Active Pre-formed Fibrils for Neurodegeneration Research

Active Pre-formed Fibrils for Neurodegeneration Research

StressMarq supplies cutting-edge research products for the study of neurodegenerative diseases.  This includes our new alpha synuclein, beta synuclein, gamma synuclein, tau, SOD, and TTR proteins.  Certain fibrils and filaments can induce protein aggregation, inducing disease pathology in vitro and in vivo. These proteins can be used to study diseases such as Alzheimer’s, Parkinson’s, ALS, and amyloidosis.

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.

Active Pre-formed Fibrils for Neurodegeneration Research

Rat primary hippocampal neurons treated with Type 1 mouse alpha synuclein PFFs show Lewy body inclusions

Active Pre-formed Fibrils for Neurodegeneration Research

IHC analysis of rat brain injected with Type 1 mouse alpha synuclein PFFs (SPR-324) shows alpha synuclein pathology.

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 alpha synuclein proteins available. This table summarizes the differences between them:

Catalog NoProteinMutantMonomer sourceEndotoxinThioflavin T ActivityPrimary Cells ActivityIn Vivo Activity
SPR-322Type 1 PFFsWild-typeSPR-32110-20 EU/mL>12 000 fluorescent units when combined with SPR-321 monomer. 1300 fluorescent units when combined with SPR-316 monomerInduces alpha synuclein phosphorylation and Lewy body pathology in primary rat and mouse neuronsInduces alpha synuclein pathology within 30 days of injection into rat brain
SPR-324Type 1 PFFsWild-typeSPR-32310-20 EU/mL> 3100 fluorescent units when combined with SPR-323 monomerInduces alpha synuclein phosphorylation and Lewy body pathology in primary rat neurons and primary mouse neuronsInduces alpha synuclein pathology within 30 days of injection into rat brain.
SPR-326Type 1 PFFsA53TSPR-32510-20 EU/mL28 000 fluorescent units when combined with SPR-325 monomerInduces alpha synuclein phosphorylation and Lewy body pathology in primary rat neuronsUnder Investigation
SPR-317Type 2 PFFsWild-typeSPR-316<2 EU/mL3000 fluorescent units when combined with SPR-321 monomer. 400 fluorescent units when combined with SPR-316 monomerDoes not induce Lewy body pathology in primary rat cells in short-term experiments, but can induce cell deathUnder Investigation
SPR-448Type 3 PFFs (Treated with alternative scaffold)1Wild-typeSPR-316<2 EU/mL1000 fluorescent units when combined with SPR-321 monomerUnder investigationUnder Investigation
SPR-450Soluble Filaments2Wild-typeSPR-32110-20 EU/mLUnable to seed SPR-321 monomer (measured by turbidity). Forms insoluble fibrils in 7 days when incubated at 37C with shaking at 700 rpm.Under InvestigationUnder Investigation

Beta synuclein, like alpha synuclein, is found in presynaptic nerve terminals. It is believed to play a role in maintaining vesicular membrane curvature1 and does not aggregate under physiological conditions.2 WT beta synuclein inhibits the aggregation of alpha synuclein3 and mutant (V70M and P123H) beta synuclein has been associated with Dementia with Lewy Bodies (DLB).4 Human beta synuclein PFFs do not induce endogenous alpha synuclein aggregation in primary rat hippocampal neurons, meaning they could be useful as a control in alpha synuclein experiments.

Catalog No.ProteinSpeciesMutantMonomer sourceEndotoxin
SPR-457Type 1 PFFsHumanWild-typeSPR-405Made with LPS, endotoxin not removed
Active Pre-formed Fibrils for Neurodegeneration Research

TEM of Active Human Recombinant Beta Synuclein Protein Preformed Fibrils (Type 1) (SPR-457)

Active Pre-formed Fibrils for Neurodegeneration Research

Primary rat hippocampal neurons treated with human beta synuclein preformed fibrils (SPR-457) do not show Lewy body inclusion formation. Fibrils were sonicated 1 hour and added at 1 ug/mL concentration. A) Hoechst. B) pSer129. C) Merge

1. Westphal, C.H., Chandra, S.S. (2013). J Biol Chem. 288(3):1829-40.
2. Yamin, F. et al. (2005) Biochemistry. 44, 9096-9107.
3. Hashimoto, M. et al. (2001) Neuron. 32,213-223.
4. Ohtake, H. et al. (2004). Neurology. 63(5):805-11

Gamma synuclein is associated with neurodegenerative diseases as well as certain cancers. Gamma synuclein can seed alpha synuclein aggregation,1 and its oxidation and aggregation is seen in Dementia with Lewy bodies,2 Parkinson’s Disease,2 and ALS.3

