Short Article on Alzheimer's Disease: #5 Tau Aggregation and Propagation

The axons of neurons are made of microtubules that serve as tracks for neuronal transport. They are formed from tubulin and are widely believed to be stabilised by a protein called tau. Post-translational modifications of tau (e.g. phosphorylation) alter its structure and conformation and as a result impact its ability to bind to microtubules. In Alzheimer’s disease, several factors will promote these changes and the resulting modified tau proteins will start to self-aggregate into neurofibrillary tangles, which are thought to contribute to the degeneration of neurons.

Tau Aggregation

Structural biology studies have shown that the main components of neurofibrillary tangles in AD are paired helical and straight filaments and are mostly composed of abnormally hyperphosphorylated tau proteins.[1] The tangles form by aggregation of monomeric hyperphosphorylated tau into higher-order molecular species and assemble first as dimers then oligomers followed by (Paired Helical Filaments) PHFs and finally into the characteristic neurofibrillary tangles of AD.

Tau Propagation
Recent evidences suggest that tau aggregates are formed first in a small number of brain cells and then propagate to other neuroanatomically connected regions via a “prion-like mechanism” [2]. The idea is that tau “seeds” or fibrils, when entering adjacent cells, might recruit endogenous tau and seed further aggregation and as a result spread pathology. This idea is gaining popularity and several studies have demonstrated its occurrence in cell culture and animal studies.[3]

StressMarq Biosciences has recently released the first commercially available Active Tau Monomers and Pre-Formed Fibrils (PFFs) to facilitate research on tau aggregation. This follows the recent launch of its first active human and mouse alpha-synuclein monomers and pre-formed fibrils for Parkinson's research. All products are available in the UK and Ireland from Newmarket Scientific.

The new active tau proteins are available as two sets of monomers and PFFs:

• from a full-length tau protein (2N4R or Tau-441) with a P301S mutation encoded by exon 10 to prevent microtubule assembly [4]

Active Human Recombinant Tau441 (2N4R), P301S mutant Protein Monomer (Cat. No. SPR-327)
Active Human Recombinant Tau441 (2N4R), P301S mutant Protein Pre-formed fibrils (Cat. No. SPR-329)

• and from a truncated form of human tau (K18) containing only the 4 microtubule binding repeats with a P301L (PL) mutation that promotes beta-sheet formation and the formation of PHFs.[4]

Active Human Recombinant Tau (K18), P301L mutant Protein Monomer (Cat. No. SPR-328)

Active Human Recombinant Tau (K18), P301L mutant Protein Pre-formed fibrils (Cat. No. SPR-330)

N.B: Both P301S and P301L mutant transgenic mouse models are used in tau research.

These active preformed fibrils have been shown to seed aggregation by recruiting monomers to form larger fibrils as demonstrated in the thioflavin T assays below.

Although full-length PFFs (T441) may be more effective in seeding fibrillisation than its truncated form (K18), a combination of both can be highly toxic to neurons.[5]

Further reading
- First Commercially Available Active Human and Mouse ASYN Proteins from StressMarq
- Short Articles on Alzheimer’s Disease:
#1 Amyloid beta formation
#2 Amyloid beta accumulation, imbalance of the production and clearance of Abeta
#3 Microglia
#4 Tau Phosphorylation

References
[1] Roles of tau protein in health and disease Guo, T., Noble, W., & Hanger, D. P. (2017). Acta Neuropathologica, 133(5), 665-704.
[2] Propagation of Tau aggregates, Michel Goedert and Maria Grazia Spillantini,
Molecular Brain, 201710:18
[3] What is the evidence that tau pathology spreads through prion-like propagation? Mudher A et al, Acta Neuropathol Commun. 2017; 5: 99.
[4] alzforum.org/mutations/mutation-position-table/mapt-p301-mutations
[5] Ozcelic, S. et al. Mol Psychiatry. 2016, 21(12): 1790–1798.

Written by Magalie Dale
If you like my post why not connect to me on LinkedIn.

Short Article on Alzheimer's Disease: #4 Tau Phosphorylation

Tau is a major microtubule-associated protein primarily located in axons that contributes to the proper function of neurons. It associates with tubulin to promote their assembly into microtubules and also acts as a stabiliser for these structures. Upon mutations, post-translational modifications, oxidative stress or truncation, the binding of tau with microtubules becomes impaired and tau proteins dissociate and starts to self-aggregate into neurofibrillary tangles (NFT), one of the hallmarks of Alzheimer’s disease and other tauopathies.[1]

Hyperphosphorylation of Tau
Tau can undergo several post-translational modifications such as O-glycosylation, ubiquitination, SUMOylation, nitration, glycation, acetylation, conformational alteration, proteolytic cleavage and phosphorylation. [2]

Phosphorylation of tau proteins is normal and happens in healthy brains as the phosphorylation state of tau helps regulate its binding with tubulin. However, in Alzheimer’s disease, it happens multiple times and excessively. Over 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed with several hyperphosphorylated tau being identified in NFTs. [3] In particular, it has been shown that pathological phosphorylation of Tau at Ser396 or Ser404 decreases the binding activity of the Tau proteins to microtubules.[2]

