Claims:
1. A method to screen and identify test compounds that can restrict the trans-cellular propagation of the neurotoxic tau aggregates, the method comprising the steps of:
a) selecting a line of Drosophila flies and raising them;
b) supplementing the food of the Drosophila flies with a test compound and ageing the Drosophila flies to obtain treated flies;
c) de-capitating heads of the treated flies and sectioning the head samples;
d) incubating the head samples obtained in (c) with anti-human tau primary antibody and immunostaining the tau protein-antibody complex;
e) visualizing the tau protein-antibody complex under a microscope and measuring the distance of the propagated neurotoxic tau aggregates;
f) comparing the distance of the propagated neurotoxic tau aggregates in treated flies with the distance of the propagated neurotoxic tau aggregates in control flies,
characterised in that the distance of the propagated neurotoxic tau aggregates is measured from the mid-point of the outer lamina to the tau aggregates that has migrated the farthest.

2. The method as claimed in claim 1, wherein the control flies are given food not supplemented with the test compound.

3. The method as claimed in claim 1, wherein the food for Drosophila flies is selected from cornmeal, agar, yeast food media.

4. The method as claimed in claim 1, wherein the test compound may be selected from drugs, chemical compounds and molecules of interest.

5. The method as claimed in claim 1, wherein the line of Drosophila flies is selected from UAS-tauWT Drosophila line expressed under GMR-Gal4 driver line and UAS-tauV337M Drosophila line expressed under GMR-Gal4 driver line.

6. The method as claimed in claim 1, wherein the Drosophila flies are aged for 1, 5, 10 and 15 days under separate batches.

7. The method as claimed in claim 1, wherein the stained antibody complex is visualised under a microscope selected from fluorescence, apotome, and confocal microscope.

Description:
Field of Invention
The present invention relates to a method of screening and identification of drugs in a Drosophila model. Particularly, the present invention relates to screening of drugs/compounds/molecules which restrict or minimize the age dependent trans-cellular propagation of the neurotoxic tau aggregates.

Background and Prior art
Neurodegenerative disorders are age onset illnesses which develop due to degeneration of specific neuronal cells of the brain. Tauopathies represent a group of such human brain disorders and include several diseases such as Alzheimer’s disease, Parkinson’s disease, Fronto-temporal dementia, Pick’s disease, etc. These neurodegenerative disorders are characterized by an accumulation of aggregated tau protein in the specific area(s) of brain and impair memory, cognition, and movement (Williams, 2006; Ferrer et al., 2014; Kovacs, 2017). They manifest themselves through mechanisms which are not fully revealed yet. Here it also worth noting that World Health Organization (WHO) has also predicted that neurodegenerative diseases will emerge as the second-most widespread cause of death in the next 20 years. According to the Alzheimer’s and Related Disorders Society of India-2018, the number of cases of dementia in India was 3.1 million in 2005, 4.4 million in 2015 and would increase to 14.3 million in 2050.

Dementia causing human neurodegenerative disorders, broadly classified as tauopathies, such as Alzheimer’s, Parkinson’s diseases, frontotemporal dementia with parkinsonism-17 (FTDP-17), etc. pose a severe burden on the health services and economy every year worldwide. The population suffering from these neurological disorders is predicted to grow enormously in size by 2030 in India (Kumar et al., 2020), thus, further increasing the burden on the health services and economy. The major problem with these complex disorders remains their poor diagnoses, lack of effective treatment measures and unavailability of effective drug molecules. At present, no effective therapy is available to cure tauopathies and the available treatments only manage the symptoms. Hence, there is an urgent need to identify the effective drugs/molecules/compounds to develop treatments strategies for this kind of disease.

Age-dependent trans-cellular propagation or spreading of the neurotoxic protein aggregates such as tau containing neurofibrillary tangles (NFTs) and Paired Helical Filaments (PHFs) has emerged as a major cause of the age-dependent progression of dementia causing tauopathies such as Alzheimer’s, Parkinson’s diseases (Clavaguera et al., 2020; Vogel et al., 2020). Therefore, screening and characterization of potential drugs/compounds/molecules with properties to restrict the propagation of such disease-causing tau aggregates can prove to be enormously beneficial for the development of effective therapeutic approaches against Alzheimer’s, Parkinson’s diseases. However, due to lack of suitable in-vivo model system, at present it is extremely difficult to screen the drugs/compounds/molecules which could efficiently restrict the transcellular propagation of neurofibrillary tangles (NFTs) and/or Paired Helical Filaments (PHFs).

