Alzheimer's Disease 1

Alzheimer’s

• belongs to a larger category of diseases known as Dementia 

• 46 million people diagnosed as of 2015, prediction that by 2050 there will be 131million  

• Not the only form of dementia (covers 65%), others like Huntington’s and some come from cerebrovascular disease (20%) 

• Most obvious effects are memory loss, decline in thinking, difficulties in communication and behavioural and personality changes 

• Prominent shrinking of the cerebral cortex, enlargement of the ventricles and disturbance of the hippocampus at the macro level 

• At the micro level shows abnormal neurons —> one of the hallmarks is conglomerates of protein in the EC space and accumulation of protein aggregates inside the cell 

Protein aggregates

• Outside the cell, large amyloid beta plaques 

• Plaques present in 100% of Alzheimer’s but can be found in the walls of the blood vessels in other diseases 

• Intracellular aggregates are tau tangles of (NFD) —> not exclusively inside the cell 

Amyloid beta

• Key component is a plasma membrane protein APP (amyloid precursor protein) which is commonly expressed in neurons, in glial cells to a lesser extent 

• Normal brain function undergoes 2 types of processing  

Non amyloidogenic  

• EC part of the protein cleaved right next to a membrane by a secretase (ADAM9,10 or 17) which is part of the alpha secretase family 

• This does not result in Alzheimers because the protein is soluble 

Amyloidogenic  

• Beta secretase cuts of the EC part of the protein but in a different place, further out from the membrane  

• This allows the protein to be cleaved again, this time inside ethe membrane, by a gamma secretase complex  

• Results in AICD product which leads to the accumulation of AICD and A beta peptide  

• Usually these two processes both occur in normal neurons but in Alzheimers theres a shift in the balance of the amyloidogenic pathway leading to excess A beta 

A beta

• Initially released as a monomer but starts to clump as a dimer and oligomers forming aggregates 

• When aggregates the protein is highly insoluble and changes its conformation  

What is the physiological role of APP in a non-disease state?

• When APP is cleaved by an alpha secretase in the non-amyloidogenic pathway, it forms sAPPalpha (released outside the cell) and C83 (remains in the membrane)

• sAPPalpha has several benefical roles in the brain including:

  • helping neurons resist stress and injury

  • enhances communication between neruons and supporting the learning process

  • promotes growth and repair of neuronal projections

  • stabilise neural activity

• C83 fragment can be cleaved further by the gamma secretase, producing the p3 peptide which is not prone to aggregation and AICD which bnds to transcriptional complexes and actas as a co-regulator of transcription and act as an intracellular signalling molecule to influence

  • kinase pathways

  • calcium signalling

  • cytoskeletal organisation

Evidence linking Abeta with AD

• cleavage of APP

• Accumulation of APP

Cleavage of APP 

• Genetic mutation in the APP (amyloid precursor protein) called the London mutation in the region that the gamma secretase cuts  

• Point mutation from V—>I 

• Definitively linked the production of Abeta with Alzheimer’s —> resulted in Alzheimer’s in 100% of cases 

• Further mutations identified but all mutations in the site that the gamma or beta secretase cleave the protein  

• Change in charge in the protein can make the secretase more or less efficient at protein cleavage 

• However, there have also been studies which show the opposite: mutations in the APP can also decrease the incidence of Alzheimer’s —> Icelandic mutation A673T resulted in an almost 50% reduction in amyloid beta production  

Evidence linking Abeta with AD

• cleavage of APP

• Accumulation of APP

Accumulation of APP 

• APP is located on c21 which is additionally copied in downsynsdrome 

• Close to 100% of prevalence found in Down’s syndrome patients  

• Can release more A beta extracellularly due to additionally copy of APP which led to increased cleavage 

Mouse models of AD

• Race to create mouse models to understand the underlying mechanisms  

• Most mouse models based on overexpression of mutant proteins known to cause AD  

• Advantage of working with mice is that you can intervene with the pathology and generate transgenic models 

• We can also purify protein extracts from human patienst and insert it into the mice  

Types of mouse models: 

• APP23 one of the first mutants —> double AA change in APP protein (Swedish mutation  

• 3xTg has mutations in APP, preseneline enzyme which forms part of the gamma secretase complex and Tau which leads to aggregation fof Tau  

• 5xFAD is a very strong phenotype – has overexpression of APP with the Swedish mutation and London mutation, as well as 2 pathogenic mutation in presenilin 

Human studies

• can do post mortem studies, brain imaging, cognitive tests  

• Afetr this can do pathological analysis to identify amyloid and tau proteins

Pathology

• amyloid plaques

• synapse loss

• compromised synapses

• cognitive impairment

• Cellular correlate of memory

Amyloid plaques 

• One of the hallmarks is the presence of Ab plaques 

• In both human and mouse studies, 3-month-old mice reflect humans in stage A of disease, 6 month old mice reflect humans in stage B and 9-month-old mice represent patients in stage C (for number of plaques) 

Pathology

• amyloid plaques

• synapse loss

• compromised synapses

• cognitive impairment

• Cellular correlate of memory

Synapse loss 

• Slice of mouse brain hippocampus taken and incubated with dye to stain the neurons 

