In SITU seeding amplification assay

Abstract

The invention relates to in situ methods for detecting the presence of misfolded proteins in samples (e.g. tissue samples). The invention also relates to the use of said methods in the detection of a proteinopathy or an increased risk thereof in a subject and to methods of determining the efficacy of therapeutic interventions.

Description

[0001] IN SITU SEEDING AMPLIFICATION ASSAY

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The invention relates to in situ methods for detecting the presence of misfolded proteins in samples, such as tissue samples. The invention also relates to the use of said methods in the detection of a proteinopathy or an increased risk thereof in a subject and to methods of determining the efficacy of therapeutic interventions.

[0004] BACKGROUND OF THE INVENTION

[0005] Many neurodegenerative diseases (NDDs), including prion disorders, Parkinson's, and Alzheimer's, are caused by the misfolding and subsequent propagation of misfolding of endogenous proteins, such us prion protein, alpha-synuclein, tau, beta-amyloid, and TAR DNA-binding protein 43 (TDP43). Misfolded proteins propagate their misfolded conformation by recruiting endogenous proteins through 'templating' the pathological conformation and transmitting the misfolding to the endogenous protein. Misfolded proteins aggregate and can infect other molecules and cause cellular dysfunction and eventually cell death.

[0006] This property of NDD-associated proteins has been exploited in seeding amplification assays which detect the presence of self-propagating misfolded host proteins (seeds) in a sample by contacting the sample with recombinant monomeric protein and detecting subsequently formed misfolded protein aggregates. Two commonly used seeding amplification assays are protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC) assays. PMCA assays employ cyclic sonication and RT-QuIC assays employ a combination of temperature-control and high intensity shaking to break down aggregates and produce more seeds which can induce exponential aggregation of monomeric protein into the misfolded form (Figure 1). In the absence of seed, no monomeric protein aggregation occurs.

[0007] A current limitation of seeding amplification assays is that they are limited to in vitro detection of protein aggregation in fluid samples or homogenised tissue samples. Homogenisation of tissue samples prevents the identification of any spatial information relating to the starting seeds. As a result, a major limitation of current seeding amplification assays is the inability to identify where in a sample the seeding aggregation originates, e.g. in which anatomical area the seeding aggregation originates or whether the seed is intracellular or extracellular. As such, it is not currently possible to identify the area of tissue or the specific cells or subcellular compartments from which seeding originates. A further limitation of these methods is the inability to determine how extensive aggregate formation, and therefore protein misfolding, is throughout a tissue sample. There is an urgent and unmet need for seeding amplification assays which can be used to identify both the presence and the localisation of self-propagating misfolded host proteins.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention is based on the surprising discovery that the formation of protein aggregates can be detected in situ, e.g. in (but not limited to) intact tissue samples; and, moreover, that the present invention can provide spatial information on the formation of protein aggregates. Importantly, the method of the invention can be used to determine whether a sample contains misfolded protein seeds, and also where in the sample misfolded protein seeds exist and / or originate, e.g. whether the seed is intracellular or extracellular and the specific cell types and / or subcellular compartments in which misfolded protein seeds occur. The methods of the invention can also be used to determine how extensive protein misfolding is throughout a sample (e.g. a tissue sample) and to identify pathological lesions which have seeding potential. The methods of the invention can also be used to determine the seeding potential of a protein.

[0010] The methods of the invention may also be used to convert protein signal into DNA barcoding to achieve sequencing-based protein quantitation, for example, by chemically coupling a DNA barcode to an anti-tag antibody (e.g. such as in Kim, S.C., Haliburton, J.R., Gartner, Z.J., Abate, A.R. (2022). Single-Cell Protein Profiling by Microdroplet Barcoding and Next-Generation Sequencing. In: Ooi, A.T. (eds) Single-Cell Protein Analysis. Methods in Molecular Biology, vol 2386. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-l-0716-1771-7_7).

[0011] The methods of the invention may also allow detection of proximity of seeding signal to other protein markers, for example to determine cell type identity, by preforming an in situ proximity ligation assay using antibodies against the tag and an antibody for another protein, such as a cell type marker.

[0012] The invention provides an in situ method for detecting misfolded protein in a sample, the method comprising:

[0013] (a) contacting the sample with recombinant monomeric protein;

[0014] (b) incubating the sample under conditions which are:

[0015] (i) suitable for aggregation of the recombinant monomeric protein with misfolded protein; but

[0016] (ii) not suitable for aggregation of the recombinant monomeric protein in the absence of misfolded protein; and

[0017] (c) detecting the formation of protein aggregates in the sample; wherein the presence of protein aggregates in the sample is indicative of the presence of misfolded protein.

[0018] In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tissue sample obtained from a subject.

[0019] In some embodiments, the method comprises detecting the abundance and / or location of misfolded endogenous protein in the sample by detecting the abundance and / or location of protein aggregates.

[0020] In some embodiments, the method comprises fixing the sample after step (b) and prior to step (c).

[0021] In some embodiments, the method comprises agitating the sample during incubation. In some embodiments, agitating the sample comprises shaking and / or sonication.

[0022] In some embodiments, detecting the formation of protein aggregates in the sample comprises contacting the sample with a protein aggregation marker. In some embodiments, the protein aggregation marker is selected from Thioflavin T, Thioflavin S, Thioflavin X, ANS (1-anilinonaphthalene- 8-sulfonate), Bis-ANS (4,4'-bis-l-anilinonaphthalene-8-sulfonate), Congo Red, and SYPRO Orange.

[0023] In some embodiments, detecting the formation of protein aggregates in the sample comprises contacting the sample with an antigen binding molecule specific to the protein aggregate. In some embodiments, the antigen binding molecule is selected from an antibody or an antigen binding fragment thereof and an aptamer.

[0024] In some embodiments, detecting the location of the protein aggregates comprises:

[0025] (a) detecting the cell type in which the protein aggregates occur; and / or

[0026] (b) detecting the subcellular location in which the protein aggregates occur.

[0027] In some embodiments, detecting the protein aggregates comprises immunohistochemistry and / or microscopy-based methods.

[0028] In some embodiments, the misfolded protein is an endogenous misfolded protein.

[0029] In some embodiments, the misfolded protein is a recombinant misfolded protein.

[0030] In some embodiments, the misfolded protein comprises one or more of misfolded alpha-synuclein, misfolded prion protein, misfolded tau, misfolded beta-amyloid, misfolded TAR DNA-binding protein 43 (TDP43), misfolded superoxide dismutase 1 (SOD1), misfolded huntingtin (HTT), misfolded ABri peptide, misfolded Adan peptide, misfolded fragments of immunoglobulin light chains, misfolded fragments of immunoglobulin heavy chains, misfolded serum amyloid A, misfolded transthyretin, misfolded p-2 microglobulin, misfolded apolipoprotein Al, misfolded apolipoprotein All, misfolded apolipoprotein AIV, misfolded apolipoprotein C-ll, misfolded apolipoprotein C-ll, misfolded gelsolin, misfolded lysozyme, misfolded fibrinogen a chain, misfolded cystatin C, misfolded amylin, misfolded calcitonin, misfolded atrial natriuretic factor, misfolded prolactin, misfolded insulin, misfolded lactadherin / medin, misfolded lactoreansferrin / lactoferrin, misfolded odontogenic ameloblast- associated protein, misfolded pulmonary surfactant-associated protein C, misfolded leukocyte cell- derived chemotaxin-2, misfolded galectin-7, misfolded corneodesmosin, misfolded TGBBI / keratoepithelin, misfolded semenogelin-1, misfolded proteins S100A8 / A9, and misfolded enfuvirtide.

[0031] In some embodiments, the misfolded protein is in the form of an aggregate.

[0032] In some embodiments, the misfolded protein is in the form of a monomer, dimer, trimer, and / or a tetramer.

[0033] In some embodiments, the recombinant monomeric protein comprises one or more of recombinant monomeric alpha-synuclein, recombinant monomeric prion protein, recombinant monomeric tau, recombinant monomeric beta-amyloid, recombinant monomeric TDP43, recombinant monomeric SOD1, recombinant monomeric HTT, recombinant monomeric ABri peptide, recombinant monomeric ADan peptide, recombinant monomeric fragments of immunoglobulin light chains, recombinant monomeric fragments of immunoglobulin heavy chains, recombinant monomeric serum amyloid A, recombinant monomeric transthyretin, recombinant monomeric p-2 microglobulin, recombinant monomeric apolipoprotein Al, recombinant monomeric apolipoprotein All, recombinant monomeric apolipoprotein AIV, recombinant monomeric apolipoprotein C-ll, recombinant monomeric apolipoprotein C-ll, recombinant monomeric gelsolin, recombinant monomeric lysozyme, recombinant monomeric fibrinogen a chain, recombinant monomeric cystatin C, recombinant monomeric amylin, recombinant monomeric calcitonin, recombinant monomeric atrial natriuretic factor, recombinant monomeric prolactin, recombinant monomeric insulin, recombinant monomeric lactadherin / medin, recombinant monomeric lactoreansferrin / lactoferrin, recombinant monomeric odontogenic ameloblast-associated protein, recombinant monomeric pulmonary surfactant- associated protein C, recombinant monomeric leukocyte cell-derived chemotaxin-2, recombinant monomeric galectin-7, misfolded corneodesmosin, misfolded TGBBI / keratoepithelin, recombinant monomeric semenogelin-1, misfolded proteins S100A8 / A9, and recombinant monomeric enfuvirtide.

