StressMarq Biosciences

New Active Alpha Synuclein Protein Fibrils and Monomers

 

We have developed active alpha synuclein protein fibrils that can be used to induce endogenous alpha synuclein phosphorylation and subsequent Lewy body inclusion formation in neuronal cell culture or for in vitro oligomerization studies.

Our active alpha synuclein protein fibrils seed the formation of new fibrils from active alpha synuclein monomers.

 

Type 1 human and mouse preformed fibrils (PFFs) and monomers have been tested in cell culture assays and thioflavin T assays. Human and mouse PFFs have been tested in vivo and produced alpha synuclein pathology in rat brains within 30 days of injection.  A53T alpha synuclein monomers and PFFs are also available.  The A53T mutation has been linked to early-onset Parkinson’s Disease and increased alpha synuclein fibrillization.

Alpha synuclein oligomers are increasingly thought to be the toxic species in synucleinopathies. Dopamine and EGCG can be used to stabilize alpha synuclein in its oligomeric forms, preventing further aggregation into fibrils.

StressMarq is in the process of developing new types of fibrils as well as thoroughly characterizing our current products. We currently have several types of alpha synuclein proteins available. This table summarizes the differences between them:

 

Catalog No	Protein	Species	Mutant	Monomer source	In Vitro Activity	In Vivo Activity
SPR-322	Type 1 PFFs	Human	Wild-type	SPR-321	Positive for aggregation activity in seeded ThT assay. Induces alpha synuclein phosphorylation and Lewy body pathology in primary rat and mouse neurons and iPSC-derived neurons.	Induces alpha synuclein pathology within 30 days of injection into rat brain.
SPR-322-A594	ATTO 594-Conjugated Type 1 PFFs	Human	Wild-type	SPR-321	Positive for aggregation activity in seeded ThT assay.	Under Investigation
SPR-324	Type 1 PFFs	Mouse	Wild-type	SPR-323	Positive for aggregation activity in seeded ThT assay. Induces alpha synuclein phosphorylation and Lewy body pathology in primary rat neurons,  primary mouse neurons, and N27 rat dopaminergic cell line.	Induces alpha synuclein pathology within 30 days of injection into rat and mouse brain.
SPR-326	Type 1 PFFs	Human	A53T	SPR-325	Positive for aggregation activity in seeded ThT assay. Induces alpha synuclein phosphorylation and Lewy body pathology in primary rat neurons.	Under Investigation
SPR-317	Type 2 PFFs	Human	Wild-type	SPR-316	Does not induce Lewy body pathology in primary rat cells in short-term experiments, but can induce cell death.	Under Investigation
SPR-332	N-Acetylated Type 1 PFFs	Human	Wild-type	SPR-331	Positive for aggregation activity in seeded ThT assay.	Under Investigation
SPR-448	Type 3 PFFs (Treated with alternative scaffold)1	Human	Wild-type	SPR-316	Positive for aggregation activity in seeded ThT assay.	Under Investigation
SPR-450	Soluble Filaments2	Human	Wild-type	SPR-321	Under Investigation	Under Investigation
SPR-466	Dopamine-Stabilized Oligomers	Human	Wild-type	SPR-316	MTT and LDH assays show toxicity in primary mouse neurons.	Under Investigation
SPR-469	EGCG-Stabilized Oligomers	Human	Wild-type	SPR-316	Under investigation	Under investigation
SPR-482	PFFs	Rat	Wild-type	SPR-481	Positive for aggregation activity in seeded ThT assay.	Under investigation

1.Alpha Synuclein Type 3 PFFs have similar structure and activity as Type 1 PFFs, but are activated with a different scaffold and have lower endotoxin levels.
2.May contain small amounts of insoluble fibrils depending on handling.

 

Learn more by watching our webinar: Fibrillar and Oligomeric Constructs of Neurodegenerative Disease Associated Peptides and their Utility in Disease Modelling

Alpha Synuclein Publications

Domain-Independent Inhibition of CBP/p300 Attenuates α-Synuclein Aggregation.

• Hlushchuk, et al. (2021) ACS Chemical Neuroscience. Online ahead of print.

Suppression of aggregate and amyloid formation by a novel intrinsically disordered region in metazoan Hsp110 chaperones.

• Yakubu, U.M., Morano, K.A. (2021) bioRxiv. 296:100567.
• Read more on our blog.

The SUMO conjugase Ubc9 protects Dopaminergic cells from cytotoxicity and enhances the stability of α-synuclein in Parkinson’s disease models.

• Verma, D.K. et al. (2020) eNeuro. 0134-20.2020.
• Read more on our blog.

