynamics of CCSs in presence of TDP43 aggregates. Hemocytes used for all experiments were isolated from third instar larvae. Single larvae were dissected with the help of fine tweezers, in Schneider's medium (S2 cell medium) and hemocytes were collected in a 35 mm glass bottom dish.

[0058] 1.2 Image acquisition: Time lapse imaging of Hemocytes was performed along the X- Y plane close to the coverslip to look at the dynamics of Clathrin light chain. All the live cell imaging was done for five minutes at an interval of five seconds. Confocal microscope was used for all imaging purposes. All the images were taken using lOOx plan apo objective having numerical aperture of 1.49, using manufacturer’s software.

[0059] 1.3 Image analysis:

[0060] Directionality and velocity of vesicles were quantified by PIV analysis. All kymographs were generated using multi kymograph plugin in Fiji. To generate the kymograph, a single horizontal line was drawn from the periphery of the cell and the vesicle was tracked.

[0061] 1.4 Results:

[0062] Movement of individual CCSs were tracked and kymographs representing the spatial position of individual CCSs over time were generated. Kymograph analysis shows that movement of Clathrin coated structures (CCSs) was observed in cells isolated from wild type animals (or HttQ15) whereas there was no movement of CCSs in the cells isolated from Huntingtin aggregate-containing (HttQ138) animals (Fig 1 A). Further analysis revealed that CCSs in WT hemocytes appeared to follow a defined radial path towards the centre of the cell with a certain speed (Fig IB), whereas CCSs in cells containing Huntingtin aggregates do not show any directional movement (Fig 1C) indicating that Clathrin dynamics and movement of Clathrin coated vesicles are compromised in presence of Huntingtin aggregates.

[0063] Example 2: Validation of the screening method of the Present invention

[0064] 2,1 Utility and validation of assay:

[0065] The assay of the present invention can be used to screen compounds, small molecules, and genetic interactors. The readout in all these cases will be focused on, but not restricted to the restoration of directional movement and speed of CCSs in the presence of pathogenic forms of neurogenerative disorders. This example focusses on the disorder Huntington’s disease.

[0066] HTTQ138 forms aggregates inside the cell due to misfolding of protein and its toxicity and aggregation was reported to be modulated by DNAJ chaperones. Previously, Drosophila Mrj, a homologue of mammalian DnaJB6 was observed to rescue polyglutamine associated toxicity in Drosophila. In a screen for polyQ modifiers, DNAJB6, a member of the DNAJ (HSP40) chaperone family was found to efficiently suppress polyQ aggregation in cells. Brain-specific co-expression of DNAJB6 in a mouse model of Huntington’s disease delayed symptoms and increased the lifespan of Huntington mice. Previous studies in mice and Drosophila have also shown that polyglutamine toxicity and aggregation can be modulated by DNAJ chaperones. The DNAJ like protein Mrj which is highly enriched in the brain can effectively suppress polyQ toxicity. The assay was validated by overexpressing Mrj in the presence of HTTQ138. Cells expressing Mrj or Arp2 / 3 will serve as a positive control. CCS movement, speed and directionality were rescued when Mij was overexpressed in the presence of HttQ138 (Fig. 2a, b), thus demonstrating that our endocytosis-based assay can be suitably used for screening molecules that regulate Huntingtin toxicity. Further, overexpression of actin modulating proteins, such as Arp3 (component of the Arp2 / 3 complex) can rescue CCS movement in the presence of HttQ138 aggregates (Fig. 2c). This is a novel mechanism which works without reducing the size or the number of Huntingtin aggregates. The inventors of the present invention propose to include various other proteins and molecules that modulate the dynamics of the actin cytoskeleton.

[0067] Example 3: Comparison against Prior Art Assay

[0068] Prior art includes methods for screening of small molecules such as Camptothecin, OH- Camptothecin, 18b-Glycyrrhetinic acid, and Carbenoxolone, and found out that these molecules act as suppressors of polyQ toxicity. Additionally, authors have conducted an siRNA screen to identify the suppressor of polyQ toxicity and found that Ikbl, an upstream kinase in the mTOR / Insulin pathway, acts as a suppressor of polyQ toxicity (Schulte, J et al, 2011). However, what happens to other cellular processes has been poorly characterized. The present invention indicates that clathrin-mediated endocytosis (CME), a key pathway for receptor internalization has been severely compromised in presence of pathogenic HTTQ138 and TDP43 aggregates, and this can be used as a method for screening.

[0069] Example 4: Treatment with Camptothecin and Etoposide

[0070] Camptothecin has been shown to suppress Huntingtin aggregate formation and improve primary Drosophila neuronal culture (US20130303562). Etoposide was also tested in these previous studies. Camptothecin improved the primary neuronal culture morphology at a concentration of 56 pM. However, the inventors of the present invention observed the use both Camptothecin and Etoposide at a concentration of 2 pM on Drosophila hemocytes expressing HttQ138 and clathrin light chain tagged with GFP. Brief treatment with either compound improved clathrin-coated vesicle movements in Drosophila larval hemocytes expressing HttQ138 aggregates (Fig. 3). This indicates that drugs previously identified to reduce aggregate formation, such as Etoposide and Camptothecin can also rescue the movement of CCS. Since the movement of CCS is rescued after acute treatment, this can likely serve as a screening assay for drugs which can then be taken further into long-term assays.

[0071] Example 5: Overexpression of actin-interacting proteins or chaperones can rescue neurodegeneration even in the presence of pathogenic Huntingtin aggregates

[0072] The effect of overexpression of specific proteins which interact with the actin cytoskeleton on neurodegeneration driven by pathogenic Huntingtin aggregates was tested. Overexpression of either Arp3 (a component of the Arp2 / 3 complex) or Hipl (a protein that interacts with clathrin light chain and actin) could rescue the neurodegeneration observed in Drosophila eyes caused by the overexpression of pathogenic HTT Q127 (Fig. 4). This indicates that mechanisms resulting in actin reorganization may contribute largely to the neurodegeneration observed in Huntington’s disorder. Further, increasing the availability of proteins involved in actin reorganization are capable of restoring CME even in the presence of pathogenic aggregates.

Claims

WE CLAIM:

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. 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. 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. 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. 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. 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. The in vitro method for screening compounds as claimed in claim 1, wherein the test compound exhibits endocytic activity in neurodegenerative disease.

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. 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. 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. 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. 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. The in vitro method for screening compounds as claimed in claim 12, wherein the non-pathogenic protein is HTTQ15.

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. 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. 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. 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. 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. The kit as claimed in claim 18, wherein wild type cell is selected from the group comprising Drosophila hemocyte origin, mammalian neurons.

20. The kit as claimed in claim 18, wherein non-pathogenic protein HTTQ15.

21. The kit as claimed in claim 18, wherein pathogenic protein selected from the group comprising Huntington protein, HTTQ138, TDP43, A(beta)42, Alpha synuclein.

22. The kit as claimed in claim 18, 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.

23. The kit as claimed in claim 18, wherein the solvent is a polar solvent selected from the group comprising DMSO, distilled water or ethanol.

24. The kit as claimed in claim 18, wherein the imaging of cell is through light microscopy, and 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.

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