[0034] In some embodiments, the recombinant monomeric protein comprises a tag, optionally an epitope tag. In some embodiments, the sample is a tissue sample derived from a biopsy, a cerebrospinal fluid sample, or a blood sample.

[0035] In some embodiments, the sample is a fixed sample, a frozen sample, or a free-floating sample.

[0036] In some embodiments, the sample comprises cultured cells.

[0037] The invention also provides a method of screening for the presence of a proteinopathy or an increased risk thereof in a subject, the method comprising detecting misfolded endogenous protein in a tissue sample obtained from the subject by a method of the invention described herein, wherein the presence of misfolded endogenous protein in the tissue sample is indicative of the presence of a proteinopathy or an increased risk thereof.

[0038] The invention also provides a method of diagnosing a proteinopathy in a subject, the method comprising:

[0039] (i) detecting the presence, abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject by a method of the invention described herein; and

[0040] (ii) comparing the presence, abundance and / or location of the misfolded endogenous protein in the tissue sample with the presence, abundance and / or location of the misfolded protein in a control associated with a distinct proteinopathy, wherein:

[0041] (A) a difference between the presence, abundance and / or location of the misfolded endogenous protein in the tissue sample and the presence, abundance and / or location of the misfolded protein in the control indicates the absence of the distinct proteinopathy associated with the control; or

[0042] (B) no difference between the presence, abundance and / or location of misfolded endogenous protein in the tissue sample and the presence, abundance and / or location of the misfolded protein in the control indicates the presence of the distinct proteinopathy associated with the control.

[0043] In some embodiments, the method comprises comparing the presence, abundance and / or location of misfolded endogenous protein in the tissue sample with the presence, abundance and / or location of misfolded protein in more than one control, wherein each control is associated with a distinct proteinopathy.

[0044] The invention also provides a method of monitoring the progression of a proteinopathy in a subject, the method comprising: (i) comparing the abundance and / or location of misfolded endogenous protein in a first tissue sample obtained from the subject at a first time point with the abundance and / or location of misfolded endogenous protein in a second tissue sample obtained from the subject at a second time point; and

[0045] (ii) determining whether the proteinopathy has progressed between the first and second time point based on the comparison performed in step (i); wherein the abundance and / or location of misfolded endogenous protein in the first and second tissue samples is determined by a method of the invention described herein.

[0046] The invention also provides a method for determining the efficacy of a therapeutic intervention in a patient having a proteinopathy, the method comprising:

[0047] (i) comparing the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a first time point prior to administration of the therapeutic intervention with the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a second time point after administration of the therapeutic intervention; and

[0048] (ii) determining the efficacy of the therapeutic intervention based on the comparison performed in step (i); wherein the abundance and / or location of misfolded endogenous protein in the first and second tissue samples is determined by a method of the invention described herein.

[0049] The invention also provides an in vitro method for determining the efficacy of a therapeutic intervention against a proteinopathy, the method comprising:

[0050] (i) contacting a sample comprising misfolded protein associated with the proteinopathy with the therapeutic intervention;

[0051] (ii) detecting the abundance and / or location of misfolded protein in the sample by a method of the invention described herein; and

[0052] (ill) comparing the abundance and / or location of misfolded protein in the sample to a control; wherein the efficacy of the therapeutic intervention is determined based on the comparison performed in step (iii).

[0053] In some embodiments, the control is associated with a therapeutic intervention that is known to be efficacious against the proteinopathy.

[0054] In some embodiments, the proteinopathy is selected from amyloidosis, a synucleinopathy, a prion disease, and a tauopathy. In some embodiments, the proteinopathy is selected from Dementia with Lewy bodies, Parkinson's disease, Alzheimer's disease, frontotemporal dementia, vascular dementia, Creutzfeldt-Jakob disease, a trinucleotide repeat disorder (such as Huntington's disease), Amyotrophic lateral sclerosis (ALS), limbic predominant age-related TDP-43 encephalopathy (LATE), and multiple system atrophy.

[0055] In some embodiments, the therapeutic intervention comprises administration of a therapeutic agent, optionally wherein the therapeutic agent is selected from a small molecule, a peptide, an aptamer, and a peptidomimetic.

[0056] DESCRIPTION OF THE FIGURES

[0057] Figure 1: RT-QuIC a-syn assay overview. Sample containing a-syn seeds incubated with monomeric a-syn induces the recruitment and conversion of a-syn monomers into a-syn aggregates which elongate and result in amyloid formation that can be detected by Thioflavin T (ThT) fluorescence (A). ThT fluorescence intensity curve from a RT-QuIC assay. In the presence of proteopathic seed: lag phase - recruiting and forming nuclei; exponential phase - a-syn aggregation and growth into fibrillar structure; and plateau - monomer-fibril equilibrium is reached (Frost, B. and M.l. Diamond, Prion-like mechanisms in neurodegenerative diseases. Nature Reviews Neuroscience, 2010. 11(3): p. 155-159). In the absence of proteopathic seed, no a-syn aggregation is induced, resulting in absence of exponential and plateau phases (B).

[0058] Figure 2: Tissue sampling and process for tissue sections. Brain is fixed in formalin and sampled in desired regions of interest. Formalin-fixed tissue is then embedded (FFPE) and then microtome- sectioned to form slides with tissue sections.

[0059] Figure 3: Workflow for development of in situ RT-QuIC a-syn assay. FFPE sections are initially subjected to first steps common to immunohistochemistry (IHC) (dewaxing, rehydration, endogenous peroxidase blocking and antigen retrieval) (1). Then, cylinders are stuck to tissue area of interest and reaction buffer (monomeric His-tagged a-syn + ThT + PBS) is added to the cylinder (2). Slides and cylinders sealed with adhesive sealer are placed in FLUOStar Omega overnight and shaken at 37 °C, 500 rpm (3). Sealer and cylinders are removed, and protocol continues as standard IHC (normal horse serum (NHS) blocking, incubation with primary and secondary antibodies, followed by DAB and counterstaining (4).

[0060] Figure 4: Comparison of no shaking and shaking for in situ RT-QuIC a-syn assay. Amygdala from previously characterised positive control was either incubated overnight at 37 °C without shaking or with shaking. The results demonstrate that shaking improves results. Images were taken at 20x and lOx magnification, scale bar represents 100 pm.

[0061] Figure 5: Characterisation of in situ RT-QuIC a-syn assay. Amygdala from negative control (no a-syn pathology; 1), positive case (a-syn pathology; 2) and technical negative (same positive case but without His-a-syn being added; 3) were compared. An increase in ThT fluorescence was seen only in positive case (a). IHC against His-tag was performed (b) and a positive result was observed only in the positive control (2). Same case without His-a-syn resulted in no signal (3), as well as negative control being empty (1) (c).

[0062] Figure 6: Results of in situ RT-QuiC a-syn assay. A representative example of results obtained with the a-syn in situ RT-QuIC assay is displayed. Formalin-fixed paraffin embedded section of the amygdala of a Parkinson's patient labelled with in situ RTQuIC for alpha-synuclein (AS). (A) points to a neuron which cytoplasm is filled with AS seeding-competent material. A neurite filled with seeding-competent material is seen in (B). (C) and (D) point to an astrocyte and an oligodendrocyte, respectively, demonstrating that these cell types also contain AS seeding-competent material. In some areas devoid of neuronal labelling, e.g. (E), astrocytes display seeding-competent material in their cytoplasm (F).

[0063] Figure 7: Results of in situ RT-QuiC tau assay. A representative example of results obtained with the tau in situ RT-QuIC assay is displayed. Formalin-fixed paraffin embedded section of the temporal cortex of an Alzheimer's patient labelled with in situ RTQuIC for tau. Multiple neuronal perikaryal as well as smaller lesions in the neuropil demonstrate tau seeding-competent material.

[0064] DETAILED DESCRIPTION OF THE INVENTION

[0065] The inventor has demonstrated for the first time that protein aggregation can be detected in situ in intact samples, such as intact tissue samples. Importantly, the present invention enables identification of both the presence and the location of protein aggregation within a sample (e.g. tissue sample). Spatial information relating to the origin of seeding aggregation is increasingly being recognised as an essential component to improving understanding of proteinopathies, such as neurodegenerative diseases, and so the present invention is of significant clinical importance. By detecting the location of protein aggregation, the method of the invention can be used to determine where in the sample (e.g. tissue sample) misfolded protein seeds originate, e.g. whether the seed is intracellular or extracellular and the specific cell types and / or subcellular compartments in which misfolded protein seeds occur. The methods of the invention can also be used to determine how extensive protein misfolding is throughout a sample (e.g. tissue sample) and to identify pathological lesions which have seeding potential. The methods of the invention can also be used to determine the seeding potential of a protein. The methods of the invention can also be used to screen and / or validate the efficacy of therapeutic interventions that may be useful for treating protein aggregation disorders.

[0066] A key benefit of using intact tissue samples is that the methods of the invention are highly compatible with immunohistochemistry-based methods for analysing tissue samples. Beneficially, the method of the invention is also compatible with various aggregation detection methods. For example, incubating the sample (e.g. tissue sample) in the presence of an aggregation marker (e.g. a fluorescent marker such as thioflavin) enables real time tracking of aggregate formation as seed amplification progresses. Localised staining of nascent misfolded aggregates (e.g. using immunohistochemistry-based methods) can also be used to determine with spatial resolution the tissue, cell, or organelle origin of the misfolded protein. In addition, the level of detectable signal can be correlated to the abundance of misfolded protein present in the sample enabling areas (e.g. cell types and / or subcellular compartments) which exhibit a high density of misfolded protein to be identified.

