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US-12618856-B2 - Alpha-synuclein mutants and uses thereof

US12618856B2US 12618856 B2US12618856 B2US 12618856B2US-12618856-B2

Abstract

Alpha-Synuclein mutants and uses thereof are disclosed herein. The mutants of the present invention are non-self-aggregating forms of Alpha-Synuclein which make them a suitable candidate for use as substrates in aggregation assay for evaluating the presence of misfolded α-Syn protein. Also disclosed are kits and method for detection of synucleinopathies in individuals, using the mutants of the present invention.

Inventors

  • Samir K. Maji
  • Gadhe Laxmikant GANESHRAO
  • Rakesh Kumar
  • Soumik RAY

Assignees

  • INDIAN INSTITUTE OF TECHNOLOGY BOMBAY

Dates

Publication Date
20260505
Application Date
20220419
Priority Date
20210419

Claims (8)

  1. 1 . An invitro method for detection of Synucleinopathy in an individual, said method comprising: obtaining a sample from said individual; mixing said sample with a mutant to obtain a reaction mixture; incubating the reaction mixture to facilitate amplification of pathological α-Syn aggregates; detecting the presence of amplified pathological aggregates; and determining presence of the aggregates for detecting Synucleinopathy, wherein the mutant is an artificial mutant alpha-Synuclein (α-Syn) protein, comprising a polypeptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, said mutant having decreased or no ability to self-aggregate.
  2. 2 . The method as claimed in claim 1 , wherein said detecting step comprises mixing said amplified pathological aggregates and Congo red dye, and incubating; placing the mixture on suitable reaction paper; and detecting based on retention of said Congo red dye on the paper.
  3. 3 . The method as claimed in claim 1 , wherein said Synucleinopathy is at least one condition selected from the group consisting of Parkinson's disease, Dementia with Lewy bodies, Lewy body variant of Alzheimer disease, Multiple system atrophy, and Autonomic dysfunction.
  4. 4 . The method as claimed in claim 1 , wherein the step of incubating further includes sonicating the reaction mixture.
  5. 5 . The method as claimed in claim 1 , wherein the pathological α-Syn aggregates are present in the body fluids of said individuals.
  6. 6 . The method as claimed in claim 1 , wherein the sample is a biological sample consisting of body fluids such as blood, blood components, plasma, serum, saliva, and cerebrospinal fluid, obtained from a said individual.
  7. 7 . The method as claimed in claim 1 , wherein the incubation step is performed by subjecting the mixture to seed amplification assays.
  8. 8 . The method as claimed in claim 1 , wherein said detecting of pathological α-Syn aggregate includes at least one method selected from a group consisting of Thioflavin T fluorescence assay, western blotting method, immunoassays, immunostaining, Congo red dot assay, and enzyme-linked immunosorbent assay (ELISA).

Description

CROSS REFERENCE TO RELATED APPLICATION This application is based on and derives the benefit of Indian Provisional Application 202121018108 filed on the 19 Apr. 2021, the contents of which are incorporated herein by reference. TECHNICAL FIELD The present invention relates to alpha-Synuclein mutants and more particularly to alpha-Synuclein mutants having modified aggregation properties. Further, it relates to a kit and method for detection of Synucleinopathies such as Parkinson's disease (PD) using alpha-Synuclein mutants. INCORPORATION BY REFERENCE The sequence listing provided in the file entitled SequenceListingv2GPB.txt, which is an ASCII text file that was created on Apr. 19, 2022, and which comprises 8,290 bytes, is hereby incorporated by reference in its entirety. BACKGROUND Neurological disorders have risen substantially in the past 30 years, and it is a leading source of disability globally (Feigin V L et al. The global burden of neurological disorders: translating evidence into policy. Lancet Neurol. 2020 March; 19(3):255-265. doi: 10.1016/S1474-4422(19)30411-9. Epub 2019 December 5. PMID: 31813850). Parkinson's disease (PD) is the second most common progressive neurological disorder after Alzheimer's disease (AD). The prevalence of PD is 160/100000 in Western Europe (Davie, C. A. (2008) A review of Parkinson's disease. Br. Med. Bull. 86, 109-127) and it affects 1-2 per 1000 of population at any time. The prevalence of PD increases up to ˜1% of the population for age groups more than 60 years (Tysnes, O. B., and Storstein, A. (2017) Epidemiology of Parkinson's disease. J. Neural Transm. 124, 901-905). According to the global burden of disease study 2016, the overall number of people affected by PD is 6.1 million globally and Parkinson's disease is the fastest growing neurological disorder. Parkinson's disease is characterized by shaking, rigidity, bradykinesia, and difficulties with walking. Julian M et al reported Parkinson's disease is primarily characterized by neuronal deaths mainly in Striatum and Substantia nigra (SN) regions of the brain (Fearnley J M, and Lees A J (1991) Ageing and Parkinson's disease: substantia nigra regional selectivity. Brain. 114, 2283-2301). These neurons mainly play a role in the synthesis of dopamine neurotransmitters. Degeneration of dopaminergic neurons of substantia nigra results in impairment in motor functions as well as non-motor functions. Secondary symptoms may include neuropsychiatric dysfunction, sleep disorders, autonomic dysfunction, sensory symptoms, and pain, etc. The primary pathological hallmarks of PD are degeneration of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain and the deposition of a protein in the form of Lewy bodies. α-Syn protein (140 amino acid long) is a major component of Lewy bodies in PD patient brain tissue (Osterhaus, A., Groen, J., Bildt, M. Van De, Martina, B., Vos, J., and Egmond, H. Van (1997) Î-Synuclein in Lewy bodies Endogenous proviruses as “mementos”?). Isolated amyloid from Lewy body showed similar fibril-like morphology to that of in vitro fibrils of α-Syn. The α-Syn aggregation is shown to be nucleation dependent process and it can be seeded by misfolded nuclei or fibril of α-Syn (Stephen J. Wood. Et al. α-Synuclein Fibrillogenesis Is Nucleation-dependent: implications for the pathogenesis of parkinson's disease *, Journal of Biological Chemistry, Volume 274, Issue 28, 1999, Pages 19509-19512, ISSN 0021-9258, https://doi.org/10.1074/jbc.274.28.19509). The physiological function of α-Syn is unclear, but it is considered to be involved in vesicular trafficking, synaptic function, and neurotransmitter release (Abeliovich A, Schmitz Y, Fariñas I, Choi-Lundberg D, Ho W H, Castillo P E, Shinsky Verdugo J M, Armanini M, Ryan A, Hynes M, Phillips H, Sulzer D, Rosenthal A. Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron. 2000 January; 25(1):239-52, doi: 10.1016/s0896-6273(00)80886-7. PMID: 10707987). The multiplication and disease-linked point mutation in the SNCA gene lead to the acceleration of α-Syn oligomerization and fibrillation that cause the early onset of PD. α-Syn is also associated with filamentous inclusion of multiple system atrophy (MSA) and dementia with Lewy body (DLB) that have the filamentous pathology in nerve cells and glial cells. Synucleinopathy, a term used for diseases that show the common involvement of accumulated aggregates of α-Syn in nerve cells, glial cells, or nerve fibers. Mutations in the SNCA gene (such as duplications, triplications, or point mutation), which encodes α-Syn are associated with the autosomal dominant form of PD. Parkinson's disease is characterized by a variety of motor and non-motor symptoms that affect the lifestyle of the individual to a variable degree. As there is no definitive diagnostic test available, Parkinson's disease diagnosis is mainly based on clinical criteria but the pathologica