Koychev et al 2017
They did PET-CT imaging with radiolabel tracers (18[F]AV45) - ligand for AB . Found that age and period since symtpom onset did not interact significantly with PET AB values in these models.
Hardy and Higgins 1992
Came up with the amyloid cascade hypothesis This was that the accumulation of AB peptides in the brain parenchyma was the central event in the pathogenesis of AD Quote - “Our hypothesis is that deposition of amyloid B protein, the main component of plaques is the causitive agent of Alzheimer’s pathology and that the neurofibrillary tangles, cell loss, vascular damage and dementia follow as a direct result of this deposition”
Jonsson et al 2012
Searched for low-frequency vairants in the APP genes, studied the coding variants in APP in a set of whole genome sequence data from 1,800 Icelanders. Found a coding mutation in the APP gene that protects agianst AD and cognitive decline in the elderly without AD. Transfection of this mutant APP resulted in approx 40% reduction in the formation of amyloidogenic peptides in vitro in human embyronic kidney cells compared to WT. Proof that reducing cleavge of APP may protect against the disease.
Harold et al 2009
Looked at 6000 pts with AD and 10,000 controls. The most significant hit was APOE locus (we have known about this for a long time). However, they also identified 3 new signficiant genes - clusterin, PICALM and CR1 (complement receptor 1 - immune system, inflammation). Clusterin is a major brain apolipoprotein and present in amyloid plaques (this is interesting as it suggested that susceptibility genes are not randomly distributed through funcitonal pathways). PICALM is a clathrin-binding protein involved in vesicle trafficking (may result in the synapse pertubation seen in AD). No presenlins were discovered.
Castellano et al 2011
apoE4 contributes to AB accumulation in the brain not by affected AB synthesis but by affecting its clearance
- They did lumbar punctures to obtain CSF of cognitively normal individuals under the age of 70 carrying different APOE genotypes.
- They found that those that were homozygous for the e4 genotype had much lower CSF AB concentration than those with the protective e2/e3 genotype.
- Then, they analysed imaging data using a dye called Pittsburgh compound B that binds to amyloid plaques in the brain and showed that those individuals with the e4/e4 genotype bound more dye than did those with the other APOE genotypes (showed they had more plaques in their brains too) (PET)
- They then moved on to a mouse model of AD where mice expressed one of the three human apoE isoforms.
- They measured the AB concs in the interstitial fluid of these mice using in vivo microdialysis and looked at stained hippocampal sections of these mice.
- They found greater AB concentrations in both interstitial fluid and the hippocampis in mice expressing apoE4 isoform than in those expressing either the E3 or E2 isoforms.
- They then measured the clearance of AB from the interstitial fluid of young mice (using microdialysis) and showed that those with the human apoE4 isoform were less able to clear AB than those with the apoE2 or apoE3 isoforms.
- The researchers showed that processing of the amyloid precursor protein and generation of the Aβ peptide did not vary according to genotype, lending credence to the hypothesis that apoE4 affects clearance of Aβ but not its synthesis (shown synthesis was not affected using in vivo stable isotopic labelling kinetics) Caveat - did not look at AD pts?
Bien-Ly et al 2012
- Generated a transgenic mouse model expressing APP and they had the ApoE4 allele under the control of Cre-LoxP.
- In one hippocampus, they injected an AAV with the Cre enzyme - in these hippocampal cells you completely KO ApoE4 expression.
- In the other side they injected a saline placebo control so they didnt KO the ApoE4.
- They found that in the control side where they didnt KO ApoE4, there was a massive increase in AB production . In the KO side, they found there was a bit of a rescue of the phenotype - seen via decreased AB protein.
- ‘Strikingly, insoluble AB42 decreased by ~50% in Cre-injected hippocampi compared w ontrols.’
- Caveat - The AAV was targetted at astrocytes - does not reflect an effect on neurons or other cell types in the hippocampus, even those they looked at AB expression in hippocampal homogenates
- Authors conclude - The feasibility of therapeutically lowering apoE levels to substantially reduce AB accumulation - not sure about this conclusion!