[0067] Prior to the present invention, to the best of the inventor's knowledge, it was not known in the technical field that seeding amplification assays could be applied to intact samples (e.g. tissue samples) to enable in situ identification of protein aggregation. The present invention provides a remarkable contribution to the technical field which significantly increases the clinical utility of seeding amplification assays.

[0068] The invention provides an in situ method for detecting misfolded protein in a sample (e.g. a tissue sample), the method comprising: (a) contacting the sample (e.g. tissue sample) with recombinant monomeric protein; (b) incubating the sample under conditions which are: (i) suitable for aggregation of the recombinant monomeric protein with misfolded protein; and (ii) not suitable for aggregation of the recombinant monomeric protein in the absence of misfolded protein; and (c) detecting the formation of protein aggregates in the sample; wherein the presence of protein aggregates in the sample is indicative of the presence of misfolded protein.

[0069] The sample is typically a tissue sample, such as a tissue sample obtained from a subject. The invention is not limited to tissue samples, and may be applied to other types of samples, e.g. artificial cellular or artificial acellular samples.

[0070] In some embodiments, the method comprises detecting the abundance and / or location of misfolded endogenous protein in the sample (e.g. tissue sample) by detecting the abundance and / or location of protein aggregates. Suitable methods for detecting the abundance and / or location of protein aggregates are known in the art and include, but are not limited to, those described herein. Misfolded proteins present in the sample propagate their misfolded conformation by recruiting the recombinant monomeric protein and transmitting the misfolded conformation to said recombinant monomeric proteins. Misfolded proteins form aggregates which can be detected using various methods known in the art. Misfolded proteins which are capable of transmitting their misfolded conformation to monomeric proteins are referred to herein as "misfolded protein seeds", "proteopathic seeds" or "seeds". As used herein, the term "aggregate" embraces all forms of protein entities that may be present during protein aggregation, including, but not limited to, oligomers, protofibrils, filamentous aggregates (fibrils), amorphous aggregates, tangles, plaques, and Lewy bodies.

[0071] In some embodiments, the misfolded protein is a misfolded endogenous protein. It will be understood that for diagnostic and prognostic methods described herein, the misfolded protein is a misfolded endogenous protein.

[0072] In some embodiments, the misfolded protein is a misfolded recombinant protein. For example, for methods of screening the efficacy of a therapeutic intervention as described herein, the misfolded protein may be a misfolded recombinant protein.

[0073] In some embodiments, the misfolded protein is a misfolded protein expressed from exogenous genetic element(s) within cell(s) (e.g. cultured cell(s) comprising a plasmid expressing the protein, or a transgenic host expressing the protein). For example, for methods of screening the efficacy of a therapeutic intervention as described herein, the misfolded protein may be a misfolded protein expressed from exogenous genetic element(s) within cell(s).

[0074] In some embodiments, the misfolded protein is in the form of a monomer, dimer, trimer, or tetramer. In some embodiments, the misfolded protein is in the form of an aggregate.

[0075] In some embodiments, the method comprises fixing the sample (e.g. tissue sample) after the sample has been contacted with recombinant monomeric protein and incubated under conditions suitable for aggregation and before detecting the formation of protein aggregates. Fixation advantageously immobilises nascent aggregates while retaining cellular and subcellular structures. Fixation may be achieved by any suitable method known in the art, for example, by formalin, ethanol, methanol or acetone fixation. In some embodiments, the sample (e.g. tissue sample) is fixed using paraformaldehyde (PFA).

[0076] In some embodiments, the sample is agitated during incubation. Without wishing to be bound by theory, the Inventor believes that agitation may provide signal amplification by disrupting protein aggregates to produce new seeds for further protein aggregate formation. Alternatively or additionally, the Inventor believes that agitation may provide energy required for the initiation or maintenance of protein aggregate formation. Agitation may result in exponential aggregation of monomeric protein into the misfolded form when seed is present. Surprisingly, the Inventor has found that agitation does not appear to result in dissipation of protein aggregates or seeds from the site at which the seeding originates ( / .e. the site of the original misfolded protein).

[0077] In some embodiments, agitation of the sample comprises physical disruption, optionally wherein the physical disruption comprises shaking and / or sonication. In some embodiments, the sample is agitated by shaking. In some embodiments, shaking comprises cyclic shaking, e.g. at a rate of at least 50 rotations per minute (rpm), at least 100 rpm, at least 150 rpm, at least 200 rpm, at least 250 rpm, at least 300 rpm, at least 350 rpm, at least 400 rpm, at least 450 rpm, at least 500 rpm, at least 600 rpm, at least 700 rpm, at least 800 rpm, at least 900 rpm, at least 1000 rpm, at least 2000 rpm, at least 3000 rpm, at least 4000 rpm, or at least 5000 rpm. In some embodiments, the sample is incubated in a plate reader and is agitated by shaking, e.g. at 500 rpm. In some embodiments, the sample is agitated by sonication.

[0078] In some embodiments, the sample is agitated continuously during incubation. In some embodiments, the sample is agitated at predetermined intervals throughout incubation. In some embodiments, the sample is agitated for at least 5 seconds, at least 10 seconds, at least 30 seconds, at least 45 seconds or at least 1 minute at predetermined intervals throughout incubation. In some embodiments, the sample is agitated at intervals of at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, or at least 1 hour throughout incubation. In some embodiments, the sample is agitated for at least 5 seconds, at least 10 seconds, at least 30 seconds, at least 45 seconds or at least 1 minute at intervals of at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, or at least 1 hour throughout incubation.

[0079] As used herein, a tissue sample comprises one or more intact cells. It will be readily understood that detecting misfolded protein in situ in a tissue sample means that the misfolded protein(s) are retained in their cellular location. As such, the tissue sample is not homogenised or otherwise processed in a way that would disrupt the cellular location of the misfolded protein(s) prior to performing the method of the invention.

[0080] In some embodiments, the tissue sample is derived from a biopsy. In some embodiments, the tissue sample is derived from a brain biopsy. In some embodiments, the tissue sample is derived from an intestinal biopsy. In some embodiments, the tissue sample is derived from cerebrospinal fluid or blood, e.g., the tissue sample is derived from plasma or serum. In some embodiments, the tissue sample is derived from extra-CNS (central nervous system) tissue or fluid. In some embodiments, the tissue sample is derived from stool, urine or saliva. In some embodiments, the tissue sample comprises cultured cells. In some embodiments, the tissue sample is an acellular tissue sample.

[0081] In some embodiments, the subject is a mammal. In some embodiments, the subject is selected from a human, a non-human primate, a mouse, a dog, a horse, or a cat. Typically, the subject is a human.

[0082] In some embodiments, the sample (e.g. tissue sample) is a fixed sample. In some embodiments, the sample is a paraffin-embedded tissue sample, optionally a formalin-fixed paraffin-embedded (FFPE) tissue sample. Fixed samples may be prepared prior to performing the method of the invention using methods consistent with standard histology. For example, the methods may comprise deparaffinising FFPE samples, e.g. using standard xylene-based solutions and a succession of alcohols, prior to performing the method of the invention. Endogenous peroxidase activity may be blocked with H2O2. Antigen retrieval for immunohistochemistry (IHC) may be achieved by heat-mediated antigen retrieval, e.g. using citric buffer at pH 6 and heating, such as in a microwave.

[0083] In some embodiments, the sample (e.g. tissue sample) is a frozen tissue sample. In some embodiments, the tissue sample is a free-floating tissue sample. Free-floating tissue samples typically comprise tissue sections floating in solution. Wherein the tissue sample is a free-floating tissue sample, the sample may be mounted on a solid support (e.g. a glass slide) after the tissue sample has been contacted with recombinant monomeric protein and incubated under conditions suitable for aggregation and before detection of the formation of protein aggregates.

[0084] In some embodiments, the misfolded protein comprises one or more of misfolded alpha-synuclein (also referred to as a-synuclein or a-syn), misfolded prion protein, misfolded tau, misfolded betaamyloid, misfolded TAR DNA-binding protein 43 (TDP43), misfolded superoxide dismutase 1 (SOD1), misfolded huntingtin (HTT), misfolded ABri peptide, misfolded Adan peptide, misfolded fragments of immunoglobulin light chains, misfolded fragments of immunoglobulin heavy chains, misfolded serum amyloid A, misfolded transthyretin, misfolded p-2 microglobulin, misfolded apolipoprotein Al, misfolded apolipoprotein All, misfolded apolipoprotein AIV, misfolded apolipoprotein C-ll, misfolded apolipoprotein C-ll, misfolded gelsolin, misfolded lysozyme, misfolded fibrinogen a chain, misfolded cystatin C, misfolded amylin, misfolded calcitonin, misfolded atrial natriuretic factor, misfolded prolactin, misfolded insulin, misfolded lactadherin / medin, misfolded lactoreansferrin / lactoferrin, misfolded odontogenic ameloblast-associated protein, misfolded pulmonary surfactant-associated protein C, misfolded leukocyte cell-derived chemotaxin-2, misfolded galectin-7, misfolded corneodesmosin, misfolded TGBBI / keratoepithelin, misfolded semenogelin-1, misfolded proteins S100A8 / A9, and misfolded enfuvirtide. The skilled person will appreciate that the above list of misfolded proteins is not exhaustive. Advantageously, the method of the invention can be used to detect more than one misfolded protein, e.g. by using two or more recombinant monomeric proteins which are tagged with different epitope tags.