Kinases involved in the hyperphosphorylation of Tau.
Phosphorylation of Tau involves the coordinated action of several kinases and phosphatase. In AD, amyloid beta not only disrupts communication between neurons, but also starts an immune response leading to inflammation. More specifically, amyloid beta causes the activation of p38 MAPK that results in the abnormal phosphorylation of tau proteins. Some tau kinases, which have been identified in AD include glycogen synthase kinase 3 beta (GSK3beta), cyclin dependant kinase CDK5, CaMKII and tyrosine kinases such as Src, Fyn and c-Abl, MARK. [4]

Phosphorylation of Ser422 inhibits the caspase cleavage of tau
Recent studies have reported that the proteolytic processing of tau by caspases, a family of cysteine proteases involved in apoptosis, generates truncated tau proteins that might play a role in the abnormal aggregation of tau. Indeed, it is known that tau protein contains several canonical sites for caspase cleavage and altered tau proteins including truncated tau proteins at the aspartic acid 421 site have been identified in tau tangles.[5,6] Also, in vitro experiments have demonstrated that caspase 3 could cleave the Asp421 site on tau with the resulting truncated tau proteins aggregating more readily than the full-length tau proteins.[1] However, this cleavage could be inhibited in vitro with tau proteins phosphorylated at the Ser422 site.[3,6]

Further reading:
Short Articles on Alzheimer’s Disease:
#1 Amyloid beta Formation
#2: Amyloid beta accumulation, imbalance of the production and clearance of Abeta
#3 Microglia
#5 Tau Aggregation and Propagation

References
[1] Structure and Pathology of Tau Protein in Alzheimer Disease, Kalarova M. et al, Int J Alzheimers Dis. 2012;2012:731526
[2] Tau Hyperphosphorylation and Oxidative Stress, a Critical Vicious Circle in Neurodegenerative Tauopathies? Alavi Naini SM et al, Oxid Med Cell Longev. 2015;2015:151979
[3] Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease Sandhu P et al, Bioorganic & Medicinal Chemistry Letters, 2017; 27: 3, 642-652
[4] Oxidative stress and the amyloid beta peptide in Alzheimer's disease, Cheignon C et al, Redox Biol. 2018; 14:450-464.
[5] Halting of Caspase Activity Protects Tau from MC1-Conformational Change and Aggregation, Kestoras ME et al, J Alzheimers Dis. 2016 Oct 18;54(4):1521-1538.
[6] Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivo, Guillozet‐Bongaarts AL et al, J Neurochem. 2006 May;97(4):1005-14

Written by Magalie Dale
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Short Article on Alzheimer’s Disease: #3 Microglia

One of the pathological hallmarks of Alzheimer’s disease is the accumulation of amyloid beta in senile plaques. This is due to an imbalance between an increased production of amyloid beta and its clearance mechanisms or its clearance mechanisms being impaired. Amyloid beta peptides have high affinity for many receptors and their clearance can occur in cells via several processes. For instance, the transcytosis of monomeric amyloid beta across the brain blood barrier can be achieved via the low-density lipoprotein receptor-related protein 1 (LRP1) as seen in a previous blog post. Another process is the internalisation of amyloid beta by the brain macrophages or microglia. Although their role is beneficial in clearing amyloid beta, constant microglia activation might further contribute to neuroinflammation.

Microglia as first line of defence in the brain
Microglia are immune cells, resident in the central nervous system (CNS) that derive from yolk sac progenitors during embryogenesis. They play a key role in brain maintenance and have recently been shown to mediate synaptic plasticity, learning and memory.[1] In their “resting” form, microglia constantly scan the environment by stretching out their long processes in order to detect immune threats while maintaining homeostasis in the CNS.[2] They are extremely plastic and are able to respond quickly to changes in their extracellular environment caused for instance by stress, trauma, diseases or upon activation by various factors such as pro-inflammatory cytokines, cell necrosis factors, lipopolysaccharide or a variation in the extracellular potassium levels.[2] These factors induce a change in their morphology and microglia will shift from a ramified to an amoeboid form that will allow their phagocytosis activity.[2,3] Iba1, a protein expressed in microglia and upregulated upon their activation, represents a useful marker for researchers to visualise these cells.

Microglia and Alzheimer's
With regard to Alzheimer’s disease, microglia have a dual role. On a positive side, they have a neuroprotective effect and will clear amyloid beta/senile plaques through phagocytosis. As a reminder, amyloid beta peptides are formed by the proteolytic cleavage of the transmembrane protein APP. Although they are first produced as monomers, they will clump together to form aggregates and senile plaques, with the smallest aggregates thought to be the most neurotoxic.

On the other hand, continuous activation of microglia also contributes to neuroinflammation by releasing pro-inflammatory cytokines. In particular, microglia are a well-known source of reactive oxygen species (ROS) in the brain and will as a result promote neuronal loss mediated by oxidative stress. Indeed, ROS are extremely reactive species and if not scavenged by antioxidants, they will damage DNA and membrane lipids to form more stable molecules such as 8-OHdG and 4-HNE, which can be useful markers in AD research.[3]

 

Newmarket Scientific, a distributor of life science reagents in the UK and Ireland with a strong focus in neuroscience research, provides products from a number of different suppliers and in particular, Biosensis and StressMarq, who provide several antibodies and ELISA kits (8-OHdG) for Alzheimer’s, oxidative stress and lipid peroxidation research.