Bis et al 2018
This was the Alzheimer’s Disease Sequencing Projected - results reported in Nature Whole exome-sequencing in 5,740 LOAD pts and 5.096 healthy controls with no cognitive impairment. APOE was biggest hit
- Also detected associations in three novel genes:
- IGHG3 – an immunoglobulin gene whose antibodies interact with β-amyloid
- AC099552.4 – a long, non-coding RNA
- ZNF655 – a zinc-finger protein
- The last two hint potential for transcriptional regulation in the pathology of AD
- They also identified some potential rare variants, including TREM2, TREML4, SORL1 and ABCA7
- TREML4 for example is expressed on the myeloid cell surface and plays a role in clearing dead cells by phagocytosis
- IMPORTANCE
- Provides new insight into disease mechanisms
- Can direct future experiments to further explore role of these genes in AD pathology
- But other deep-sequencing approaches = needed to identify variants that could be in non-coding regions, and to look at structural variants (e.g. copy number changes, larger insertions and deletions) to AD risk
Rapoport et al 2002
Took hippocampal neurons from three types of mouse and treated them with fibrillar AB.
- These mice were WT, tau KO and human tau transgenic mice.
- Morphological analysis indicated that neurons expressing either mouse or human tau proteins degenerated in the presence of AB.
- On the other hand, tau-depleted neurons showed no signs of degeneration in the presence of AB.
- This implies tau is required for AB neuronal toxicity
- Caveats Tau might have had a role in neural embryology - what if tau was heavily implicated in the development of neuronal connections? They also used very high, non-physiological concentrations of AB
Shipton et al 2011 (Wade-Martins lab)
Asked whether tau was required for AB-induced impairment of hippocampal LTP.
- Used WT mice and Mapt KO mice and recorded field potentials in acute slice preparation.
- AB42 will inhibit the formation of LTP in WT mice at 500nM. This inhibition is not seen in Mapt KO mice (no tau). (Mapt encodes tau)
- They show (using Western of hippocampal slices) that AB increases tau phosphorylation and that a specific inhibitor of GSK3 blocks (with AR014418) the increased tau phosphorylation induced by AB and prevents AB-induced impairment of LTP in WT mice.
- Propose a molecular pathway linked AB, GSK and phosphorylation of tau, which inhibits LTP
- They used synthetic AB peptides - might be different from physiological ones. Again, maybe development compensation for tau KO
Ittner er al 2010
Demonstrated that tau’s targetting of Fyn to the postsynapses facilitates AB neurotoxicity by stabilising NMDARs
- First generated transgenic mice that overexpressed a variant of tau that lacked the C terminus and could bind to Fyn but not to microtubules.
- Both this truncated mouse and tau-/- disrupt the postsynaptic targetting of Fyn.
- Then found that markedly less NMDAR subunits co-immunoprecipitated with PPSD-95 from truncated tau and tau-/- compared to WT (destabilising NMDA). Found that truncated tau expression and tau -/- prevent memory deficits in a mouse model of AD (APP23).
- THEREFORE, IN THE TAU MUTANT MICE = POSTSYNAPTIC FYN LOCALISATION IS REDUCED = DESTABILISING OF THE NMDAR/PSD-95 INTERACTION AND PROTECTION FROM EXCITOTOXICITY
- Caveat -The actual localisation of tau and Fyn within dendritic spines and their modes of actions under physiological conditions
Bence et al 2001
- “• upregulation of 2 unrelated aggregated-prone proteins (a huntingtin fragment containing a pathogenic polyglutamine repeat + a folding mutant of cystic fibrosis transmembrane conductance regulator - CFTR) in embryonic kidney cells caused nearly complete inhibition of the ubiquitin-proteasome system
- They used a mutant GFP that glowed when intact, but when degraded by the proteasome, it stopped fluorescing
- A high level of green fluorescence in these cells indicated that the proteasome was not functioning”
Wang et al 2015
Studied TREM2 deficiency in a mouse model of AD.
- Find TREM2 KO mice significantly increased AB (measured with a mAb against AB) in hippocampi.
- Further found that the TREM2 deficiency diminished the capacity of microglial to cluster around amyloid plaques (did this using confocal microscopy).
- Finally found that the R47H mutation impairs TREM2 detection of lipid ligands - they generated TREM R47H reporter cells and compared their response to identified ligands to that of TREM2 reporter cells.
- The R47H mutations considerably reduced reporter activation in response to many ligands (such as phosphatididic acid).
- Suggested R47H reduces overall capacity of TREM2 to bind anionic ligands.