[0085] In some embodiments, the recombinant monomeric protein comprises one or more of recombinant monomeric alpha-synuclein, recombinant monomeric prion protein, recombinant monomeric tau, recombinant monomeric beta-amyloid, recombinant monomeric TDP43, recombinant monomeric SOD1, recombinant monomeric HTT, recombinant monomeric ABri peptide, recombinant monomeric ADan peptide, recombinant monomeric fragments of immunoglobulin light chains, recombinant monomeric fragments of immunoglobulin heavy chains, recombinant monomeric serum amyloid A, recombinant monomeric transthyretin, recombinant monomeric p-2 microglobulin, recombinant monomeric apolipoprotein Al, recombinant monomeric apolipoprotein All, recombinant monomeric apolipoprotein AIV, recombinant monomeric apolipoprotein C-ll, recombinant monomeric apolipoprotein C-ll, recombinant monomeric gelsolin, recombinant monomeric lysozyme, recombinant monomeric fibrinogen a chain, recombinant monomeric cystatin C, recombinant monomeric amylin, recombinant monomeric calcitonin, recombinant monomeric atrial natriuretic factor, recombinant monomeric prolactin, recombinant monomeric insulin, recombinant monomeric lactadherin / medin, recombinant monomeric lactoreansferrin / lactoferrin, recombinant monomeric odontogenic ameloblast-associated protein, recombinant monomeric pulmonary surfactant- associated protein C, recombinant monomeric leukocyte cell-derived chemotaxin-2, recombinant monomeric galectin-7, misfolded corneodesmosin, misfolded TGBBI / keratoepithelin, recombinant monomeric semenogelin-1, misfolded proteins S100A8 / A9, and recombinant monomeric enfuvirtide. It will be readily understood that the recombinant monomeric protein employed will depend on the type of misfolded protein being detected. For example, in embodiments wherein the method comprises detecting misfolded alpha-synuclein, the recombinant monomeric protein comprises recombinant monomeric alpha-synuclein.

[0086] In some embodiments, the recombinant monomeric protein comprises one or more tags (e.g. one, two, three, four, five, six or more tags), optionally an epitope tag. In some embodiments, the recombinant monomeric protein comprises an epitope tag selected from a poly-histidine tag, a hemagglutinin tag and a c-Myc tag. In some embodiments, the recombinant monomeric protein comprises a tag selected from a fluorescent protein, a directly conjugated fluorophore, a conjugated isotope (mass-tag), a nano-tag, a DNA barcode, Albumin-binding protein (ABP), Alkaline Phosphatase (AP), AU1 epitope, AU5 epitope, Bacteriophage T7 epitope (T7-tag), Bacteriophage V5 epitope (V5- tag), Biotin-carboxy carrier protein (BCCP), Bluetongue virus tag (B-tag), Calmodulin binding peptide (CBP), Chloramphenicol Acetyl Transferase (CAT), Cellulose binding domain (CBP), Chitin binding domain (CBD), Choline-binding domain (CBD), Dihydrofolate reductase(DHFR), E2 epitope, FLAG epitope, Galactose-binding protein (GBP), Green fluorescent protein (GFP), Glu-Glu (EE-tag), Glutathione S-transferase (GST), Human influenza hemagglutinin (HA), HaloTag®, Histidine affinity tag (HAT), Horseradish Peroxidase (HRP), HSV epitope, Ketosteroid isomerase (KSI), KT3 epitope, LacZ, Luciferase, Maltose-binding protein (MBP), Myc epitope, NusA, PDZ domain, PDZ ligand, Polyarginine (Arg-tag), Polyaspartate (Asp-tag), Polycysteine (Cys-tag), Polyhistidine (His-tag), Polyphenylalanine (Phe-tag), Profinity eXact, Protein C, Sl-tag, S-tag, and Streptavadin-binding peptide (SBP).

[0087] In some embodiments, the recombinant monomeric protein comprises two or more tags (e.g. two, three, four, five, six or more tags) that are different to each other. Using different tags allows more than one type of protein aggregation to be detected.

[0088] In some embodiments, the recombinant monomeric protein is a modified protein comprising a mutation, truncation, and / or posttranslational modification relative to the endogenous version of that protein. In some embodiments, the recombinant monomeric protein is an isoform of an endogenous version of that protein. In some embodiments, the recombinant monomeric protein is a fusion protein. Modified proteins also enable investigation of disease mechanisms e.g. by investigating the role played by protein truncations, fragments, isoforms, peptide mimetics or other posttranslational modifications on disease mechanisms. Similarly, modified proteins may also enable differential diagnosis.

[0089] It will be understood that the conditions which are (i) suitable for aggregation of the recombinant monomeric protein with misfolded endogenous protein and (ii) not suitable for aggregation of the recombinant monomeric protein in the absence of misfolded protein will depend on various factors, e.g. the type of sample (e.g. tissue sample) being used, and the misfolded protein being detected. The conditions suitable for aggregation of the recombinant monomeric protein with misfolded endogenous protein typically comprise a temperature, time period and reaction buffer that is suitable to enable, in the presence of misfolded protein, formation of a detectable level of protein aggregates. Conditions which are not suitable for aggregation of the recombinant monomeric protein in the absence of misfolded protein typically mean that the sample is not exposed to conditions which result in the misfolding of the recombinant monomeric protein or wild-type endogenous proteins, e.g. high temperatures.

[0090] In some embodiments, the sample (e.g. tissue sample) is incubated at a temperature of at least 20 °C, at least 25 °C, at least 30 °C, at least 35 °C, at least 36 °C, at least 37 °C, at least 38 °C, at least 39 °C, or at least 40 °C. In some embodiments, the sample (e.g. tissue sample) is incubated at a temperature of at least 37 °C.

[0091] In some embodiments, the sample (e.g. tissue sample) is incubated with recombinant monomeric protein for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 60 hours or at least 72 hours.

[0092] In some embodiments, the sample is agitated throughout an incubation. In some embodiments, the sample is agitated during part of an incubation. In some embodiments, the sample is not agitated during an incubation.

[0093] In some embodiments, the method comprises contacting the sample (e.g. tissue sample) with a reaction buffer comprising recombinant monomeric protein. In some embodiments, the reaction buffer comprises recombinant monomeric protein at a concentration of at least 0.01 mg / ml, at least 0.05 mg / ml, at least 0.1 mg / ml, at least 0.15 mg / ml, at least 0.2 mg / ml, at least 0.25 mg / ml, at least 0.3 mg / ml, at least 0.35 mg / ml, at least 0.4 mg / ml, at least 0.45 mg / ml, at least 0.5 mg / ml, at least 0.6 mg / ml, at least 0.7 mg / ml, at least 0.8 mg / ml, at least 0.9 mg / ml, or at least 1 mg / ml.

[0094] Any suitable reaction buffer may be used.

[0095] In some embodiments, the reaction buffer has a pH of 4 to 8, 5 to 8, 6 to 8 or 7 to 8. In some embodiments, the reaction buffer has a pH of 4, 5, 6, 7, 7.2, 7.4, 7.6, 7.8, or 8.

[0096] In some embodiments, the reaction buffer comprises 1,4-Piperazinediethanesulfonic acid (PIPES).

[0097] In some embodiments, the reaction buffer comprises phosphate buffered saline (PBS).

[0098] In some embodiments, the reaction buffer comprises 80 mM NaCI in PBS at pH 7.4. Advantageously, reaction buffer comprising 80 mM NaCI in PBS at pH 7.4 has been found to reduce background noise and increase signal intensity. In some embodiments, the method comprises preconditioning the sample (e.g. tissue sample) by immersion in 80 mM NaCI in PBS at pH 7.4.

[0099] In some embodiments, the reaction buffer comprises 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES).

[0100] In some embodiments, the reaction buffer comprises 50 mM HEPES, 2 M NaCI, and 10 mg / ml heparin. In some embodiments, the misfolded protein comprises misfolded alpha-synuclein, or the recombinant monomeric protein comprises recombinant monomeric alpha-synuclein, and the reaction buffer comprises PBS.

[0101] In some embodiments, misfolded protein comprises misfolded tau, or the recombinant monomeric protein comprises recombinant monomeric tau, and the reaction buffer comprises HEPES.

[0102] In some embodiments, detecting the formation of protein aggregates comprises contacting the sample (e.g. tissue sample) with a protein aggregation marker. It will be readily understood that a protein aggregation marker is a protein, molecule or compound which can be used to detect protein aggregation. In some embodiments, the protein aggregation marker emits a detectable signal when bound to beta sheet-rich structures, such as protein aggregates. In some embodiments, the protein aggregation marker is a dye, optionally a fluorophore. In some embodiments, the protein aggregation marker is selected from Thioflavin (e.g. Thioflavin T, Thioflavin S or Thioflavin X), ANS (1- anilinonaphthalene-8-sulfonate), Bis-ANS (4,4'-bis-l-anilinonaphthalene-8-sulfonate), Congo Red, SYPRO Orange, Amytracker, luminescent-conjugated poly / oligo-thiophene, and Proteostat. In some embodiments, the protein aggregation marker is a marker that allows PET imaging, optionally Pittsburgh Compound-B.

[0103] In some embodiments, the method comprises contacting the sample (e.g. tissue sample) with a protein aggregation marker after the sample has been contacted with recombinant monomeric protein and incubated under conditions suitable for aggregation of the recombinant monomeric protein with misfolded protein. In some embodiments, the method comprises contacting the sample (e.g. tissue sample) with recombinant monomeric protein and a protein aggregation marker prior to incubation of the sample under conditions suitable for aggregation of the recombinant monomeric protein with misfolded protein. Advantageously, contacting the sample (e.g. tissue sample) with recombinant monomeric protein and a protein aggregation marker prior to incubation enables protein aggregation to be tracked in real time.