Further reading
- Oxidative Damage – The Damaging Effect of Reactive Oxygen Species ROS
- Amyloid Beta Plaque Staining
- Short Articles on Alzheimer’s Disease:
#1 Amyloid beta Formation
#2 Amyloid beta accumulation: imbalance of the production and clearance of Abeta
#4 Tau Phosphorylation
#5 Tau Aggregation and Propagation

References
[1] Microglia across the lifespan: from origin to function in brain development, plasticity and cognition, Tay TL et al, J Physiol. 2017 Mar 15; 595(6): 1929–1945.
[2] Microglia, Wikipedia https://en.wikipedia.org/wiki/Microglia accessed 27/07/2018
[3] Chronic stress as a risk factor for Alzheimer's disease: Roles of microglia-mediated synaptic remodeling, inflammation, and oxidative stress, Bisht K. et al, Neurobiol Stress. 2018 Nov; 9: 9–21.

Written by Magalie Dale
If you like my post why not connect to me on LinkedIn.

Short Article on Alzheimer's Disease: #2 Amyloid beta accumulation, imbalance of the production and clearance of Abeta

Alzheimer’s disease is an incurable and progressive neurodegenerative illness with the two commonly accepted hallmarks being the deposition of insoluble amyloid plaques and the aggregation of neurofibrillary tangles in the brain. There are mainly two types, early-onset Alzheimer’s which affects people as young as 30 and late-onset Alzheimer’s which begins after the age of 65. Late-onset Alzheimer’s (sporadic) is the most common form of the disorder affecting about 90% of AD sufferers. The causes are possibly a combination of genetic, lifestyle and environmental factors.

Accumulation of Amyloid beta
One hypothesis is that the accumulation of amyloid beta arises from an imbalance of the production and clearance of Abeta.[1] Increased production of amyloid beta is associated with pathogenic mutations in three genes, AAP gene on chromosome 21, presenilin1 (PSEN1) on chromosome 14, and presenilin 2 (PSEN2) on chromosome 1 and is most common in early-onset Alzheimer’s and familial Alzheimer’s.[2] However, recent data suggests that in most cases/sporadic AD, imbalance occurs as a result of amyloid beta clearance impairment.[1]

APOE E4, A major genetic risk factor in late-onset Alzheimer’s.
One of the major genetic risk factors known for late-onset AD is the E4 isoform of apolipoprotein E (APOE). The APOE gene has three major allelic variants, E2, E3, E4 with E3 the most common allele. Each individual possesses two alleles inherited from both parents and it is known that having one or two alleles e4 of APOE gene increases by 3-fold and 12-fold respectively the risk of developing AD.[2] However, the possession of the E4 allele is not sufficient enough nor necessary to develop AD as only half of APOE E4 carriers will develop AD by age 85 (compare to 10% of non-carriers).[3]

Function of APOE in Lipid Metabolism
The APOE gene code for APOE apolipoprotein-E lipid-transport protein, a regulator of lipid metabolism that allows lipids and cholesterol to be transported into cells via cell-surface lipoprotein receptors such as the low-density lipoproteins receptors (LDL) or LDL receptor related proteins (LRP) etc..[4] This is particularly important as cholesterol and lipids are essential for central nervous system (CNS) functions, such as neuronal growth, synaptic plasticity and neuronal maintenance and repair.

It is still not yet fully understood how APOE E4 increases AD risk but emerging data show that there is a correlation between APOE4 and increased levels of neurotoxic soluble oligomeric amyloid beta.[5] It is thought that in the CNS, the ability of APOE4 in clearing beta-amyloid across the blood brain barrier is impaired (while APOE3 and APOE2 are more efficient in performing this task), consequently contributing to the accumulation of amyloid beta in the brain. [3,4]

Newmarket Scientific is a distributor in the UK and Ireland of life science reagents with a strong focus in neuroscience research. It provides several antibodies, ELISA kits (Oligomeric amyloid-beta; apolopoprotein E/Beta-amyloid complex) and peptides for Alzheimer's research:

Further reading:
- Amyloid beta plaque staining
- Short Articles on Alzheimer’s Disease:
#1 Amyloid beta Formation
#3 Microglia
#4 Tau Phosphorylation
#5 Tau Aggregation and Propagation

References:
[1] Evidence for impaired amyloid β clearance in Alzheimer's disease, Wildsmith KR et al,  Alzheimers Res Ther. 2013 Jul 12;5(4):33
[2] Alzheimer’s Disease, Masters C. et al. Nature reviews Disease Primers 1, article number 15056, 2015
[2] APOE genotype and cognition in healthy individuals at risk of Alzheimer's disease: A review, O'Donoghue MC et al, Cortex. 2018 Jul;104:103-123.
[3] Apolipoprotein E: Structure and Function in Lipid Metabolism, Neurobiology, and Alzheimer’s Diseases Huang Y et al, Neurobiol Dis. 2014 Dec; 72PA: 3–12. 
[4] Soluble apoE/Aβ complex: mechanism and therapeutic target for APOE4-induced AD risk, Tai LM et al, Mol Neurodegener. 2014; 9: 2.

Written by Magalie Dale
If you like my post why not connect to me on LinkedIn.