Due to the several limitations associated with human as a model system, unavailability of human brain tissues and absence of flexible genetic/molecular tools in mammalian model systems, it is difficult to screen and characterize effective drug molecules which can suppress the pathogenesis and progression of these devastating human neurodegenerative disorders. As per the current understanding, aetiology of human tauopathies begins with the pathogenic aggregation of tau monomers to form toxic insoluble filaments known as paired helical filaments (PHFs) and mature neurofibrillary tangles (NFTs) (Kovacs, 2017). Formation of NFTs has been considered as a hallmark of the tauopathies. The abundance and distribution of these neurotoxic NFTs in brain show a direct correlation with the disease phenotypes (Serrano-Pozo et al., 2011; Bejanin et al., 2017). Interestingly, transcellular spreading of NFTs from the diseased cells to adjacent healthy cells has emerged as one of the key mechanisms for age-dependent progression of tauopathies in human (Vogel et al., 2020; Brunello et al., 2020; Ruan et al., 2021; Wegmann et al., 2019). Therefore, age-dependent progression of tauopathies can be significantly curtailed by restricting trans-cellular spreading of tau aggregates and/or NFTs from affected to the healthy neurons. Also, a major limitation attached with the mammalian models is that they require injection of human tau aggregates in the brain which exceeds the natural amount of tau aggregates and do not reflect an ideal in-vivo condition (Vogel et al., 2020).

Therefore, there is a need of a methodology to screen potential drugs/ compounds/molecules against tau propagation.

Singh, Sandeep Kumar et al. “Overview of Alzheimer's Disease and Some Therapeutic Approaches Targeting Aß by Using Several Synthetic and Herbal Compounds.” Oxidative medicine and cellular longevity vol. 2016 (2016): 7361613. doi:10.1155/2016/7361613 discloses Drosophila melanogaster as a transgenic Model of Alzheimer’s Disease. The document further discloses that Drosophila disease model can be used for novel drug screening against AD. However, the document does not disclose screening drugs to reverse tau protein aggregates propagation.

Shim KH, et.al., “Small-molecule drug screening identifies drug Ro 31-8220 that reduces toxic phosphorylated tau in Drosophila melanogaster”. Neurobiol Dis. 2019 Oct; 130:104519. doi: 10.1016/j.nbd.2019.104519. Epub 2019 Jun 22. PMID: 31233882 discloses the ability of small therapeutic compounds (a custom library) to improve tau-induced rough-eye phenotype in a Drosophila melanogaster model of frontotemporal dementia. The document assessed the tau phosphorylation in vivo and selected hit compounds. Among the potential hits, Ro 31-8220, described earlier as a potent PKCa inhibitor was investigated as it robustly improved the rough-eye phenotype, reduced phosphorylated tau species in vitro and in vivo, reversed tau-induced memory impairment, and improved the fly motor functions.

WO2011140997A1 discloses transgenic Drosophila model and method for screening therapeutic drugs for Alzheimer's disease. However, the document does not disclose drugs against tau propagation.

US20040137522A1 discloses identification of several genes that either enhance or suppress Tau-related neurodegeneration when expressed. The document also discloses an assay for screening a test compound for its ability to increase the expression of a DNA that suppresses Tau-related neurotoxicity. Specifically, a test compound is screened for its ability to increase or decrease the expression of a gene that modulates Tau-related neurotoxicity by incubating cells that express the gene in the presence of the test compound and measuring expression using standard techniques. The document uses a Drosophila tauopathy model based on the GAL4-UAS expression system, in which a human tau transgene downstream of a yeast upstream activating sequence (UAS) is controlled by driver lines which express the GAL4 transcriptional activator in particular spatial and temporal patterns. Specifically, a genotype UAS- tauV337M /+; GMR-GAL4/+, with a moderately rough eye was chosen to identify both enhancers and suppressors of Tau toxicity.

Here it is important to note that although age-dependent trans cellular propagation of tau aggregate is the major cause of the disease progression (Clavaguera et al., 2020; Vogel et al., 2020), however none of the above-mentioned Drosophila based prior arts facilitates the screening or identification of explicit drugs/compounds/molecules which could restrict or minimize the propagation of the neurotoxic tau aggregates.