[0104] In some embodiments, protein aggregation may be detected using a commercially available protein aggregation kit (e.g. ProteoStat Protein Aggregation Assay Kit from Enzo).

[0105] In some embodiments, the method comprises contacting the sample (e.g. tissue sample) with a reaction buffer comprising a protein aggregation marker. In some embodiments, the reaction buffer comprises protein aggregation marker at a concentration of at least 1 pM, at least 2 pM, at least 3 pM, at least 4 pM, at least 5 pM, at least 6 pM, at least 7 pM, at least 8 pM, at least 9 pM, or at least 10 pM. In some embodiments, the reaction buffer comprises protein aggregation marker at a concentration of at least 5 pM. In some embodiments, the reaction buffer comprises thioflavin (e.g. thioflavin T, thioflavin S or thioflavin X) at a concentration of at least 1 pM, at least 2 pM, at least 3 pM, at least 4 pM, at least 5 pM, at least 6 pM, at least 7 pM, at least 8 pM, at least 9 pM, or at least 10 pM, optionally at least 5 pM.

[0106] In some embodiments, detecting the formation of protein aggregates in the sample (e.g. tissue sample) comprises contacting the sample with an antigen binding molecule specific to the protein aggregate. In some embodiments, the antigen binding molecule is selected from an antibody or antigen binding fragment thereof or an aptamer. An aptamer is typically a short oligonucleotide that binds a specific target molecule.

[0107] Any suitable detection method may be used. In some embodiments, the method comprises immunostaining.

[0108] In some embodiments, the method comprises detecting the formation of protein aggregates by immunohistochemistry (IHC). Suitable IHC methods are known in the art and typically comprise contacting the sample with an antigen binding molecule and detecting binding of the antigen binding molecule. In the context of the present invention, the antigen binding molecule may be specific to the recombinant monomeric protein. In some embodiments, the antigen binding molecule is specific to an epitope tag present on the recombinant monomeric protein.

[0109] In some embodiments, the method comprises detecting the formation of protein aggregates by immunofluorescence (IF). Suitable IF methods are known in the art and typically comprise contacting the sample with an antigen binding molecule and detecting binding of the antigen binding molecule. In the context of the present invention, the antigen binding molecule may be specific to the recombinant monomeric protein. In some embodiments, the antigen binding molecule is specific to an epitope tag present on the recombinant monomeric protein.

[0110] In some embodiments, detecting the formation of protein aggregates comprises: (i) contacting the sample with an antigen binding molecule which is specific to the protein aggregates; and (ii) detecting binding of the antigen binding molecule. In some embodiments, the recombinant monomeric protein comprises an epitope tag and detecting the formation of protein aggregates comprises: (i) contacting the sample with an antigen binding molecule which is specific to the epitope tag; and (ii) detecting binding of the antigen binding molecule.

[0111] The antigen binding molecule which is specific to the protein aggregate or epitope tag may be referred to herein as a primary antigen binding molecule. In some embodiments, detecting binding of the primary antigen binding molecule comprises contacting the sample with a secondary antigen binding molecule which is specific to the first antigen binding molecule.

[0112] In some embodiments, the primary and / or secondary antigen binding molecules are conjugated to a detectable marker and detecting binding of the primary and / or secondary antigen binding molecules comprises detecting the marker. In some embodiments, the detectable marker is a fluorescent marker. In some embodiments, the detectable marker is an enzyme marker.

[0113] In some embodiments, detecting binding of the primary and / or secondary antigen binding molecules comprises peroxidase-based staining or alkaline phosphatase-based staining. Methods of peroxidase-based or alkaline phosphatase-based staining are known in the art and typically include contacting the sample with an antigen binding molecule which is conjugated to peroxidase (e.g. horseradish peroxidase (HRP)) or alkaline phosphatase and a reporter substrate (e.g. a chromogen or fluorophore) which is activated by the enzyme to produce a detectable signal. In positive samples comprising peroxidase or alkaline phosphatase linked antigen binding molecules, addition of appropriate reporter substrates leads to the precipitation of insoluble, coloured precipitates at the site of antigen binding, thereby enabling the location of the antigen to be determined. Advantageously, enzymatic activation of the reporter molecule provides an amplified signal enabling detection of even low levels of antigen.

[0114] In some embodiments, detecting the formation of protein aggregates comprises contacting the sample with: (i) a primary and / or a secondary antigen binding molecule conjugated to peroxidase; and (ii) a reporter molecule which is configured to produce a detectable signal following contact with peroxidase. In some embodiments, the reporter molecule is selected from DAB (3,3'- Diaminobenzidine) and AEC (Aminoethyl carbazole). HRP catalyses the oxidation of DAB to produce a localised brown precipitate which can be detected by light microscopy. In some embodiments, the reporter molecule is a dye-labelled tyramide. HRP catalyses activation of the tyramide resulting in the production of a localised tyramide derivative signal (e.g. fluorescence). It will be understood that methods comprising peroxidase-based signal amplification typically comprise a step of blocking endogenous peroxidase activity, e.g. using hydrogen peroxide (H2O2).

[0115] In some embodiments, detecting the formation of protein aggregates comprises contacting the sample with: (i) a primary and / or a secondary antigen binding molecule conjugated to alkaline phosphatase; and (ii) a reporter molecule which is configured to produce a detectable signal following contact with alkaline phosphatase. In some embodiments, the reporter molecule is Fast Red. In some embodiments, the primary and / or secondary antigen binding molecule is conjugated to biotin and antigen binding is detected by contacting the sample with enzyme-linked streptavidin. In some embodiments, antigen binding is detected by contacting the sample with peroxidase-linked streptavidin or alkaline phosphatase-linked streptavidin.

[0116] In some embodiments, the method further comprises counterstaining the tissue sample with antigen binding molecules and / or dyes specific to one or more cell types. In some embodiments, the method further comprises counterstaining the tissue sample with antigen binding molecules and / or dyes specific to one or more cellular components, such as the nucleus, cytosol, or cytoskeleton.

[0117] In some embodiments, detecting binding of the primary and / or secondary antigen binding molecules comprises microscopy, optionally light microscopy and / or fluorescence microscopy.

[0118] In some embodiments, the primary and / or secondary antigen binding molecules are each selected from an antibody or an antigen binding fragment thereof and an aptamer.

[0119] In some embodiments, wherein if protein aggregates are detected in the sample (e.g. tissue sample), the method further comprises detecting the location of the protein aggregates within the sample (e.g. tissue sample). In some embodiments, identifying the location of the aggregates comprises identifying the cell type in which aggregates occur. In some embodiments, the method comprises detecting whether aggregation occurs in one or more of types of cells, for example neurons, neuron subtypes, endothelial cells, pericytes, meningothelial cells, ependymal cells, choroid plexus, neuroendocrine cells, inflammatory cells, cell types within the retina, cell types within lymphoid tissue and glial cells for example astrocytes, oligodendrocytes, microglia, satellite cells or Schwann cells. In some embodiments, identifying the location of the aggregates comprises identifying the subcellular compartment in which aggregates occur. In some embodiments, the method comprises detecting whether aggregation occurs in one or more subcellular organelles or locations, for example the nucleus, endoplasmic reticulum, Golgi apparatus, synaptic vesicles, exosomes, multivesicular bodies and lysosomes of the cell. In some embodiments, the method comprises detecting whether aggregation occurs in parts of a neurons such as the soma and / or neurites, neuritic spines, synapses, axons or axonic boutons, myelin sheath of neurons. In some embodiments, the method comprises detecting whether aggregates are intracellular and / or extracellular or relationship to specific extracellular compartments such as for example synaptic cleft or perivascular space.

[0120] In some embodiments, the method comprises detecting whether aggregation occurs in inclusions or pathological lesions such as Lewy bodies, Lewy neurites, tangles, plaques, tumours, or cancerous tissue. In some embodiments, the method comprises detecting whether aggregation occurs in the intestinal lumen. In some embodiments, the method comprises detecting whether aggregation occurs in the enteric nervous system, optionally enteric nervous neurons.

[0121] It will be readily understood that cell types and subcellular compartments may be identified using a variety of methods known in the art. For example, cell types and subcellular compartments may be identified using microscopy-based methods. Cell types and subcellular compartments may also be identified using IHC-based methods.

[0122] In some embodiments, the method further comprises quantifying the abundance of aggregate formation in the sample (e.g. tissue sample). It will be readily understood that the method used to quantify the abundance of aggregate formation will depend on the protein aggregate detection method used. For example, wherein the method comprises using a protein aggregation marker to detect aggregate formation, the abundance of aggregate formation may be determined by correlating the level of marker to the abundance of protein aggregates present in the sample. The abundance of aggregate formation can in turn be correlated to the abundance of misfolded protein present in the starting sample.

[0123] Embodiments described herein in the context of in situ methods for detecting misfolded protein apply equally to the diagnostic, prognostic and screening methods described herein. Said methods include methods of screening for the presence of a proteinopathy or an increased risk thereof in a subject, methods of diagnosing proteinopathies, methods of monitoring the progression of a proteinopathy in a subject, and methods of determining the efficacy of a therapeutic intervention.