Short Article on Alzheimer’s Disease: #1 Amyloid beta Formation

Alzheimer’s disease is a devastating condition with currently no cure or treatment to halt its progression. It is an unremitting neurodegenerative disorder, causing slow and progressive cognitive impairment and is the major known cause of dementia. Although some treatments exist to temporarily relieve some of the symptoms such as memory loss and co-morbid illnesses such as cerebrovascular diseases, patients mostly rely heavily on the support received from their social network. This condition was first described by Alois Alzheimer in 1907 and later histological analyses of brain tissues of patients with these symptoms showed proteinaceous aggregates, also called senile plaques, containing insoluble forms of amyloid beta. [1]

Formation of Amyloid beta from APP

Amyloid beta is derived from amyloid precursor protein or APP, which is found in the membrane of neurons and plays a important role in neuron growth and repair after an injury. It is usually processed and cleaved sequentially by two enzymes, alpha and gamma secretases. The resulting fragments from this cleavage are soluble, non-toxic to neurons and in healthy brains are broken down and eliminated. However, if the cleavage by alpha secretase is inhibited, APP is cleaved by beta-secretase (BACE) and gamma secretase consisting of the proteins presenilin 1/presenilin 2, nicastrin, PEN-2 and APH-1.[2] This results in the formation of the fragments amyloid beta 40-42 with the most aggregation-prone fragment amyloid beta 42, to form amyloid plaques in the extracellular space. These plaques weaken the communication and plasticity at synapses and can also deposit around blood vessels in the brain causing amyloid angiopathy and hence increase the likelihood of haemorrhages.[1]

The Multiple Forms of Amyloid beta [3-6]
Amyloid beta peptides are intrinsically disordered proteins (IDPs), meaning they are extremely flexible with no fixed or three-dimensional structures. They undergo rapid conformation changes and fast aggregation processes and as a result exist as multiple forms with distinct polymorphic structures. It is believed that amyloid beta oligomers are the most neurotoxic species, however their study is challenging as different preparation methods might lead to the generation of different oligomeric intermediates which are hard to compare between studies. Several conformation-dependant antibodies exist and are able to recognise generic epitopes that are associated with specific aggregation states on amyloid-forming proteins and this independently of the amino acid sequence. For instance, A11 antibodies can recognise out-of-register anti-parallel beta sheet structures, whereas OC antibodies detect in-register parallel beta sheets.[5]

Newmarket Scientific, a distributor of Life Science reagents in the UK and Ireland, represents several suppliers that provide high quality reagents for Alzheimer's disease research. Below are some product highlights for amyloid beta research.

Further reading
- Amyloid beta plaque staining
- Short Articles on Alzheimer’s Disease:
#2 Amyloid beta accumulation, imbalance of the production and clearance of Abeta
#3 Microglia
#4 Tau Phosphorylation
#5 Tau Aggregation and Propagation
 
References
[1] Alzheimer Disease, Kuma A. et al, Treasure Island (FL): StatPearls Publishing; 2018 Jan
[2] Alzheimer’s Disease, Masters C. et al. Nature reviews Disease Primers 1, article number 15056, 2015
[3] Insights into the Molecular Mechanisms of Alzheimer’s and Parkinson’s Diseases with Molecular Simulations: Understanding the Roles of Artificial and Pathological Missense Mutations in Intrinsically Disordered Proteins Related to Pathology, Coskuner-Weber O. et al., Int J Mol Sci. 2018 Feb; 19(2): 336.
[4] Structural Classification of Toxic Amyloid Oligomers, Glabe C.G, J Biol Chem. 2008 Oct 31; 283(44): 29639–29643.
[5] Amyloid-β Receptors: The Good, the Bad, and the Prion Protein, Jorosz-Griffiths H.H. et al, J Biol Chem. 2016 Feb 12; 291(7): 3174–3183.
[6] Crucial role of protein oligomerization in the pathogenesis of Alzheimer’s and Parkinson’s Diseases, Choi M.L et al, FEBS J. 2018 Jun 20.
[7] A Generic Method for Design of Oligomer-Specific Antibodies, Brännström et al., PLoS One. 2014 Mar 11;9(3):e90857.
[8] Intraneuronal Aβ detection in 5xFAD mice by a new Aβ-specific antibody, Youmans K. et al., Mol Neurodegener. 2012; 7: 8.

Written by Magalie Dale
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Photosynthesis - How do Plants Grow?

Photosynthesis is a chemical process occurring in plants and single celled algae using sunlight to convert water and carbon dioxide to six carbon sugars that will be used for plant growth or metabolised for energy. Another important “by-product” of photosynthesis is oxygen with roughly half of the oxygen in the atmosphere being produced by single celled algae in oceans and the rest from trees in tropical rain forests. How oxygen is formed is a well-understood mechanism and will be described below as well as a brief description of the processes happening in photosynthesis.

For scientists working in this area, global warming offers many challenges that need to be overcome. Plant productivity depends significantly on the speed and efficiency of photosynthesis, specific environmental conditions and the availability of nutrients essential for plant growth. With the planet undergoing environmental changes, such as increased temperature, acidification of oceans due to an increased uptake of carbon dioxide, understanding photosynthesis and how it is affected by these environmental changes is of tremendous importance for our existence.