Hence, there is a need in the art for screening of specific drugs/compounds/molecules which could restrict or minimize the age-dependent trans-cellular propagation of the tau aggregates. The present invention facilitates a rapid and precise screening or identification of the drugs/compounds/molecules for their ability to restrict the propagation of tau aggregates, which is otherwise not possible with any of the prior arts as of now.

Object of the invention
An object of the present invention is to overcome the drawbacks of the prior art.

An object of the present invention is to provide a method for screening drugs/compounds/molecules which restrict or minimize the trans-cellular propagation of the neurotoxic tau aggregates in a Drosophila model.

Another object of the present invention is to provide a rapid screening and identification of potential drugs/compounds/molecules to restrict the trans-cellular spreading/ propagation of dementia causing neurofibrillary tangles (NFTs) and/or tau aggregates.

Yet another object of the present invention is to provide potential drugs/compounds/molecules that can restrict the trans-cellular spreading/ propagation of dementia.

Summary of the Invention
The following disclosure presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

In an aspect of the present invention, there is provided a method to screen and identify test compounds that can restrict the trans-cellular propagation of the neurotoxic tau aggregates, the method comprising the steps of: a) selecting a line of Drosophila flies and raising them; b) supplementing the food of the Drosophila flies with a test compound and ageing the Drosophila flies to obtain treated flies; c) de-capitating heads of the treated flies and sectioning the head samples; d) incubating the head samples obtained in (c) with anti-human tau primary antibody and immunostaining the tau protein-antibody complex; e) visualizing the tau protein-antibody complex under a microscope and measuring the distance of the propagated neurotoxic tau aggregates; f) comparing the distance of the propagated neurotoxic tau aggregates in treated flies with the distance of the propagated neurotoxic tau aggregates in control flies,
characterised in that the distance of the propagated neurotoxic tau aggregates is measured from the mid-point of the outer lamina to the tau aggregates that has migrated the farthest.

Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses exemplary embodiments of the invention.

Brief Description of Accompanying Drawings
The above and other aspects, features and advantages of the embodiments of the present disclosure will be more apparent in the following description taken in conjunction with the accompanying drawings, in which:

Figure 1 illustrates a flow chart of the method for screening of drugs/compounds/ molecules which restrict or minimize the trans-cellular propagation of the neurotoxic tau aggregates.

Figure 2 illustrates data collection represented graphically for control and treated group in an age-dependent manner.

Figure 3 illustrates method for measuring distance of the propagated tau aggregates/NFTs.

Figure 4 illustrates disodium cromoglycate mediated restriction of the age-dependent propagation of tau aggregates/NFTs in Drosophila in adult heads. A-D' represent the confocal images of adult eye sections stained with 5A6 antibody (total tau) across different ages. (A) 1-day old control section depicts minimal propagation; (A') 1-day old treated head section shows similar propagation; however, decreased abundance of aggregates can be noted. (B) The control section of 5 days old head section displays the beginning of the migration of tau tangles; (B') which are seen to be restricted in the age-matched treated head section. (C) The 10 days old head section exhibits extensive propagation in the control group; (C') which has successfully been suppressed in the age-matched treated counterparts. (D) 15 days old head section shows the maximum propagation with aggregates reaching up to the central brain region; (D') however, disodium cromoglycate treatment effectively restricts the spreading of the tau aggregates/NFTs in the age-matched adult head. (E) Graph depicting the comparative distance propagated by the tau aggregates from the outer lamina in control vs treated tissues across different age group. (F) The schematic representation of the adult fly eye representing the various regions. Statistical analysis has been performed by unpaired student’s t-test. (Scale bar: A- D'= 50 µm; *p?=?0.0191, **p =?0.0069 and ****p<0.0001)

Detailed Description of the Invention
The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of various embodiments of the present disclosure as defined by the claims and their equivalents. It includes various specific details to assist in that understanding, but these are to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the various embodiments described herein can be made without departing from the scope and spirit of the present disclosure. In addition, descriptions of well-known functions and constructions may be omitted for clarity and conciseness.

The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the present disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of various embodiments of the present disclosure is provided for illustration purpose only and not for the purpose of limiting the present disclosure as defined by the appended claims and their equivalents.

All terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which various embodiments belong. Further, the meaning of terms or words used in the specification and the claims should not be limited to the literal or commonly employed sense but should be construed in accordance with the spirit of the disclosure to most properly describe the present disclosure.