[0124] The invention provides a method of screening for the presence of a proteinopathy or an increased risk thereof in a subject, the method comprising detecting misfolded endogenous protein in a tissue sample obtained from the subject by a method of the invention, wherein the presence of misfolded endogenous protein in the tissue sample is indicative of the presence of a proteinopathy or an increased risk thereof.

[0125] In some embodiments, the method comprises detecting the location and / or abundance of misfolded protein in the sample (e.g. tissue sample) by a method of the invention and comparing the location and / or abundance of aggregate formation in the sample with a control. In some embodiments, the control is a predetermined location and / or abundance of misfolded protein that is known to be associated with the presence of a proteinopathy or an increased risk thereof. In some embodiments, the control is a predetermined location and / or abundance of misfolded protein that is known to be associated with the absence of a proteinopathy or a decreased risk thereof, e.g. the control may be a predetermined location and / or abundance of misfolded protein that is known to be associated with a healthy control.

[0126] As used herein, subjects identified as being at an increased risk of a proteinopathy are more likely to develop a proteinopathy in the future than subjects who exhibit (a) presence, location and / or abundance of misfolded protein that is not associated with the presence of a proteinopathy or an increased risk thereof; and / or (b) subjects who exhibit presence, location and / or abundance of misfolded protein that is associated with the absence of a proteinopathy or a decreased risk thereof.

[0127] In some embodiments, the method comprises monitoring subjects who are identified as being at an increased risk of developing a proteinopathy. In some embodiments, monitoring subjects identified as being at an increased risk of developing a proteinopathy comprises repeating the method of the invention with tissue samples obtained from the subject at specified time intervals, e.g. every 6 months, every year, or every two years.

[0128] The invention also provides a method of diagnosing a proteinopathy in a subject, the method comprising: (i) detecting the presence, abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject by a method of the invention; and (ii) comparing the presence, abundance and / or location of misfolded endogenous protein in the tissue sample with the presence, abundance and / or location of misfolded protein in a control associated with a distinct proteinopathy, wherein: (A) a difference between the presence, abundance and / or location of misfolded endogenous protein in the tissue sample and the presence, abundance and / or location of misfolded protein in the control indicates the absence of the distinct proteinopathy associated with the control; or (B) no difference between the presence, abundance and / or location of misfolded endogenous protein in the tissue sample and the presence, abundance and / or location of misfolded protein in the control indicates the presence of the distinct proteinopathy associated with the control.

[0129] Advantageously, the methods of the invention enable differential diagnosis of proteinopathies by comparing the presence, abundance and / or location of misfolded endogenous proteins between a patient derived tissue sample with the presence, abundance and / or location of misfolded proteins in controls associated with distinct proteinopathies. Thus, in some embodiments, the method comprises comparing the presence, abundance and / or location of misfolded endogenous protein in the tissue sample with the presence, abundance and / or location of misfolded protein in more than one control, wherein each control is associated with a distinct proteinopathy. In some embodiments, the method comprises determining the control to which the presence, abundance and / or location of misfolded endogenous protein in the tissue sample is most similar. In diagnosis methods of the invention, the subject may be known to have or be suspected of having a proteinopathy, e.g. based on screening methods of the invention and / or diagnostic methods known in the art (such as PET or SPECT scans or symptom analysis or familial gene mutations of pathogenic nature). Alternatively, the subject may have been identified as being at an increased risk of developing a proteinopathy, e.g. by a method of the invention and / or diagnostic methods known in the art.

[0130] The invention also provides a method of monitoring the progression of a proteinopathy in a subject, the method comprising: (i) comparing the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a first time point with the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a second time point; and (ii) determining whether the proteinopathy has progressed between the first and second time point based on the comparison performed in step (i); wherein the abundance and / or location of misfolded endogenous protein in the tissue samples is determined by an in situ method of the invention.

[0131] In some embodiments, a difference in the abundance and / or location of misfolded protein between the first time point and the second time point indicates that the proteinopathy has progressed, whereas no difference in the abundance and / or location of misfolded protein between the first time point and the second time point indicates that the proteinopathy has not progressed. In some embodiments, the method comprises comparing the abundance and / or location of misfolded protein at multiple time points to track the progression of the proteinopathy over time. It will be understood that detecting the abundance and / or location of misfolded protein may comprise detecting the extent of protein misfolding throughout a tissue sample, e.g. by determining the proportion of cells within a tissue sample which exhibit protein misfolding.

[0132] Typically, the greater the extent of protein misfolding, the more severe the proteinopathy becomes. Thus, a patient identified as having a proteinopathy that has progressed between the first and subsequent time point(s) may exhibit more severe symptoms. A patient identified as having a proteinopathy that has not progressed between the first and subsequent time point(s) may exhibit a similar level of symptom severity as the first time point.

[0133] The invention also provides a method for determining the efficacy of a therapeutic intervention in a subject having a proteinopathy, the method comprising: (i) comparing the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a first time point prior to administration of the therapeutic intervention with the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a second time point after administration of the therapeutic intervention; and (ii) determining the efficacy of the therapeutic intervention based on the comparison performed in step (i); wherein the abundance and / or location of misfolded endogenous protein in the first and second tissue samples is determined by an in situ method of the invention.

[0134] Advantageously, the methods of the present invention have use in personalised medicine, whereby biopsy-derived tissue samples (e.g. biopsy-derived tissue samples) can be used to screen therapeutic interventions for therapeutic efficacy against subject specific aggregates.

[0135] In some embodiments, a difference in the abundance and / or location of misfolded protein between the first and second time points indicates that the therapeutic intervention is efficacious. For example, a lower abundance of misfolded protein at the second time point as compared to the first time point may indicate that the therapeutic intervention has high efficacy. In some embodiments, no difference in the abundance and / or location of misfolded protein between the first and second time points indicates that the therapeutic intervention has low or no efficacy.

[0136] The invention also provides an in vitro method for determining the efficacy of a therapeutic intervention against a proteinopathy, the method comprising: (i) contacting a sample (e.g. tissue sample) comprising misfolded protein with the therapeutic intervention; (ii) detecting the abundance and / or location of misfolded protein in the sample by the in situ method of the invention; and (ill) comparing the abundance and / or location of misfolded protein in the sample to a control; wherein the efficacy of the therapeutic intervention is determined based on the comparison performed in step (iii).

[0137] In some embodiments, the control is the abundance and / or location of misfolded protein in a corresponding sample (e.g. tissue sample) that has not been treated with a therapeutic intervention. In this embodiment, a difference in the abundance and / or location of misfolded protein between the sample and the control indicates that the therapeutic intervention is efficacious. For example, a lower abundance of protein aggregates in the sample as compared to the control typically indicates that the therapeutic intervention is efficacious. In some embodiments, no difference in the abundance and / or location of misfolded protein between the sample and the control indicates that the therapeutic intervention has low or no efficacy.

[0138] In some embodiments, the control is the abundance and / or location of misfolded protein in a corresponding sample (e.g. tissue sample) that has been treated with a therapeutic intervention which is known to be efficacious against that proteinopathy. In this embodiment, a difference in the abundance and / or location of misfolded protein between the sample and the control indicates than the tested therapeutic intervention has a different level of efficacy than the therapeutic intervention associated with the control. For example, a higher abundance of protein aggregates in the sample as compared to the control may indicate that the therapeutic intervention has lower efficacy than the therapeutic intervention associated with the control whereas a lower abundance of protein aggregates in the sample as compared to the control may indicate that the therapeutic intervention has higher efficacy than the therapeutic intervention associated with the control. In some embodiments, no difference in the abundance and / or location of misfolded protein between the sample and the control indicates that the tested therapeutic intervention has the same level of efficacy as the therapeutic intervention associated with the control.

[0139] The skilled person will readily understand that the therapeutic intervention will depend on the particular proteinopathy in question. In some embodiments, the therapeutic intervention comprises administration of a therapeutic agent to the subject. In some embodiments, the therapeutic agent is selected from a small molecule, a peptide, an aptamer, and a peptidomimetic. In some embodiments, the therapeutic intervention is predicted to be useful in the treatment of a proteinopathy, e.g. based on in vitro or animal models of said proteinopathy.

[0140] As used herein, the term "proteinopathy" refers to diseases and conditions associated with the formation of misfolded protein aggregates. In some embodiments, the proteinopathy is selected from a synucleinopathy, a prion disease or a tauopathy. In some embodiments, the proteinopathy is a neurodegenerative disease. In some embodiments, the proteinopathy is selected from amyloidosis, a synucleinopathy, a prion disease, and a tauopathy. In some embodiments, the proteinopathy is selected from Dementia with Lewy bodies, Parkinson's disease, Alzheimer's disease, frontotemporal dementia, vascular dementia, Creutzfeldt-Jakob disease, a trinucleotide repeat disorder (such as Huntington's disease), Amyotrophic lateral sclerosis (ALS), limbic predominant age-related TDP-43 encephalopathy (LATE), and multiple system atrophy.

[0141] In some embodiments, the subject has not previously been diagnosed with a proteinopathy. In some embodiments, the subject is suspected of having a proteinopathy based on known diagnostic methods, e.g. symptoms analysis or medical imaging (such as PET / SPECT scans). In some embodiments, the subject exhibits proteinopathy-associated symptoms. In some embodiments, the subject has been diagnosed with a proteinopathy.

[0142] EXAMPLES

[0143] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and are in no way limiting. EXAMPLE 1

[0144] Summary

[0145] Described herein are methods for the in situ amplification and visualisation of protein aggregates which are capable of seeding. The methods described herein advantageously enable preservation of the morphological and spatial context of these protein aggregates. The methods include, but are not limited to, the use of tagged monomeric proteins, in situ seeding amplification using shaking, monitoring in situ aggregate amplification using a dye (e.g. Thioflavin T), fixation in situ of nascent amplified aggregates and / or detection in situ of nascent amplified aggregates using e.g. immunohistochemistry for the protein tag.