Agrisera, a Swedish company, provides an extensive list of antibodies for the detection of plant and algal proteins with a significant number being suitable for photosynthesis/Rubisco research. Agrisera is represented in the UK and Ireland by Newmarket Scientific and products can be viewed on the following website: www.newmarketscientific.com/Agrisera

Photosynthesis in brief

Photosynthesis occurs in large organelles found in leaf cells named chloroplasts and more particularly on the thylakoid membrane which is the third innermost chloroplast membrane. This membrane is a lipid bilayer, formed into stacks of flat disk shape membranes termed grana and in which are embedded large complexes of proteins and chlorophyll, a pigment molecule similar to heme with a magnesium atom Mg2+ in its centre. These complexes are called photosystems.

Plants and algae utilise two photosystems called PSI and PSII with both having distinct functions. They both contain two chlorophyll a molecules (Chl-a) but due to different protein environments, these chlorophylls in the two reactions centres differ in their light absorption maxima (680 nm for PSII and 700 nm for PSI).

So how does photosynthesis work? Photosynthesis is a combination of different processes that aim to form NADPH and ATP so that plants have the necessary energy and electrons to convert CO2 and water to six carbon sugars.

Photosynthesis can be briefly described as follows.

Light absorbed by chlorophylls attached to proteins in the thylakoid membrane raises the chlorophyll molecules to a higher energy state or excited state which is unstable. In the photosystem PSII, this triggers the transfer of an electron from a P680 Chl-a molecule to an acceptor quinone on the stromal surface. This results in (1) the formation of a positively charged P680 Chl-a molecule, which becomes the strongest oxidising agent able to oxidise water to oxygen (2H2O--> O2 + 4H+ + 4e-), (2) the protons being formed as a result of the oxidation of water to remain in the thylakoid membrane and (3) the electrons on the acceptor quinone to move to the electron-donor site on the luminal surface of the PSI reaction centre.

PSI will then use the energy from an absorbed photon to transfer an electron to ferredoxin where NADP+ will be reduced to NADPH.

It should be noted that the electron transport between the two photosystems PSII and PSI occurs through a chain of carriers in the thylakoid membrane. Cytochrome b6f plays an important role in this process and will simultaneously pump protons from the stroma to the thylakoid space hence creating a pH gradient across the thylakoid membrane. Protons will then move down their concentration gradient from the thylakoid lumen to the stroma via ATP synthase (CF0CF1 complex), hence converting ADP and Pi to ATP.

These reactions are light-dependent hence called “light” reactions and in oxygenic photosynthesis, the overall reaction of these “light” reactions is:

2H2O +2NADP+ + 3ADP + 3Pi -->O2 + 2NADPH + 3ATP

Finally, the last process in photosynthesis is the fixation of CO2 and the conversion of CO2 into glucose utilising previously formed ATP and NADPH. These reactions are independent of light energy and are sometimes described as “dark reactions”. An enzyme of interest, called RuBisCO, is involved in one of the first major steps of carbon fixation and shall be discussed into more details in a subsequent blog post.

Written by Magalie Dale

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Concentrating exosome samples with a tangential flow filtration system

The two main membrane filtration methods are Direct Flow Filtration (DFF) and Tangential Flow Filtration (TFF). Both methods can be used for the concentration of exosomes

Direct Flow Filtration vs Tangential Flow Filtration

DFF also called “dead-end filtration”, has a feed stream that is perpendicular to the filtration membrane, so the feed must pass straight through the membrane to be filtered.

In DFF the pores of the membrane may become blocked by larger particles making it difficult for the smaller particles to pass through the filter. As a result, this can cause the feed flow to reduce dramatically, requiring increased pressure to be applied to the fluid so that smaller particles can eventually pass through the membrane.

TFF has a feed stream that passes parallel to the filtration membrane, so the feed passes across the surface of the fliter..

In TFF, as the feed passes across the surface of the membrane, this constant agitation in the feed flow prevents blockage of the membrane, making TFF a fast and efficient method for the purification, concentration and buffer exchange of exosome samples.

In TFF what passes through the membrane is called the permeate and what is retained is called the retentate. The retentate can then be recirculated to the feed reservoir until the desired concentration of the sample has been achieved.

Concentrating EV samples with HansaBioMed TFF-easy.
 
HansaBioMed TFF-Easy is a filter cartridge made from polysulfone hollow fibres with 20 nm pores and based on the tangential flow filtration methodology as described above. It allows the concentration and the removal of small proteins and molecules from diluted matrices (cell conditioned media, urine, etc..) prior to the EV purification and EV dialysis for changing buffer conditions. Water and small molecules (<20 nm) pass through the hollow fibre pores and are collected in the collection bag and extracellular vesicles can be easily recovered from the hollow fibre cartridge with a syringe.

The protocol is simple, using a syringe containing the sample at one end and a clean empty syringe at the other. Then to start the concentration process, simply push the two syringes alternately until the desired volume has been obtained.


Comparison between TFF-easy and MWCO spin concentrator.
When compared with the MWCO spin concentrator in an ELISA assay assessing the expression of CD81 in LnCAP CCM, TTF-easy showed better performance and low variability between samples as seen in the graph below.

Combine TFF-easy with the HansaBioMed Size exclusion Chromatography Columns to obtain Pure Exosomes and Extracellular Vesicles.

