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
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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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