[0146] Methods

[0147] Regions of formalin-fixed postmortem human brain were sampled and processed to paraffin blocks in the standard way (Figure 2). Formalin-fixed paraffin embedded (FFPE) tissue sections at 5 pm thickness were placed in a 55-60 degrees oven for a minimum of 45 min and then dewaxed and rehydrated in xylene (10 min total) and sequential changes of EtOH from 100% to 70% for 5 min each and then washed with distilled water. Endogenous peroxidase activity was blocked in 0.3% H2O2 for 30 min. Thereafter slides containing tissue were subjected to total heat cycles at maximum potency for 10 min in the microwave in citrate buffer Ph 6.0 and cooled on ice. Then hydrophobic pen (PAP pen) was drawn around the tissue to retain the liquid added in the tissue section. Slides were washed with PBS and tapped off, with a cylinder being stuck in the area of interest within the tissue. A reaction buffer of PBS, 0.1 mg / ml of substrate and ThT was added to the cylinder. The alpha-synuclein assay used 0.1 mg / ml commercial monomeric His-tagged a-syn as substrate and 5 Mm ThT in PBS to a total volume of 100 pl per cylinder. A corresponding approach, achieving similar results, was taken using 0.05mg / ml and 0.025mg / ml alpha-synuclein.

[0148] Optionally, a buffer containing 80Mm NaCI in PBS, Ph 7.4 (plus the monomeric protein and ThT) can be used, which may reduce background and increase signal intensity. In this case, preconditioning the slides before the cylinder is applied by immersion in 80Mm NaCI in PBS, Ph 7.4 is recommended.

[0149] The slides (Figure 3) were fixed with glue to a 96 well plate lid which has suitable dimensions for a plate reader. Optionally, cylinders can also be glued to the slides. The plate lid with the slides is then sealed completely, e.g. using cling film of PCR adhesive. FLUOstar BMG Omega plate reader was used which allows double orbital shaking at 500rpm at 37 °C overnight (o / n). Addition of ThT allowed real time aggregation of substrate to be tracked by detecting the fluorescence signal during incubation in the plate reader. After o / n shaking, the adhesive sealer, cylinder and slides are removed from the lid and 4% PFA added for lOmin. This may be achieved by adding 100 pl PFA to the reaction buffer inside the cylinder, removing the cylinder and then adding an extra 100 pl PFA to the whole tissue section to add total 200 pl PFA. Fixation allows the nascent aggregates / fibrils formed to remain in situ.

[0150] Slides were washed with PBS then blocked with 10% normal horse serum (NHS) for lh at RT. Primary antibody anti-6x His tag was added at 1:500 concentration for 2h at RT. Optionally, the antibody can be diluted to up to 1:5000 with overall same signal and decrease in background. Slides were washed with PBS and incubated for 30min at RT with ImmPress HRP horse anti-rabbit IgG Polymer and washed again with PBS prior to DAB chromogen for visualisation of signal. Optionally, 0.05-1% PBS-Tween 20 for washing slides and for blocking steps can be used, which may reduce background. Slides are counterstained with hematoxylin for 2min and dehydrated with sequential EtOH washes from 90- 100% and xylene.

[0151] Results

[0152] An example of the typical results obtained with the alpha-synuclein in situ aggregate detection method of the invention is displayed in Figures 4, 5 and 6. Pathology detected included Lewy bodies (LB), neurites, astrocytic and oligodendrocytic a-syn and the core and some dystrophic neurites of beta-amyloid (A ) plaques. Positive signal was detected in the amygdala of a dementia with Lewy bodies (DLB) case, which had a-syn pathology as detected by immunohistochemistry (IHC) with anti- a-syn antibody, while signal was absent in a negative control (i.e. brain tissue with no a-syn pathology). No signal was detected in a consecutive section of amygdala from the DLB case in which the His-tagged a-syn protein was omitted ("technical negative"). Standard IHC with anti-his tag antibody on the DLB or control slides did not result in any signal. Shaking significantly improved the size of the signal produced by the positive control (Figure 4).

[0153] ThT signal (Figure 5(a)) increased exponentially in positive cases only, as observed in the graph illustrating ThT fluorescence over time. ThT signal was detected 9h-10h post-shaking. Figure 5(b) illustrates the position of the cylinders on the slides. The ThT signal was observed on the areas corresponding to positive tissue only (Figure 5(a)), providing macroscopic real-time spatial information. After performing IHC against His-tag (Figure 5(c)), as well as right image on Figure 4 and Figure 6), alpha-synuclein seeding-competent aggregates are visualised with microscopic spatial resolution.

[0154] Figure 6 displays a representative higher magnification example of results obtained with the a-syn in situ RT-QuIC assay. Formalin-fixed paraffin embedded section of the amygdala of a Parkinson's patient were labelled with in situ RTQuIC for alpha-synuclein (AS). (A) points to a neuron which cytoplasm is filled with AS seeding-competent material. A neurite filled with seeding-competent material is seen in (B). While neurons are the cell types that are currently considered to be responsible for the prion-like spread of seeding-competent AS material, (C) and (D) point to an astrocyte and an oligodendrocyte, respectively, demonstrating that these cell types also contain AS seeding-competent material. Moreover, in some areas devoid of neuronal labelling, e.g. (E), astrocytes display seeding-competent material in their cytoplasm (F).

[0155] Conclusion

[0156] Described herein for the first-time are seed amplification assay methods which allow visualisation in situ of protein aggregate seeding activity with preservation of the morphological and spatial context. Using a-synuclein as a model system, the method reveals a-synuclein seeding capacity in situ in FFPE human post-mortem brain tissue sections. Also described are various optional method developments to further improve the efficiency of the assay. It will be readily understood that the methods described herein can be applied to reveal the seeding capacity and / or activity for other proteinopathies (e.g. tau, beta-amyloid, TDP43). In addition, the method is not limited to a particular type of tissue, but could be applied to murine tissue and other animal models, cultured cell lines, traditional seeding amplification assay fluid samples (CSF / blood spotted on slides, cyto-spins or cell blocks), or acellular in vitro systems. Importantly, the method provides the ability to track seed amplification in real time.

[0157] EXAMPLE 2

[0158] The process as described in Example 1 was repeated using tau protein as the substrate. Regions of formalin-fixed postmortem human brain were sampled and processed to paraffin blocks in the standard way. Formalin-fixed paraffin embedded (FFPE) tissue sections at 5 pm thickness were placed in a 55-60 degrees oven for a minimum of 45 min and then dewaxed and rehydrated in xylene (10 min total) and sequential changes of EtOH from 100% to 70% for 5 min each and then washed with distilled water. Endogenous peroxidase activity was blocked in 0.3% H2O2 for 30 min. Thereafter slides containing tissue were subjected to total heat cycles at maximum potency for 10 min in the microwave in citrate buffer Ph 6.0 and cooled on ice. Then hydrophobic pen (PAP pen) was drawn around the tissue to retain the liquid added in the tissue section. A reaction buffer of 50 mM HEPES, 2 M NaCI, 10 mg / ml heparin, ddH2O, 0.75 mg / ml tau306 protein substrate (purified according to Saijo et al., Methods Mol Bio, 2019;1873:19-37. Doi: 10.1007 / 978- 1-4939-8820-4_2. PMID: 30341601; defrosted at room temperature and filtered before using), and 500 uM ThT to a total volume of 100 pl per cylinder was added to the cylinder. Alternatively, the method may be performed using commercially available recombinant tau protein.

[0159] The slides were fixed with glue to a 96 well plate lid which has suitable dimensions for a plate reader. Optionally, cylinders can also be glued to the slides. The plate lid with the slides is then sealed completely, e.g. using cling film of PCR adhesive. FLUOstar BMG Omega plate reader was used which allows double orbital shaking at 500rpm at 42 °C overnight (o / n). Addition of ThT allowed real time aggregation of substrate to be tracked by detecting the fluorescence signal during incubation in the plate reader. After two overnights shaking, the adhesive sealer, cylinder and slides are removed from the lid and 4% PFA added for lOmin. This may be achieved by adding 100 pl PFA to the reaction buffer inside the cylinder, removing the cylinder and then adding an extra 100 pl PFA to the whole tissue section to add total 200 pl PFA. Fixation allows the nascent aggregates / fibrils formed to remain in situ.

[0160] Slides were washed with PBS then blocked with 10% normal horse serum (NHS) for lh at room temperature. Primary antibody anti-6x His tag was added at 1:1000 concentration for 2h at RT. Slides were washed with PBS and incubated for 30min at RT with ImmPress HRP horse anti-rabbit IgG Polymer and washed again with PBS prior to DAB chromogen for visualisation of signal. Optionally, 0.05-1% PBS-Tween 20 for washing slides and for blocking steps can be used, which may reduce background. Slides are counterstained with hematoxylin for 2min and dehydrated with sequential EtOH washes from 90-100% and xylene.

[0161] An example of the typical results obtained with the tau in situ aggregate detection method of the invention is displayed in Figure 7. Positive ThT signal is detected by IHC with anti-tau antibody when tau is present.