Ultracentrifugation is currently the gold standard methodology for Extracellular Vesicle isolation from biological fluids or cell conditioned media. However, it does not isolate EVs efficiently, tends to alter the vesicle shape and functionality, requires expensive equipment and is time-consuming. To address these issues, HBM-LS has developed optimised tools for the isolation of total or specific EV populations.

In particular, HansaBioMed-LS has developed different classes of Size Exclusion Chromatography columns from different sample matrices and volumes (from 100ul and up to 20 mL). Size exclusion chromatography is currently one of the best methods to isolate exosomes and EVs. As a result, using TFF-easy to concentrate samples before using the PureEV SEC columns from HansaBioMed allows researchers to isolate easily and efficiently highly pure exosomes and EVs for further studies.

Written by Magalie Dale
If you like my post why not connect to me on LinkedIn.

First Commercially Available Active Human and Mouse ASYN Proteins from StressMarq

Alpha-synuclein is a 140 amino acid protein which in humans, is encoded by the SNCA gene. It is expressed predominantly in the brain and particularly in presynaptic nerve terminals.

Due to its structural flexibility, alpha-synuclein can adopt several conformations and depending on the environment and the binding partners, exists as a dynamic balance between monomeric unfolded and an amphipathic alpha-helix (membrane binding) states.

In healthy brains, quality control systems ensure the correct assembly of alpha-synucleins and intracellular alpha-synuclein homeostasis is controlled via the ubiquitin-proteasome system and the lysosomal autophagy system, with the latter being involved in clearing oligomer assemblies. Other synucleins are also able to inhibit and control the oligomerisation of alpha-synuclein.

Failure in these systems, oxidative stress, pH changes are, to name a few, examples of triggers that can lead to the overproduction and accumulation of alpha-synuclein. In addition, post-translational modifications of alpha-synuclein can lead to a change in conformation resulting in the alpha-synuclein proteins being more susceptible to aggregation. In Lewy bodies, it has been shown that phosphorylated Ser129 alpha-synuclein is the most abundant form of alpha-synuclein present in these aggregates.

In pathological conditions such as Parkinson’s disease, soluble alpha-synuclein monomers associate to form oligomers that will combine further to generate protofibrils that subsequently aggregate to form large and insoluble aggregates, the main component of Lewy body inclusions.[1] These neurotoxic aggregates will cause the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, causing the motor symptoms in PD. [2]

StressMarq has recently released the first commercially available active Human and Mouse Alpha Synuclein Monomers and Pre-formed Fibrils:

- Active Human Recombinant Alpha Synuclein Protein Monomer (Cat No. SPR-321)
- Active Human Recombinant Alpha Synuclein Pre-Formed Fibrils (Cat No. SPR-322).
- Active Mouse Recombinant Alpha Synuclein Protein Monomer (Cat No. SPR-323)
- Active Human Recombinant Alpha Synuclein Pre-Formed Fibrils (Cat No. SPR-324).

The active alpha synuclein pre-formed fibrils (Cat No. SPR-322) in presence of the active alpha-syn monomer (SPR-321) has been shown to induce Lewy body inclusion formation in neuronal cell culture, a characteristic of Parkinson’s Disease.

This was demonstrated using T-thioflavin, a fluorescent dye that binds to beta sheet-rich structures, such as those in α-Syn aggregates and which upon binding, experiences a red-shift in its emission spectrum and increased fluorescence intensity. As seen in the images below, it was shown that 10 nM of active α-Syn aggregate (SPR-322) combined with 100 µm of active α-Syn monomer (SPR-321) could induce aggregation, as compared to active α-Syn aggregate (SPR-322) and active α-Syn monomer (SPR-321) alone.

A similar experiment was also performed to show the activity of the active mouse pre-formed fibrils and monomers (SPR 323 and SPR324).

In addition, StressMarq has also launched control alpha synuclein protein monomers (Cat No. SPR-316) and control alpha synuclein protein aggregates (Cat No. SPR-317) which are inactive as shown in the image below.

These active alpha synuclein proteins and the related alpha synuclein antibodies below can be purchased in the UK and Ireland via Newmarket Scientific, the distributor of StressMarq.

StressMarq alpha synuclein antibodies:	Cat. No.
Alpha synuclein, clone 3C11	SMC-530
Alpha synuclein, clone 10H7	SMC-531
Alpha synuclein, clone 3F8	SMC-532
Alpha synuclein, clone 4F1	SMC-533
Alpha synuclein pSer129	SPC-742

References:
[1] Alpha-Synuclein: From Early Synaptic Dysfunction to Neurodegeneration, Ghiglieri V. et al, Front Neurol. 2018; 9 : 295

[2] Linking Neuroinflammation and Neurodegeneration in Parkinson’s Disease, Gelders G. et al, J Immunol Res., 2018: 4784268. doi: 10.1155/2018/4784268. eCollection 2018.

Written by Magalie Dale

If you like my post why not connect to me on LinkedIn.

Migraine: The culprits behind the headache?