The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising" used herein specify the presence of stated features, integers, steps, operations, members, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, members, components, and/or groups thereof.

The present disclosure will now be described more fully with reference to the accompanying drawings, in which various embodiments of the present disclosure are shown. The present disclosure may, however, be embodied in many different forms and should not be construed as being limited to the various embodiments set forth herein, rather, these various embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the present disclosure.

The present invention provides a Drosophila based in-vivo method which facilitates rapid screening of drugs/compounds/ molecules which restrict or minimize the trans-cellular propagation of the neurotoxic tau aggregates.

The method for screening of drugs/compounds/ molecules which restrict or minimize the trans-cellular propagation of the neurotoxic tau aggregates includes the following steps:

a) Untreated/control group of GMR-Gal4>UAS-tauWT or GMR-Gal4>UAS-tauV337M flies are taken where no drugs/ compounds / molecules of interest in supplemented in standard Drosophila food

b) Treated/experimental group of GMR-Gal4>UAS-tauWT or GMR-Gal4>UAS-tauV337M flies are taken where Drugs/ compounds / molecules of interest are added in standard Drosophila food

c) The adult flies of both the groups are aged for 1, 5, 10 and 15 days in separate batches at 22-25 0C. The flies are transferred to fresh food vial at alternative days.

d) De-capitation of differentially aged adults is done and paraffin embedded moulds are prepared

e) The moulds containing adult heads from untreated and treated groups are sectioned to generate 10-22 µm thick sections followed by immunostaining with anti-Tau (5A6) antibody

f) Fluorescence/apotome/confocal microscopic observation of stained tissues and quantification of propagated NFTs/tau aggregates in an age-dependent manner is carried out.

The method is illustrated by way of a flowchart in Figure 1. The observation and data collection are done as shown in Figure 2. The distance of migration of NFTs/tau aggregates in an age-dependent manner are measured and mapped.

To execute the method, the present inventors have utilised UAS-tauWT Drosophila line which expresses wild-type form of human tau transgene under UAS promoter (Wittmann et al., 2001). Upon driving with GMR-Gal4 driver line (Hay et al., 1994), flies replicate the characteristic features of human neuronal tauopathies such as accumulation of abnormal tau and NFTs and progressive neurodegeneration (Chanu and Sarkar, 2017).

To establish the methodology, the present inventors have used disodium cromoglycate, which is conventionally an anti-allergic agent based on cromolyn (Sinniah et al., 2017). Interestingly, cromolyn has also been reported to be an inhibitor of Gsk3ß (Motawi et al., 2013). Gsk3ß is a major contributor to tau pathogenesis as it is one of the most potent kinases that brings about tau hyperphosphorylation (Lauretti et al., 2020).

The method described above was followed to examine the impact of disodium cromoglycate on trans-cellular propagation of NFTs/tau aggregates in variably aged adult Drosophila heads. The method revealed that disodium cromoglycate is enormously proficient in limiting the propagation of tau aggregates/NFTs up to 93.025 ± 7.86µm distance in the treated group (as compared to an average of 116.7± 13.447µm travelled by the aggregates in the control group even in the 15 days old flies.

The present invention therefore provides disodium cromoglycate as a promising drug which can efficiently restrict age-dependent trans-cellular migration of the neurotoxic NFTs/tau aggregates.

Therefore, the present invention relates to a method to screen and identify various drugs/compounds/molecules that can restrict the propagation of NFTs and tau aggregates, and eventually suppress the age-dependent disease progression.

The present invention provides a method to screen and identify test compounds that can restrict the trans-cellular propagation of the neurotoxic tau aggregates, the method comprising the steps of:

a) selecting a line of Drosophila flies and raising them;
b) supplementing the food of the Drosophila flies with a test compound and ageing the Drosophila flies to obtain treated flies;
c) de-capitating heads of the treated flies and sectioning the head samples;
d) incubating the head samples obtained in (c) with anti-human tau primary antibody and immunostaining the tau protein-antibody complex;
e) visualizing the tau protein-antibody complex under a microscope and measuring the distance of the propagated neurotoxic tau aggregates;
f) comparing the distance of the propagated neurotoxic tau aggregates in treated flies with the distance of the propagated neurotoxic tau aggregates in control flies,
characterised in that the distance of the propagated neurotoxic tau aggregates is measured from the mid-point of the outer lamina to the tau aggregates that has migrated the farthest.

As provided herein the control flies are given food not supplemented with the test compound. The rest of the method is the same.