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 gamma synuclein PFFs available. This table summarizes the differences between them:

Catalog No.ProteinSpeciesMutantMonomer sourceEndotoxinThioflavin T
SPR-459Type 1 PFFsHumanWild-typeSPR-407Made with LPS, 10-20 EU/mLUnder investigation
SPR-460Type 1 PFFsMouseWild-typeSPR-408Made with LPS, 10-20 EU/mL>800 fluorescent units when combined with SPR-408 monomer
Active Pre-formed Fibrils for Neurodegeneration Research

TEM of Active Human Recombinant Gamma Synuclein Protein Preformed Fibrils (Type 1) (SPR-459)

  1. Surguchov A: Synucleins: are they two-edged swords? J Neurosci Res 2013, 91(2):16
  2. Galvin, J.E. Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains a-, b-, and g-synuclein. Proc Natl Acad Sci USA. 1999 Nov 9; 96(23): 13450–13455.
  3. Peters OM, Millership S, Shelkovnikova TA, et al. Selective pattern of motor system damage in gamma-synuclein transgenic mice mirrors the respective pathology in amyotrophic lateral sclerosis. Neurobiol Dis 2012;48: 124–131.

Cu/Zn superoxide dismutase 1 (SOD1) misfolding and aggregation into neurotoxic species is implicated in Amyotrophic Lateral Sclerosis (ALS). Superoxide dismutase (SOD) is an endogenously produced intracellular enzyme present in almost every cell in the body (3). It works by catalyzing the dismutation of the superoxide radical O2ˉ to O2 and H2O2, which are then metabolized to H2O and O2 by catalase and glutathione peroxidase (2,5). In general, SODs play a major role in antioxidant defense mechanisms (4). There are two main types of SOD in mammalian cells. One form (SOD1) contains Cu and Zn ions as a homodimer and exists in the cytoplasm. The two subunits of 16 kDa each are linked by two cysteines forming an intra-subunit disulphide bridge (3). SOD1 misfolding and aggregation into neurotoxic species is implicated in Amyotrophic Lateral Sclerosis (ALS).

1. Adachi T., et al. (1992). Clin. Chim. Acta. 212: 89-102.
2. Barrister J.V., et al. (1987). Crit. Rev. Biochem. 22:111-180.
3. Furukawa Y., O’Halloran T. (2006). Antioxidants & Redo Signaling. Vol 8, No 5,6.
4. Gao B., et al. (2003). Am J Physiol Lung Cell Mol Physiol 284: L917-L925.
5. Hassan H.M. (1988). Free Radical Biol. Med. 5: 377-385.

Active Pre-formed Fibrils for Neurodegeneration Research

StressMarq’s Active Tau Proteins

StressMarq is excited to announce the launch of new 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.

Active Pre-formed Fibrils for Neurodegeneration Research

Immunohistochemistry analysis of P301L mouse hippocampus injected with K18 P301L tau PFFs (SPR-330) shows seeding of tau pathology at injection site. AT8 (pSer202/pThr205) tau antibody shows tangle-like inclusions. Experiments performed at reMYND N.V.

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.

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.

Active Pre-formed Fibrils for Neurodegeneration Research

TEM of active recombinant Tau441 (2N4R), P301S mutant preformed fibrils (PFFs) (SPR-329).

Active Pre-formed Fibrils for Neurodegeneration Research

TEM of active recombinant Tau (K18), P301L mutant preformed fibrils (PFFs) (SPR-330).

Active Pre-formed Fibrils for Neurodegeneration Research

TEM of Human Recombinant Tau Protein Filaments (SPR-463)

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.

 

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.ProteinSpeciesLength/FragmentMutantMonomer sourceFibrillization Scaffold
SPR-3292N4R, P301S PFFs Human2N4R (441-amino acid) Full lengthP301SSPR-327Heparin
SPR-330K18, P301L PFFsHumanK18 (4R) TruncatedP301LSPR-328Heparin
SPR-461dGAE PFFsHumandGAE (AA297-391) TruncatedWTSPR-444
SPR-462dGAE, C322A PFFsHumandGAE (AA297-391) TruncatedC322ASPR-445
SPR-4632N4R, P301S FilamentsHuman2N4R (441-amino acid) Full LengthP301SSPR-327Linear Anionic Scaffold
SPR-4752N4R, P301S PFFsMouse2N4R (441-amino acid) Full LengthP301SSPR-474Heparin
SPR-477K18, K280 Deletion PFFsHumanK18 (4R) TruncatedΔK280SPR-476