Migraine is not just a simple headache. Very often it is accompanied by various symptoms such as nausea, sensitivity to light, noise and visual disturbances, it is characterised by a unilateral throbbing pain sensation that can last for up to 72 hours and is accentuated during physical exercise. 1 in 7 people suffers from migraine and although it is not correlated with age or social background, women tend to suffer twice as much than men. In the UK alone, it is estimated that migraine costs £2 billion a year, hence extensive research is being carried out to develop effective treatments.[1]

Headaches in Migraine – The Crucial Role of CGRP

The headache phase in the migraine occurs when the dilated blood vessels mechanically activate the perivascular trigeminal sensory nerve fibres, the principle sensory nerve in the head. This triggers a pain response with several neurotransmitters, such as substance P and CGRP (calcitonin gene–related peptide), being released to transmit the nociceptive signals from the trigeminal sensory afferents to second-order neurons. CGRP, a potent vasodilator, will aggravate the dilation of cranial blood vessels, cause mast-cell degranulation and initiate neurogenic inflammation within the meninges. As a result, prolonged activation of the meningeal trigeminal nerves, vessels and mast cells causes sensitisation of secondary and tertiary-order neurons which could explain symptoms associated with migraine such as allodynia (skin sensitivity) and photosensitivity.[2]

The role played by CGRP in migraine headaches is widely accepted. Indeed, it has been observed that during a migraine attack, the levels of CGRP in blood and saliva were increased.[3] It was also shown that administrating CGRP to patients prone to migraines resulted in inducing migraine-like headaches.[4]

The Controversial Role of TRPV1 in Migraine Pathology

More recently, it has been shown that the cation channel TRPV1, a nonselective cation channel that is activated by stimuli such as high temperature and capsaicin could also be involved in migraine pathophysiology. Colocalised with CGPR in trigeminal ganglion neurons, TRPV1 when activated promotes the release of CGPR.

It is also known that patients suffering from chronic migraine show elevated levels of nerve growth factor (NGF) in the cerebral spinal fluid and it has been suggested that a NGF-dependent mechanism could lead to the insertion of TRPV1 into the plasma membrane hence increasing the number of TRPV1 channels available for activation on the nociceptor surface membrane. In addition, several studies have also demonstrated that through different pathways, prostaglandins, ATP, BK and possibly NGF reduce the TRPV1 activation threshold by phosphorylating TRPV1 on S502 and S800 and hence causing sensitisation.[5]

Based on this, TRPV1 antagonists seemed to be attractive targets for the treatment of migraine and SB-705498, a TRPV1 antagonist showed promising results in cats. However, in the case of humans, the clinical trial was terminated early due to a lack of efficacy in treating acute migraine.[6] More recently, two TRPV1 receptor antagonists have been shown to be effective in two different experimental models of migraine suggesting that further clinical trials using different TRPV1 antagonists should be performed to better understand the role played by TRPV1 in migraine pathology.[6]

Working with TRPV1? Have a look at our products:

Biosensis Antibodies to TRPV1	Cat. No.
Rabbit antibody to human capsaicin receptor (531-541): whole serum	R-053-100
Rabbit antibody to human capsaicin receptor (608-621): whole serum	R-076-100
Mouse monoclonal antibody to rat capsaicin receptor (VR1, TRPV1, 819-838), [Clone BS397]: IgG	M-1714-100
StressMarq Small Molecules	Cat. No.
Capsaicin (TRPV1 opener)	SIH-322
BCTC (TRPV1 blocker)	SIH-307
SB-366791 (TRPV1 blocker)	SIH-321

Current and Emerging Treatments

Current migraine treatments include the use of nonsteroidal anti-inflammatory drugs (NSAID) such as aspirin, ibuprofen, naproxen and didofenac potassium. When the pain is moderate to severe, triptans (highly selective serotonin 5-HT1B and 5-HT1D receptor agonists) are considered to be the first line of treatment. Indeed, serotonin vasoconstricts the nerve endings and blood vessels and consequently affects nociceptive pain. [7]

However, because of the presence of the 5-HT1B receptors on blood vessels and the vascular risks associated with the use of these drugs, ditans, a novel class of chemicals selectively targeting the 5-HT1F receptors expressed in the trigeminal nerve pathway but lacking the vasoconstrictive properties, are being developed. Currently, lasmiditan is in phase III clinical trials in the US. [8]

As seen previously, CGRP plays an important role in the pathogenesis of migraine and as a result, several small molecules, CGRP receptor antagonists called gepants have been developed. In particular, telcagepant and MK-3207 were shown to be effective treatments for acute migraine, but were terminated due to their liver toxicity.[2] Other drugs based on monoclonal antibodies are currently being developed. These new treatments target either CGRP or the CGRP receptors and currently show promising results in human clinical trials. Aimovig (erenumab) developeed by Amgen and Novartis has recently been FDA-approved as a preventive migraine treatment by blocking the activity of CGRP receptors.[9]

Migraine Research: ImmunoStar Antibodies can help

ImmunoStar has developed an excellent range of antibodies for neuroscience research. These antibodies are put through extensive testing before release to ensure they are both high quality and high titer. This provides excellent reliability and lot-to-lot consistency. They have also been specifically tested for use in immunohistochemistry. They are currently available to purchase through Newmarket Scientific.

