FIELD OF THE DISCLOSURE

This is related to the field of α-synucleinopathies, specifically to methods for rapid detection of misfolded α-Synuclein (αSyn) and diagnosis of α-synucleinopathies.

BACKGROUND

Many neurodegenerative diseases are caused, at least in part, by the accumulation of specific misfolded proteins. These deposits are typically identified upon post-mortem analysis of brain tissue, allowing diagnoses to be made based on specific neuropathological and molecular findings. Currently, prior to death, less certain diagnoses can be proffered based on combinations of clinical signs and biomarker levels. In addition, discrimination of causative agents in some diseases can be complicated by variations and overlaps in diagnostic indicators.

With Parkinson disease (PD), diagnoses are usually made late in the disease course with less accuracy by general practitioners and neurologists (50-75%) than movement disorder specialists (90%) (Hughes et al., 2002, Brain 125:861-70; Hughes et al., 1992, J. Neurol. Neurosurg. Psychiatry 55:181-184; Alder et al., 2014, Neurology 83:406-12). At first visit the diagnostic accuracy of a clinical diagnosis of PD varies between 26% and 88% (Alder et al., 2014, supra). Lewy Body dementias (including Dementia with Lewy bodies (DLB)), and multiple systems atrophy (MSA) are synucleinopathies that involve the pathological accumulation of α-Synuclein (αSyn). Attempts have been made to determine if cerebrospinal fluid (CSF) levels of total, phosphorylated or oligomeric αSyn can be used for diagnosis (reviewed in Parnetti et al., 2013, Nat Rev Neurol 9:131-40). To date, the results have been variable between studies, and the diagnostic utility of immunoassays for αSyn in CSF has not been demonstrated (Mollenhauer et al., 2017, Mov Disord doi:10.1002/mds.27090.; Sancesario and Bernardini, 2015, Crit Rev Clin Lab Sci 52:314-26) A need remains for highly sensitive assays for αSyn.

SUMMARY OF THE DISCLOSURE

While amplification reactions for detecting αSyn seeding activity have been developed, these methods are slow, and must be performed over 5 to 13 days. It is disclosed herein that using specific mutated recombinant alpha synuclein (rαSyn) substrates, a more rapid αSyn RT-QuIC assay was developed. Misfolded αSyn present in a sample was quickly detected, with excellent sensitivity and specificity. These methods disclosed herein can be used to detect misfolded αSyn in a biological sample, or fraction thereof, and can be used to diagnose synucleinopathies.

In some embodiments, methods are disclosed for determining whether a subject has a synucleinopathy. These methods include performing an αSyn seeding assay on a biological sample from the subject, or a fraction thereof, wherein the seeding assay includes: i) contacting the biological sample, or fraction thereof, with a soluble mutated rαSyn to form a reaction mixture, wherein the soluble mutated rαSyn comprises an amino acid sequence a) comprising one to eight point mutations in SEQ ID NO: 1, wherein the point mutations are at residues K23, D2, K21, K45, T59, G67, V77, and/or A78 and/or b) a deletion of up to ten amino acids in amino acids 2-11 of SEQ ID NO: 1; ii) incubating the reaction mixture to permit coaggregation of misfolded αSyn aggregates present in the biological sample, or fraction thereof, with the mutated rαSyn; iii) maintaining incubation conditions that promote coaggregation of the mutated rαSyn with the misfolded αSyn aggregates to result in a conversion of the soluble mutated rαSyn to mutated rαSyn aggregates while inhibiting spontaneous aggregation of soluble mutant rαSyn; and iv) agitating mutated rαSyn aggregates formed during step iii), wherein the conditions comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. Misfolded mutated rαSyn aggregates are detected in the reaction mixture, wherein detection of misfolded mutated rαSyn aggregates in the reaction mixture indicates that the subject has the synucleinopathy.

Methods are also disclosed for detecting misfolded αSyn in a biological sample or a fraction thereof. These methods include mixing the biological sample or fraction thereof with a purified soluble mutated rαSyn to make a reaction mix, wherein the soluble mutated rαSyn comprises an amino acid sequence a) comprising one to eight point mutations in SEQ ID NO: 1, wherein the point mutations are at residues K23, D2, K21, K45, T59, G67, V77, and/or A78 and/or b) with a deletion of up to ten amino acids in amino acids 2-11 of SEQ ID NO: 1. An amplification reaction is performed that includes: (i) incubating the reaction mix to permit coaggregation of the soluble mutated rαSyn with misfolded αSyn that may be present in the reaction mix, and maintaining incubation conditions that promote coaggregation of the soluble mutated rαSyn with the misfolded αSyn and result in a conversion of the soluble mutated rαSyn to misfolded rαSyn aggregates initiated by the presence of misfolded αSyn in the sample, while inhibiting development of spontaneously arising rαSyn aggregates; and (ii) agitating aggregates formed during step (i), in shaking cycles, wherein each shaking cycle of the shaking cycles comprises a period of rest and a period of shaking, wherein agitating is performed in the absence of sonication. Misfolded rαSyn aggregates are then detected in the reaction mix, and detection of misfolded rαSyn in the reaction mix indicates that misfolded αSyn was present in the biological sample.

Protein amino acid residues are referred to throughout by their amino acid identity and position number in the protein. For example, with regard to SEQ ID NO: 1, “D2” refers to the aspartic acid at position 2 of the protein described in SEQ ID NO: 1; “K21” refers to the lysine at position 21 of the protein described in SEQ ID NO:1; “K23” refers to the lysine at position 23 of the protein described in SEQ ID NO:1 and so forth. Mutations to protein residues are referred to throughout by the amino acid originally present in the protein, the position number of the amino acid in the protein, followed by the new amino acid replacing the original amino acid in the mutated form of the protein. For example, “K23Q” indicates that the lysine at position 23 has been changed to a glutamine “K23X” indicates that the lysine at position 23 has been changed to any other amino acid except lysine.

The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