The food for Drosophila flies is selected from cornmeal, agar, yeast food media.
The test compound may be selected from drugs, chemical compounds and molecules of interest.

The test compounds are dissolved or diluted in appropriate solvent to make a stock solution. An appropriate amount of the stock solution is added to Drosophila food to achieve desired working concentration(s). Concentration of the new drugs/compounds/molecules being tested should be standardized with every new screening process as per protocols in the state of art.

In one embodiment, the method described herein uses a line of Drosophila carrying UAS-tauWT transgene expressed under GMR-Gal4 driver line. Another Drosophila line, UAS-tauV337M can also be utilized for the screening process.

As provided herein, the Drosophila flies are aged up to 15 days in identical conditions. In a preferred embodiment, the flies are aged for 1, 5, 10 and 15 days under separate batches. The flies are aged at a temperature of 22-25°C.

Incubation with anti-human tau primary antibody (5A6) and immunostaining of the tau protein-antibody complex is carried out by methods known in the art. Another antibody, anti-tau rabbit monoclonal antibody (catalog # M00097-1. Boster, USA) can also be utilized.

The stained antibody complex is visualised under a microscope selected from fluorescence, apotome, and/or confocal microscope.

The images of stained tissues of all the age groups (control and treated) under fluorescence/apotome/confocal microscope are captured using appropriate filters/lasers. The distance of the propagated tau aggregates/NFTs are measured from the mid-point of the outer lamina to the tau aggregate/NFT that has migrated the farthest using suitable manual and/or software-based quantification method.

The distance of the propagation of tau aggregates in treated groups are compared to the distance of the propagation of tau aggregates in the control group of the same age. Where the distance in the treated group is less than the control group, the test compound added in the food of the respective treated group is identified to restrict the trans-cellular propagation of the neurotoxic tau aggregates.

The method as described herein is allows rapid screening of the potential drug molecules within the span of 35-45 days, with accuracy and reproducibility.

Advantages:
• The developed method allows rapid screening and identification of potential drugs/compounds/ molecules to restrict trans-cellular spreading/ propagation of dementia causing neurofibrillary tangles (NFTs) and/or tau aggregates. Identification of such drugs/compounds/molecules would be immensely helpful in developing the effective treatment strategies against human neurodegenerative tauopathies.
• This method allows screening of large number of drugs/ compounds/ molecules within a span of about 35-45 days.
• The method allows large number of sampling/ data analysis within a short period to ensure the actual efficacy of the identified drugs/ compounds/ molecules.
• This method also allows quantification of the relative cellular abundance of neurofibrillary tangles (NFTs) and/or tau aggregates in control and test samples.
• The method is very economical as this does not involve any sophisticated and/or expensive tool and/or material.

The present invention is now being illustrated by way of non-limiting examples. The examples are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. Efforts have been made to ensure accuracy with respect to numbers used, but some experimental errors and deviations should be accounted for.

Examples
A. Detailed material and methods
1. Fly stocks, rearing condition, genetic crosses and aging
Live Drosophila (a non-hazardous well-established insect model system) which is publicly available for procurement from the Bloomington Drosophila Stock Centre, USA, for research and invention purposes is used in the present method.

The fly stocks are raised on standard cornmeal/agar/yeast media at 22-25 0C under 12:12 hour dark:light cycle. A genetic cross is set between w-; +/+; UAS-tauWT/UAS-tauWT or UAS-tauV337M; +/+; +/+ and w-; GMR-Gal4/GMR-Gal4; +/+ flies on normal food and collect the F1 progenies with w-; GMR-Gal4/+; UAS-tauWT/+ (GMR-Gal4>UAS-tauWT) or UAS-tauV337M; GMR-Gal4/+; +/+ (GMR-Gal4>UAS-tauV337M) genotype on the first day of their eclosion. The w-; +/+; UAS-tauWT/UAS-tauWT transgenic Drosophila line expresses human-tau, UAS-tauWT (0N4R variant - with four microtubule binding domains; no N-terminal region) transgene under UAS promoter (Wittmann et al., 2001). The GMR-Gal4 line (Bloomington stock #1104; Hay et al., 1994) drive the expression of tauWT transgene in the eye region (Wittmann et al., 2001). The flies are aged in independent batches on normal food and drug-supplemented food (in appropriate amount/ concentration) in identical conditions and sacrificed once the desired aging is completed. During the course of aging, the flies are transferred to fresh food vials on every alternative day to maintain healthy culture conditions.