Migraine related articles using antibodies from the ImmunoStar range can be found below:

References	ImmunoStar antibodies
Neural mechanism for hypothalamic-mediated autonomic responses to light during migraine, Noseda R et al, PNAS, 2017, 114 (28): E5683-E5692	Tyrosine hydroxylase, oxytocin, vasopressin 1
Hypothalamic and basal ganglia projections to the posterior thalamus: Possible role in modulation of migraine headache and photophobia; Kagan R et al, Neuroscience 2013, 248, 359-368	Tyrosine hydroxylase, cholecystokinin octapeptide (CCK8)
Neurochemical Pathways That Converge on Thalamic Trigeminovascular Neurons: Potential Substrate for Modulation of Migraine by Sleep, Food Intake, Stress and Anxiety, Noesda R et al, Plos one 2014, 9 (8): e103929	Tyrosine hydroxylase
Dopamine β-Hydroxylase Statins Decrease Expression of the Proinflammatory Neuropeptides Calcitonin Gene-Related Peptide and Substance P in Sensory Neurons, Bucelli RC et al, J. Pharmacol Exp Ther, 2008, 324 (3): 1172-1180	Substance P

References:

[1] https://www.telegraph.co.uk/news/2018/04/17/new-migraine-treatment-could-help-drugs-fail/ Accessed on 21/05/2018

[2] Migraine, Dodick DW, The Lancet, 2018, 391 (10127), 1315-1330

[3] Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Goadsby PJ et al, Ann Neurol. 1990;28:183–7.

[4] Elevated saliva calcitonin gene-related peptide levels during acute migraine predict therapeutic response to rizatriptan. Cady RK et al. Headache. 2009;49:1258–66.

[5] TRPV1 in migraine pathophysiology, Meents JE et al, Trends in Molecular Medicine, 2010, 16 (4):153-159

[6] Two TRPV1 receptor antagonists are effective in two different experimental models of migraine, Meents JE et al, The Journal of Headache and Pain, 2015, 16:57

[7] CGRP and serotonin in migraine, Aggarwal M et al, Annal od Neurosciences, 2012 19 (2): 88-94 https://www.researchgate.net/publication/265558756_Serotonin_and_CGRP_in_migraine

[8] Migraine Therapy: Current Approaches and New Horizons,  Goadsby PJ et al, Neurotherapeutics, 2018, 15 (2) :271-273
[9] https://www.genengnews.com/gen-news-highlights/amgen-novartis-set-to-launch-migraine-drug-aimovig-next-week-after-fda-approval/81255834 Accessed on 21/05/2018

Written by Magalie Dale

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Amyloid beta plaque staining

Alzheimer's disease is characterised by a gradual decline of the cognitive functions and it is accepted that one of its hallmarks is the accumulation of insoluble proteinaceous deposits called amyloid fibrils. Originally thought to be carbohydrate in nature as they could be stained in tissues with iodine, they were called amyloids as iodine was a well-known stain for polysaccharides such as amylose. It was later discovered they were actually constituted of proteins fibres (mostly amyloid beta 1-40 and amyloid beta 1-42), but for historic reasons, the name was conserved.

Histologic dyes such as Congo Red or Thioflavin S or T show affinity for the amyloid plaques. Although they are commonly used for amyloid plaques detection, they come with several limitations, firstly in their spectral properties, with both having a broad range of excitation frequencies leading to a "bleed through" when illuminated with other filters and secondly by requiring harsh chemicals making it less compatible with subsequent use of antibodies.

Amylo-Glo RTD (Cat. No. TR-300-AG) is a new histological marker developed by Biosensis (Thebarton, South Australia) that shows clear superiority for visualising amyloid plaques over these conventional markers (Schmued et al., 2012).

1. Amylo-Glo shows the same affinity as conventional probes (Congo red, ThioflavinS and Pan abeta 40) and is suitable to use with fresh, frozen, and formalin-fixed immunohistochemistry or cytochemistry.
2.  Amylo-Glo has a unique emission/excitation profile. Only excitable with UV light and emits a bright blue/yellow colour.
3.  Amylo-Glo only uses mild conditions making it ideal for subsequent immunohistochemistry labelling.
4.  Its chemical and spectral properties allow for multi-labelling studies. Its specific blue fluorescence contrasts effectively with red and green immunofluorescence labelling.
5. Amylo-Glo is perfect for low magnification studies. Indeed, its unusual brightness (5-6 times brighter than conventional probes) allows to generate strong signals at extremely low magnification (x2).

This triple exposure allows for the simultaneous localization of Amylo-Glo® positive amyloid plaques (blue), GFAP positive hypertrophied astrocytes (green) and activated microglia (red) in the hippocampus of the AD/Tg mouse. Combined UV, blue and green light illumination.

Amylo-Glo UV illumination only

Datasheet available on www.newmarketscientific.com.

Biosensis Amylo-Glo RTD is also available with an ethidium bromide counter stain (Cat. No TR-400 AG) for a quick and effective way to visualise amyloid plaques as well cell nuclei and cell bodies of cells while under UV illumination in one step.

Both products are currently available to purchase in the UK and Ireland through Newmarket Scientific, the new distributor of Biosensis.

Reference:
Schmued L. et al, Journal of Neuroscience Methods 209 (2012), 120-126

To learn more about Alois Alzheimer:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614590/


Written by Magalie Dale

If you like my post why not connect to me on LinkedIn.