• In control cells the density of dendritic spines increases across 5-15 days  

• If we have the introduction of amyloid monomers (reflecting early stage Alzheimer’s), we do not change the number of dendritic spines  

• However, the oligomers have a huge affect on the dendritic spine density resulting in loss and fewer synapses 

 

Pathology

• amyloid plaques

• synapse loss

• compromised synapses

• cognitive impairment

• Cellular correlate of memory

Compromised synapses 

• In AD there are less synpases but they display dysfunction as well 

• Can take out the mouse hippocampus, the pathways in the hippocampus are very well characterised which allows you to stimulate these pathways with an alectrode and record the potential  

• APP Indiana mutation mice have less of a reduction in transmission in early stages (3-4wk and 2-4 month) but hugely increased in later stages 

• However, compared to controls, the fact that the transmission is decreased in these early stages, shows that there is something going wrong even before we are able to observe amyloid plaques and symptoms 

Pathology

• amyloid plaques

• synapse loss

• compromised synapses

• cognitive impairment

• Cellular correlate of memory

Cognitive impairment 

• Morris water maze: platform submerged in translucent water with visual cues around the room  

• Allow the mice to learn where the platform is and then a few days later, remove the platform and time how long they spend trying to find the platform  

• Mice with the Swedish/Indiana mutation shows much more random swimming patterns compared to the wildtype 

Pathology

• amyloid plaques

• synapse loss

• compromised synapses

• cognitive impairment

• Cellular correlate of memory

Long term potentiation – Cellular correlate of memory 

• Can be studies in the same way you study synaptic transmission: take out the CA3—>CA1 projections of the hippocampus and stimulate with an electrode and record with recording electrode 

• Stimulate at a high frequency to induce plasticity response 

• In human patients slices of brain taken and lysed then stain with an antibody for amyloid beta 

  • All the Alzheimer’s patients had high Abeta and controls had little 

  • Two types of Abeta: one that is high Mw (dimer), one that is low Mw (monomer) 

• Then leads us to think: can the cellular correlate of memory be affected differently by the monomers vs the dimers 

• The cellular correlate of memory (synaptic plasticity) is only decreased in patients with Alzheimer’s —> later proved that this was due to the dimers and not the monomers 

Mouse models of AD

• Even though 100% of patients show accumulation of A beta, in some mice models, you can start to see symptoms like cognitive impairment occurring before the incidence of plaque formation —> indicated that there is some signalling that something is wrong even before these plaques can be observed 

• All of them show cognitive impairments, but some of the other features like the formation of Tau tangles is not a common feature 

Tau

• Protein that usually exists associates with microbtubules in the axons  

• Usually controls the dynamics of the axon: stabilises and facilitates the growth of the axons in normal pateins  

• In AD tau is hyperphosphorylated which prevents it binding to the microtubules and starts to accumulate, forming neurofibular tangles 

• Shared pathology across other ND diseases like Parkinsons and temporal dementia 

Evidence that links tau and Abeta in the pathogenesis

• staining of mice brains

• cerebral blood flow for brain activity

• Morris water maze

• Lifespan of mice

Staining of mice brains 

• Discovered a mutation in tau (P301L) which prevented the binding of tau to the microtubules which resulted in its aggregation and formation of tau tangles 

• Mutation induced the tau pathology which they visualised with an antibody stain: black signals in the soma can be observed which represents the accumulation of tau  

• In the Swedish mutant mice alone you don’t have this accumulation of tau 

• They then crossed this mouse with a transgenic mice with Swedish mutation (causing abeta aggregation) which resulted in this massive increase in tau tangles  

• Evidence that A beta potentiates the formation of Tau tangles  

Evidence that links tau and Abeta in the pathogenesis

• staining of mice brains

• cerebral blood flow for brain activity

• Morris water maze

• Lifespan of mice

Cerebral blood flow for brain activity 

• Patients who did not yet have Alzheimers diagnosis but had pathological markers which resulted in mild cognitive impairments 

• The cerebral blood flow (proxy for activity in the brain, therefore neuronal function) measured via imaging —> only places where they observed abnormalities in blood flow/activity was the entorhinal cortex and parahippocampus 

• In mice models very early on before pathology expressed have the formation of these tau tangles but only in the mice which had the mutations in APP and the P301L mutation in the tau protein  

• Mice who only had the P301L tau mutation showed no early tau tangle expression  

Evidence that links tau and Abeta in the pathogenesis

• staining of mice brains

• cerebral blood flow for brain activity

• Morris water maze

• Lifespan of mice

Morris water maze  

• Morris water maze experiments done in mice with wildtype tau and mice   without tau  showed that you can reverse the effects of APP mutations by removing Tau proteins 

• Tau mutations worsens the pathological phenotype but this can be reversed by the removal of tau 

• Not only can you see this with the Morris water maze memory test but also observe this with the cellular correlate of memory  

• In normal conditions, observe a potentiation of signal after repeated stimulation 

• With the amyloid beta protein this potentiation is depleted  

• If you use the amyloid protein but the reverse sequence, then interestingly, the potentiation effect resembles the control (normal plasticity) because it does not aggregate 