Claims

CLAIMS1. An in situ method for detecting misfolded protein in a sample, the method comprising:(a) contacting the sample with recombinant monomeric protein;(b) incubating the sample under conditions which are:(i) suitable for aggregation of the recombinant monomeric protein with misfolded protein; but(ii) not suitable for aggregation of the recombinant monomeric protein in the absence of misfolded protein; and(c) detecting the formation of protein aggregates in the sample; wherein the presence of protein aggregates in the sample is indicative of the presence of misfolded protein.

2. The method of claim 1, wherein the sample is a tissue sample.

3. The method of claim 1 or claim 2, wherein the method comprises detecting the abundance and / or location of misfolded endogenous protein in the sample by detecting the abundance and / or location of protein aggregates.

4. The method of any preceding claim, wherein the method comprises fixing the sample after step (b) and prior to step (c).

5. The method of any preceding claim, wherein the method comprises agitating the sample during incubation.

6. The method of claim 5, wherein agitating the sample comprises shaking and / or sonication.

7. The method of any preceding claim, wherein detecting the formation of protein aggregates in the sample comprises contacting the sample with a protein aggregation marker.

8. The method of claim 7, wherein the protein aggregation marker is selected from Thioflavin T, Thioflavin S, Thioflavin X, ANS (l-anilinonaphthalene-8-sulfonate), Bis-ANS (4,4'-bis-l- anilinonaphthalene-8-sulfonate), Congo Red, and SYPRO Orange.

9. The method of any preceding claim, wherein detecting the formation of protein aggregates in the sample comprises contacting the sample with an antigen binding molecule specific to the protein aggregate.

10. The method of claim 9, wherein the antigen binding molecule is selected from an antibody or an antigen binding fragment thereof and an aptamer.

11. The method of any of claims 3-10, wherein detecting the location of the protein aggregates comprises:(a) detecting the cell type in which the protein aggregates occur; and / or(b) detecting the subcellular location in which the protein aggregates occur.

12. The method of any preceding claim, wherein detecting the protein aggregates comprises immunohistochemistry and / or microscopy-based methods.

13. The method of any preceding claim, wherein the misfolded protein is an endogenous misfolded protein.

14. The method of any of claims 1-12, wherein the misfolded protein is a recombinant misfolded protein.

15. The method of any preceding claim, wherein the misfolded protein comprises one or more of misfolded alpha-synuclein, misfolded prion protein, misfolded tau, misfolded beta-amyloid, misfolded TAR DNA-binding protein 43 (TDP43), misfolded superoxide dismutase 1 (SOD1), misfolded huntingtin ( HTT), misfolded ABri peptide, misfolded ADan peptide, misfolded fragments of immunoglobulin light chains, misfolded fragments of immunoglobulin heavy chains, misfolded serum amyloid A, misfolded transthyretin, misfolded p-2 microglobulin, misfolded apolipoprotein Al, misfolded apolipoprotein All, misfolded apolipoprotein AIV, misfolded apolipoprotein C-ll, misfolded apolipoprotein C-ll, misfolded gelsolin, misfolded lysozyme, misfolded fibrinogen a chain, misfolded cystatin C, misfolded amylin, misfolded calcitonin, misfolded atrial natriuretic factor, misfolded prolactin, misfolded insulin, misfolded lactadherin / medin, misfolded lactoreansferrin / lactoferrin, misfolded odontogenic ameloblast-associated protein, misfolded pulmonary surfactant-associated protein C, misfolded leukocyte cell-derived chemotaxin-2, misfolded galectin-7, misfolded corneodesmosin, misfoldedTGBBI / keratoepithelin, misfolded semenogelin-1, misfolded proteins S100A8 / A9, and misfolded enfuvirtide.

16. The method of any preceding claim, wherein the misfolded protein is in the form of an aggregate.

17. The method of any of claims 1-16, wherein the misfolded protein is in the form of a monomer, dimer, trimer, and / or a tetramer.

18. The method of any preceding claim, wherein the recombinant monomeric protein comprises one or more of recombinant monomeric alpha-synuclein, recombinant monomeric prion protein, recombinant monomeric tau, recombinant monomeric beta-amyloid, recombinant monomeric TDP43, recombinant monomeric SOD1, recombinant monomeric HTT, recombinant monomeric ABri peptide, recombinant monomeric ADan peptide, recombinant monomeric fragments of immunoglobulin light chains, recombinant monomeric fragments of immunoglobulin heavy chains, recombinant monomeric serum amyloid A, recombinant monomeric transthyretin, recombinant monomeric p-2 microglobulin, recombinant monomeric apolipoprotein Al, recombinant monomeric apolipoprotein All, recombinant monomeric apolipoprotein AIV, recombinant monomeric apolipoprotein C-ll, recombinant monomeric apolipoprotein C-ll, recombinant monomeric gelsolin, recombinant monomeric lysozyme, recombinant monomeric fibrinogen a chain, recombinant monomeric cystatin C, recombinant monomeric amylin, recombinant monomeric calcitonin, recombinant monomeric atrial natriuretic factor, recombinant monomeric prolactin, recombinant monomeric insulin, recombinant monomeric lactadherin / medin, recombinant monomeric lactoreansferrin / lactoferrin, recombinant monomeric odontogenic ameloblast-associated protein, recombinant monomeric pulmonary surfactant-associated protein C, recombinant monomeric leukocyte cell-derived chemotaxin-2, recombinant monomeric galectin-7, misfolded corneodesmosin, misfolded TGBBI / keratoepithelin, recombinant monomeric semenogelin-1, misfolded proteins S100A8 / A9, and recombinant monomeric enfuvirtide.

19. The method of any preceding claim, wherein the recombinant monomeric protein comprises a tag, optionally an epitope tag.

20. The method of any preceding claim, wherein the sample is a tissue sample derived from a biopsy, a cerebrospinal fluid sample, or a blood sample.

21. The method of any preceding claim, wherein the sample is a fixed sample, a frozen sample, or a free-floating sample.

22. The method of any preceding claim, wherein the sample comprises cultured cells.

23. A method of screening for the presence of a proteinopathy or an increased risk thereof in a subject, the method comprising detecting misfolded endogenous protein in a tissue sample obtained from the subject by the method of any of claims 1-22, wherein the presence of misfolded endogenous protein in the tissue sample is indicative of the presence of a proteinopathy or an increased risk thereof.

24. A method of diagnosing a proteinopathy in a subject, the method comprising:(i) detecting the presence, abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject by the method of any of claims 1-22; and(ii) comparing the presence, abundance and / or location of the misfolded endogenous protein in the tissue sample with the presence, abundance and / or location of the misfolded protein in a control associated with a distinct proteinopathy, wherein:(A) a difference between the presence, abundance and / or location of the misfolded endogenous protein in the tissue sample and the presence, abundance and / or location of the misfolded protein in the control indicates the absence of the distinct proteinopathy associated with the control; or(B) no difference between the presence, abundance and / or location of misfolded endogenous protein in the tissue sample and the presence, abundance and / or location of the misfolded protein in the control indicates the presence of the distinct proteinopathy associated with the control.

25. The method of claim 24, wherein the method comprises comparing the presence, abundance and / or location of misfolded endogenous protein in the tissue sample with the presence, abundance and / or location of misfolded protein in more than one control, wherein each control is associated with a distinct proteinopathy.

26. A method of monitoring the progression of a proteinopathy in a subject, the method comprising:(i) comparing the abundance and / or location of misfolded endogenous protein in a first tissue sample obtained from the subject at a first time point with the abundance and / or location of misfolded endogenous protein in a second tissue sample obtained from the subject at a second time point; and(ii) determining whether the proteinopathy has progressed between the first and second time point based on the comparison performed in step (i); wherein the abundance and / or location of misfolded endogenous protein in the first and second tissue samples is determined by the method of any of claims 3-22.

27. A method for determining the efficacy of a therapeutic intervention in a patient having a proteinopathy, the method comprising:(i) comparing the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a first time point prior to administration of the therapeutic intervention with the abundance and / or location of misfolded endogenous protein in a tissue sample obtained from the subject at a second time point after administration of the therapeutic intervention; and(ii) determining the efficacy of the therapeutic intervention based on the comparison performed in step (i); wherein the abundance and / or location of misfolded endogenous protein in the first and second tissue samples is determined by the method of any of claims 3-22.

28. An in vitro method for determining the efficacy of a therapeutic intervention against a proteinopathy, the method comprising:(i) contacting a sample comprising misfolded protein associated with the proteinopathy with the therapeutic intervention;(ii) detecting the abundance and / or location of misfolded protein in the sample by the method of any of claims 3-22; and(ill) comparing the abundance and / or location of misfolded protein in the sample to a control; wherein the efficacy of the therapeutic intervention is determined based on the comparison performed in step (iii).

29. The method of claim 28, wherein the control is associated with a therapeutic intervention that is known to be efficacious against the proteinopathy.

30. The method of any of claims 23-29, wherein the proteinopathy is selected from amyloidosis, a synucleinopathy, a prion disease, and a tauopathy.

31. The method of any of claims 23-30, wherein the proteinopathy is selected from Dementia with Lewy bodies, Parkinson's disease, Alzheimer's disease, frontotemporal dementia, vascular dementia, Creutzfeldt-Jakob disease, a trinucleotide repeat disorder (such as Huntington's disease), Amyotrophic lateral sclerosis (ALS), limbic predominant age-related TDP-43 encephalopathy (LATE), and multiple system atrophy.

32. The method of any of claims 27-31, wherein the therapeutic intervention comprises administration of a therapeutic agent, optionally wherein the therapeutic agent is selected from a small molecule, a peptide, an aptamer, and a peptidomimetic.

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