Alpha Synuclein Protocols and Technical Support

Handling Instructions – Learn how to handling alpha synuclein proteins safely and effectively

Thioflavin T Assay Protocol – Thioflavin T is a dye that binds to beta-sheets and allows the for the study of alpha synuclein aggregation.

Cell Culture Assay Protocol – Learn how alpha synuclein can be added to primary neuronal culture to induce Lewy body pathology.

In Vivo Assay – Alpha synuclein PFFs can be injected into rodent brains, where they produce alpha synuclein aggregation and pathology.

Frequently Asked Questions

Alpha Synuclein Protein Resources

Types of Alpha Synuclein Fibrillar Constructs – Learn about different types of our alpha synuclein fibrillar constructs.

Which Alpha Synuclein Construct Should I Use? – A guide to modelling synucleinopathies.

Alpha Synuclein Research Tools Poster – A summary of proteins and antibodies available for alpha synuclein research.

Video: Why do we Sonicate Alpha Synuclein Fibrils? – Learn why we recommend sonication of fibrils prior to use in research.

How does Sonication Affect Fibrils? – Detailed information on sonicators and fibril sonication.

A53T Alpha Synuclein – The A53T mutation can enhance alpha synuclein aggregation and has been linked to genetic forms of Parkinson’s.

Alpha Synuclein Phosphorylation at Serine 129 – Learn why pSer129 is used to detect alpha synuclein pathology and how it is relevant to PD.

N-Terminal Acetylated Alpha Synuclein – Learn about how this post translational modification affects alpha synuclein aggregation.

Oligomers – Read about the alpha synuclein oligomer toxicity in synucleinopathies

Learn More About Alpha Synuclein in Parkinson’s Disease

Alpha Synuclein Monomers – Why target them?

N-terminal Acetylated Alpha Synuclein in Parkinson’s Disease – Learn about the effects of N-terminal acetylation on aggregation.

Alpha Synuclein and Tau: Interactions in Neurodegenerative Diseases – Learn how alpha synuclein and tau can interact to promote each other’s aggregation.

Alpha Synuclein in Parkinson’s Disease – A summary of the role of alpha synuclein and how it can contribute to PD pathogenesis.

Alpha Synuclein in Parkinson’s Disease Diagnostics –  Learn how alpha synuclein can be used as a biomarker to improve disease diagnostics.

Alpha Synuclein in Parkinson’s Disease Therapeutics – Treatment approaches to targeting alpha synuclein in Parkinson’s Disease.

Alpha Synuclein Oligomers: Dopamine Stabilization and Neurodegeneration – Learn why oligomers are thought to be toxic and how dopamine is involved in their toxicity

StressMarq’s Active Tau Pre-Formed Fibrils (PFFs)

 

StressMarq is excited to offer active tau proteins to help researchers study tau aggregation, a hallmark of neurodegenerative diseases including Alzheimer’s. StressMarq is the first to offer active tau preformed fibrils (PFFs) and filaments for neuroscience research.  The process of tau aggregation can be seeded by active tau PFFs, which recruit monomers to form larger tau fibrils.  This has been demonstrated in thioflavin T assays where an increase in fluorescence, indicative of tau fibrillization, is seen when active tau PFFs are combined with active tau monomers.  Certain tau PFFs have been injected into P301L mice, where they seed tau aggregation and induce tau pathology in the hippocampus.

Monomers and fibrils are available both in the full-length isoform of the tau protein (2N4R or Tau-441) or a truncated form (K18).  Tau-441 has a molecular weight of approximately 46 kDa, whereas K18 tau has a molecular weight of approximately 15 kDa.  dGAE is a fragment of the tau protein consisting of amino acids 297-391. It is one of the core PHF subunits,1 includes both microtubule-binding domains and proline-rich regions, and assembles into PHF-like fibrils in vitro without additives or templates.2 

Proteins are available as wild-type or with a variety of mutations. P301S and P301L mutations occur in exon 10 and are associated with frontotemporal dementia.  The P301S mutation reduces tau’s ability to assemble microtubules, and the P301L mutation promotes beta-sheet formation and the formation of PHFs.  Both P301S and P301L mutant transgenic mouse models are used in tau research.  The K280 deletion mutation is also associated with frontotemporal dementia and promotes fibrillization into paired helical filaments (PHFs) in the absence of heparin and other inducers. The C322A mutation also increases tau’s ability to form PHFs.2

PFFs induce tau aggregation; full-length PFFs may be more effective in seeding fibrillization, but a combination of both can be particularly toxic to neurons. Most tau varieties are fibrillized using a heparin scaffold; soluble tau filaments are fibrillized using a linear anionic scaffold. dGAE tau and K18 K280 deletion tau both fibrillize without scaffolds.