Transthyretin (TTR) misfolding is associated with amyloid diseases. Both the L55P and Y78F mutants are amyloidogenic.1,2 L55P and Y78F TTR filaments and monomers are available. TTR aggregation can be measured via turbidity.3­

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 TTR filaments available. This table summarizes the differences between them:

Catalog No.ProteinSpeciesMutantMonomer source
SPR-464L55P FilamentsHumanL55PSPR-451
SPR-465Y78F FilamentsHumanY78FSPR-452
Active Pre-formed Fibrils for Neurodegeneration Research

TEM of Human Recombinant Transthyretin L55P Variant Protein Filaments (SPR-464)

1. Lashuel, H.A. et al. (1999) Biochem. 38(41):13560-73.
2. Terazaki, H. et al. (2006) Lab. Investigation. 86, 23-31
3. Robinson, L. Z., Reixach, N. (2014) Biochem. 53(41):6496-510

Active Pre-formed Fibrils for Neurodegeneration Research

Turbidity Assay shows increased turbidity in Human Recombinant Transthyretin L55P Variant Protein Filaments (SPR-464)

Frequently Asked Questions

Storage and Shipping

How are the PFFs shipped?

Sent frozen at 80°C and shipped on dry ice. We have a storage facility in the US, which prevents customs delays on US domestic shipments that could potentially affect product quality.

How should I store the monomer and PFFs?

Monomer and PFFs should be stored at -80°C. Please note: PFFs should not be kept at 4°C for a prolonged period of time, because instability of the fibrils will occur.

How should I thaw the monomer and PFFs?

Monomer should be thawed on ice to prevent spontaneous fibrillization, while PFFs should be thawed at room temperature. During experimental use, keep monomer on ice, and PFFs at room temperature.

Is it ok to aliquot from the 100µg vial size?

Yes, that is ok. After aliquoting the first time, it is not recommended to aliquot a second time because freeze thaw cycles may decrease fibril activity. It is also not recommended to flash freeze aliquots.

Quality Control

What kind of QC is performed on the PFFs?

Purity is determined via SDS-PAGE, and sedimentation assays are performed to make sure that most of the monomer was converted to fibril. All batches/lots are sent for EM imaging as well.

What seeding activity assays are performed on the PFFs?

We assess seeding activity of the PFFs via the ThT assay.

How is purity of the sample determined?

We use SDS-PAGE to determine the purity of the protein after purification. A sedimentation assay is also done and run on an SDS-PAGE to assess the amount of soluble alpha-synuclein present and determine how much of the monomer was converted to fibril. >90% must be in the insoluble fraction in order for it to be a successful batch. There will always be some monomer and low molecular weight oligomers present in the fibril preps but they are <10% of the total protein at the time of aliquoting (freeze/thaw cycles and sample handling may change this). We don’t run a native gel on every lot of SPR-322. As the fibrils are quite large, they end up getting stuck in the well of the stacking gel. Native gels are not part of our regular QC of this prep as the sedimentation assay tells us what we need to know.

Experimental Procedures

Will monomeric alpha-synuclein spontaneously fibrillize?

We recommend that monomeric protein be kept on ice during thawing and use, to prevent any spontaneous fibrillization. If any spontaneous fibrillization does occur if the vial is not kept on ice, the amount that aggregates will be very small, because aggregation requires shaking/agitation usually.

Have the PFFs been injected into mice?

SPR-330 has been injected into P301L transgenic mice, where it seeded endogenous tau phosphorylation and aggregation. SPR-322 and SPR-324 have been shown to seed endogenous alpha-synuclein and cause pathology in rats via injection of PFFs. The images for this experiment are found on the product pages of SPR-330, SPR-322, and SPR-324.

What control should I use with the PFFs?

It depends on the experiment being performed, but generally it is recommended to use the starting monomer source for each PFF. Eg. use SPR-316 with SPR-317, use SPR-321 with SPR-322, use SPR-323 with SPR-324. Beta synuclein PFFs can also be used as a control for alpha synuclein PFFs, since they do not seed aggregation.

Where can I find experimental protocols?

Thioflavin T, cell culture, and in vivo procedures used to generate our validation images can be found here.

Is there a sonication protocol I can refer to?

Because each lab/researcher will likely have a different model of sonicator (probe/bath etc), we cannot recommend a specific protocol to follow. Ideally, the researcher will optimize their sonication protocol and validate fibril length via EM so that they are obtaining fibrils with an average length of ~50nm. We sonicate our fibrils in a bath sonicator at room temperature.

How do I determine which fibril to use in my experiment?

You can email us at technical@stratech.co.uk with details of the experiment you are planning to set up, and we can recommend which fibril type would be best suited for your assay.

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