• after the removal of Tau you rescue the wildtype potentiation effect and show no cognitive effects even with the amyloid beta present 

• can reverse the cognitive and cellular impairments that is experienced with amyloid beta 

• These prove that tau is required to induce the mutated APP induced cognitive deficits and deficits in cellular correlates of memory  

Evidence that links tau and Abeta in the pathogenesis

• staining of mice brains

• cerebral blood flow for brain activity

• Morris water maze

• Lifespan of mice

Lifespan of mice 

• Mice who have the amyloid beta and the tau mutation have a short lifespand and mortality rate of around 85% by 6 months 

• Not only do you get correction in memory with the removal of Tau but also get increased survival when you remove Tau 

• This also holds true when you express the Swedish mutation (APP23) alone without Tau —> increased lifespan of mice 

Molecular targets and clinical trials

Eliminating the plaques is the main method of treatment —> whether if this is effective we don’t know 

How does Abeta trigger the aggregation of tau?

• Amyloid beta acts at NMDA receptors causing excess calcium influx

• the excess calcium causes the activation of kinases that modify tau, particularly:

  • GSK-3beta

  • CDK5

  • MAPkinases

• This causes the hyperphosphorylation of tau which leads to its aggregation and mislocalisation

• Secondarily, the prescence of A beta plaques leads to the activation of microglia and astrocytes, who’s cytokines can also activate tau kinases

• Furthermore, the ROS produces as a result of inflammation can drive the aggregation of tau

Vaccines

• Injecting the amyloid proteins (Abeta1-42) and allowing antibodies to be generated in response 

• Plaques almost completely disappear from the mouse models and can rescue some of the cognitive deficits 

• Unsuccessful because the injection of the peptide, leading to the generation of antibodies —> leading to massive neuroinflammation resulting in death 

• Showed no improvement in cognition in human patients, inconsistent improvement in amyloid plaques —> some patients showed complete reduction, while others showed almost no improvement  

• This lead us to think that the removal of the amyloid plaques may not be able to rescue the cognitive impairment; however, this is caveated by the neuroinflammation which may prevent cognitive function in memory tests 

Drugs

Biologics – introducing a molecule already existing in biology  

• Red = targeting amyloid, despite their previous failiures  

• Blue = targeting tau 

Solanezumab

• Directly inject the antibody into the patient —> antibody is transient and present until it degrades 

• Binds only to monomers of Abeta —> even though monomers don’t cause the pathology, if you have a lot of monomers, eventually they will start to form oligomers —> acts as a sink of monomers 

• Compared to the vaccine is a passive immunotherapy 

 

E2814

• Directly inject the antibodies against Tau MTBR (microtubule binding region) 

• However, we know that mutated Tau is unable to bind to the microtubules, therefore, an antibody against the microtubule binding domain will only mimic the disease phenotype, so what is the rationale behind this? 

• Can only act in the blood or if it crosses the BBB can act in the EC space 

• Tau is also released into the EC space visa the nerve terminal and forms NFTs in the EC space  

• Can form toxic aggregates and also enter another cell and induce the pathology in another cell  

• Antibody binds to the region that usually the protein would use to bind to microtubules and sequesters Tau, prevents the formation of aggregates and also prevent the soluble Tau from entering the other cells 

• Passive immunotherapy, targets Ec fragments of Tau and prevents spread to other cells 

AR1001

• Targets PDE5 —> is a phosphodiesterase 5 inhibitor 

• By inhibiting PDE5 you increase cGMP inside the cell  

• Increase cGMP in neurons leads to activation of pCREB, BDNF and NGF which promote neuronal survival, plasticity and neurogenesis 

• By boosting cGMP wou can also activate the autophagy pathway —> used to eliminate things that are abnormal inside the cell which clears the initial formation of neurofibular tangles or Abeta plaques  

• Also suspected to rescue memory loss phenotype and cognitive function  

Summary of drugs

• Almost all of these not yet approved 

• Nearly all of the drugs target the 2 hallmarks of Alzheimer’s: amyloid and Tau  

• Companies trying to develop antibodies which can not only recognise the monomers, but also the dimers, trimers and even plaques —> then seeing which one is most efficient 

• Also attempts to combine therapies which target both amyloid beta and tau 

• Used in very severe cases of AD where the formation of plaques is very high and Tau tangles high —> little chances of reversal 

• Currently, we don’t know if mild cognitive impairment is caused by the presence of plaques or there is another underlying cause for the impairment  

• In mild cognitive impairment, we know that these patients have a loss of synapses and synaptic function —> what if we try and target this aspect of the pathology and rescue this 

• Trends seen to target the earlier symptoms, attempts to see if they are more effective  

Targeting mild cognitive impairment

• In order to do this we need to know what happens earlier on in these stages —> number of synapses decreases in the dentate gyrus and the CA1 region  

• Specific synaptic targets: we know that theres loss of plastcicity and synapse formation so we can target molecules known to be involved in this process 

• Example: Abeta oligomers activate NMDARs which causes the increase in Ca2+ into the cell which eventually leads to cell death —> can we target this?