Tau PFFs can be expressed in baculovirus in addition to E. coli. Baculovirus tau PFFs are post-translationally modified, fibrillize without a scaffold, and are endotoxin-free*.

1. Novak, M. et al. (1993) EMBO J. 12, 365-370.
2. Al-Hilaly, Y.K. et al. (2017) J. Mol. Biol. 429(23):3650-3665.

*. Endotoxin <0.05 EU/mL (below the detectable range of the assay)

StressMarq is in the process of developing new types of fibrils for neurodegenerative disease research as well as thoroughly characterizing our current products. We currently have several types of tau PFFs and filaments available. This table summarizes the differences between them:

Catalog No.	Protein	Species	Expression System	Length/Fragment	Mutant	Monomer source	Fibrillization Scaffold	In vivo activity
SPR-329	2N4R, P301S PFFs	Human	E. coli	2N4R (441-amino acid) Full length	P301S	SPR-327	Heparin	Under Investigation
SPR-480	2N4R PFFs	Human	E. coli	2N4R (441-amino acid) Full length	WT	SPR-479	Heparin	Under Investigation
SPR-329-A488	ATTO 488-conjugated 2N4R, P301S PFFs	Human	E. coli	2N4R (441-amino acid) Full length	P301S	SPR-327	Heparin	Under Investigation
SPR-330	K18, P301L PFFs	Human	E. coli	K18 (4R) Truncated	P301L	SPR-328	Heparin	Induces tau phosphorylation and aggregation in P301L transgenic mouse hippocampus
SPR-461	dGAE PFFs	Human	E. coli	dGAE (AA297-391) Truncated	WT	SPR-444		Under Investigation
SPR-462	dGAE, C322A PFFs	Human	E. coli	dGAE (AA297-391) Truncated	C322A	SPR-445		Under Investigation
SPR-463	2N4R, P301S Filaments	Human	E. coli	2N4R (441-amino acid) Full Length	P301S	SPR-327	Linear Anionic Scaffold	Under Investigation
SPR-471	2N4R, P301S PFFs	Human	Baculovirus	2N4R (441-amino acid) Full length	P301S	SPR-473		Under Investigation
SPR-475	2N4R, P301S PFFs	Mouse	E. coli	2N4R (441-amino acid) Full Length	P301S	SPR-474	Heparin	Under Investigation
SPR-477	K18, K280 Deletion PFFs	Human	E. coli	K18 (4R) Truncated	ΔK280	SPR-476		Under Investigation

Tau Publications

Astrocyte senescence and SASP in neurodegeneration: tau joins the loop

• Ungerleider, K. et al. (2021) Cell Cycle. 1-13.
• Read more on our blog.

Aberrant AZIN2 and polyamine metabolism precipitates tau neuropathology

• Sandusky-Beltran, L.A. et al. (2021) J Clin Invest. 131(4):e126299.
• Read more on our blog.

Cornel Iridoid Glycoside Regulates Modification of Tau and Alleviates Synaptic Abnormalities in Aged P301S Mice

• Yang, Cc. et al. (2020) Curr Med Sci. 40, 1040–1046(2020).
• Read more on our blog.

Tau Protocols

Handling Instructions – Learn how to handling tau proteins safely and effectively

Thioflavin T Assay Protocol – Thioflavin T is a dye that binds to beta-sheets and allows the for the study of tau aggregation.

Tau Frequently Asked Questions

Learn about the best storage conditions and experimental methods for PFFs.

Tau Protein Resources

Types of Tau Fibrillar Constructs – Learn about our tau fibrillar constructs and their characteristics.

Which Tau Construct Should I Use? – A Guide to Modelling Tauopathies

StressMarq Tau Research Tools Poster – A summary of proteins and antibodies available for tau research.

Studying Alzheimer’s and Parkinson’s Disease using Pre-Formed Fibrils – An interview with StressMarq President & CEO Ariel Louwrier about PFFs in neurodegeneration research.

Modelling Alzheimer’s Disease in Cells and Animals – Read about different models used in AD research and their strengths and weaknesses.

Tau Mutations and their role in Neurodegenerative Disease – Learn about the role of various tau mutations in aggregation and disease progression.

Tau Phosphorylation in Alzheimer’s Disease – Learn how phosphorylation at certain sites is linked to tau aggregation.

Tau Post-Translational Modifications: An Overview of the Baculovirus Expression Vector System – What are the advantages of the baculovirus expression vector system in recombinant protein production?

Tau Post-Translational Modifications in Alzheimer’s Disease: Diagnostic and Therapeutic Opportunities – Learn about the role of tau post-translational modifications in AD and potential therapeutic strategies.