2. Drug supplementation
Appropriate amount/concentration of drugs/compounds/molecules of interest are added to the standard Drosophila cornmeal/agar/yeast food media to evaluate their post-feeding inhibitory effect on the propagation of NFTs/tau aggregates. The drugs/compounds/molecules are dissolved or diluted in appropriate solvent to make a stock solution. An appropriate amount of the stock solution is added to Drosophila cornmeal/agar/yeast food to achieve desired working concentration(s). If the drugs/compounds/ molecules are thermo-labile and/or light sensitive, they have to be prevented from direct light exposure and any potential thermo-degradation.

The disease bearing (GMR-Gal4>tauWT) freshly eclosed adult flies were raised one batch each on normal food (control group) and the food supplemented with 2 µM of disodium cromoglycate (test group) and aged up to 15 days in identical conditions as specified in the method described above.

3. Histology and immunohistochemistry
For adult head sectioning and immunohistochemistry, decapitated heads is fixed in freshly prepared 4% paraformaldehyde in 1X PBS (Phosphate Buffer Saline) (137mM NaCl, 2.68mM KCl, 1.47mM KH2PO4, 8.1mM Na2HPO4) and embed in paraffin wax (A6330, Tissue Embedding Medium, Sigma-Aldrich, USA) to prepare moulds. The differentially aged adult heads are sectioned to obtain 10-22 µm thick tissue slices on slides using a manual or automated microtome/cryotome. The tissues are de-waxed and incubated with anti-human tau primary antibody (5A6, 1:1000, DSHB, USA) for overnight at 4 °C, followed by washing with 1X PBST (137mM NaCl, 2.68mM KCl, 1.47mM KH2PO4, 8.1mM Na2HPO4, 0.1% Tween20) and incorporation of appropriate secondary antibody at room temperature for 2 hours. Counterstaining the tissues with DAPI (5µg/ml, Roche Diagnostics GmbH, Germany) is done and mount in antifade reagent (P36934, Thermo Fisher Scientific, USA) by placing a cover slip.

4. Data collection and analysis
The images of stained tissues of all the age groups (control and treated) are observed and captured under fluorescence/apotome/confocal microscope using appropriate filters/lasers. The distance of the propagated tau aggregates/NFTs is measured from the mid-point of the outer lamina to the tau aggregate/NFT that has migrated the farthest as suggested in the schematic Figure 3 using suitable manual and/or software-based quantification method.

Measuring the distance from any arbitrary point other than the mid-point of the outer lamina shall yield an erroneous distance value. Appropriate statistical tests such as student’s t-test and one-way/two-way ANOVA is applied to the raw data to determine the level of significance. Data is represented graphically for control and treated group in an age-dependent manner.

B. Test Results
The impact of disodium cromoglycate on age-dependent trans-cellular propagation of NFTs/tau aggregates was examined. An insignificant difference in the prevalence of NFTs/tau aggregates was observed in the 1-day old flies in control (distance of tau aggregates/NFTs from the mid-point outer lamina: 57.53 ± 7.07 µm) (Fig. 4A) as well as treated group (distance of tau aggregates/NFTs from the mid-point outer lamina: 53 ± 7.9 µm) (Fig. 4A'). However, on further examining the 5 days old adult head sections, the drug was noted to be effective in restricting the migration of the tau aggregates, as the distance of tau aggregates/NFTs from the mid-point of outer lamina was 72 ± 8.2 µm in control tissues (Fig. 4B) compared to 60 ± 6.89µm in the drug-fed group (Fig. 4B'). It was further noted that the drug was extremely effective in suppressing the degree of migration from 104.49 ± 7.28µm in control group (Fig. 4C) to as less as 75 ± 8.28 µm in treated (Fig. 4C') in 10 days old flies. Interestingly, the method revealed that disodium cromoglycate is enormously proficient in limiting the propagation of tau aggregates/NFTs up to 93.025 ± 7.86µm distance in the treated group (Fig. 4D) as compared to an average of 116.7± 13.447µm travelled by the aggregates in the control group (Fig. 4D') even in the 15 days old flies.

Therefore, disodium cromoglycate was identified as a promising drug which can efficiently restrict age-dependent trans-cellular migration of the neurotoxic NFTs/tau aggregates using the method as described above.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The invention is, therefore, to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.