Nixon et al 2005
Used immunogold labelling with compartmental markers and EM on neocortical biospies from AD brain to identify autophagosomes - contained amphorous undigested material. Found that autophagic vacuoles were uncommon in healthy brains. This was the first evidence that macroautophagy is extensively involved in NDD. Caveats - Post-mortem AD pts, did not measure autophagic flux (could be due to increased synthesis or reduced breakdown of autophagosomes)
Lee et al 2010
Looked at blastocysts from WT mice and PS1 KO mice. A process requiring lysosome acification is the dissociation of cation-independent mannose-6-phosphate receptor (CI-MPR) from cathepsins after their delivery to endosomes. The group used double-immunofluorescence to with antibodies to Cat D and CI-MPR - saw that most CatD-positive vesicles were CI-MPR-negative in WT cells, but were nearly all CI-MPR positive in PS1 KO cells. (indicated the dissociation was impaired). Then used LysoTracker (fluoresenct dye for tracking acidic organelles) - LysoTracker had strong fluorescence in virtually all Cat D-positive vesicles in WT- in PS1 KO cells fewer than 20% of Cat D posituve vesicles had detectable flouresence.They then went on to assess lysosomal acidification in vivo - injected a DAMP intraventricularly and used immunogold EM with the Cat D antibodies to look at the lysosomes - they found sig less acidification in the lysosomes of the KO animals
Ji et al 2005
Really simple study. They used neuro-2A cells (neural crest-derived cell line) and they upregulated ApoE4 expression within them. They showed that there was a greater lysosomal leak - used a lucifer yellow stain. When they did this with ApoE2 expression, there was significantly less of a leak. Good evidence that ApoE4 (whatever it does) seems to destabilise the lysosomal membrane
Yang 2011
transgenic mouse model of AD + deleted synstatin B (endogenous inhibitor of lysosomal cathespins) à exaggerates lysosome functioning.
o These mice had super-protection against protein misfolding. à Relative to control mice, sig lower no of giant autolysosomes (these are an indicator the system has go awry and isnt working properly). IHC revealed fewer extracellular amyloid plaques in the KO mice.
o Then went a step further and did an in vivo model model. Deletion of synstatin B improved behaviour of mice à fear conditioning paradigm – mice gets shocked in one box so avoids that box as it learns. Memory decreases with AD and so loses this fear response. KO this inhibitor of lysosomal function – they don’t lose that memory (went back to freezing)
Schonheit et al 2004
Investigated serial sections of hippocampal formation of brains with at early Braak stages (0-III) Most brains displayed both plaques and tangles. Four cases of stage ) and I, three cases with stage II and even one with stage III did not display plaques. No plaque was found in the absence of tangles. Suggests that tangles either antecede plaques or - less likely- are independently formed. In one third of their cases, tangles were seen but not plaques Limitations - Small number of total brains (n=22). Only looked at hippocampi but did early Braak staging - maybe hadnt spread there yet - only stage 3 is where involvenment of hippocampus is implicated
Schenk et al 1999
Found that immunising the PDAPP transgenic mouse (which overexpresses mutant human APP and develops hallmarks of AD) with AB protein fragments prevented AB plaque formation and hence the development of AD in the young mice. This study also importantly showed that immunisation slowed progression and reduced extent of disease in older mice, meaning this method could potentially provide a treatment rather than a prophylactic measure
The Elan Corportation trial (2000).
Disaster. Phase II trial. 6% of patients got meningeal encephalitis and the trial was abandoned in 2002. Only 20% of the treated patients developed the predetermined antibody response
CAD106 (Novartis)
short AB peptide that induces AB specific antibodies without activating AB-specific T cells. Multiple phase 1-2 studies in participants with mild AD demonstrated a strong and persitant antibody response against plaques and oligomers with initial evidence of CNA activity and no major safety issues.
Aducanumab
2015 - Completed phase Ib trial - the PRIME study - 165 AB positive pts with mild AD received 1 year of monthly injections of aducanumab or placebo. Amyloid PET imagine demonstarted a clearance of AB in a dose-dependent and time-dependent manner and stabilisation of cognitive decline as measured by MMSE - Mini-Mental State Exam (MMSE) is a widely used test of cognitive function among the elderly.
20% of pts developed amyloid related imaging abnormalities
Removal of vascular amyloid may result of vasogenic oedema - seen on MRI
Two phase III trials were discontinued for futility in 2019 (interim anayliss showed they would miss thier primary endpoints. However, in October 2019 Biogen announced that the interim analysis was wrong and there was a favorable reanalysis of of data from one of the studies (EMERGE) suggesting imporvement in clinical measures (MMSE anf so on) and amyloid brain load reduction (AB PET). On November 6 2020 FDA voted against approval - citing weaknesses in efficacy data and recommended a confirmatory trial.
Verubecestat
In Feb 2017 Merck halted its phase 2 after it was reported to have ‘virtually no chance’ of working according to an independent panel of experts
Semagacestat
Showed promise in preclinical animal studies. And was the first of its kind in phase III trials. Lilly started two Phase 3 tirals (IDENTITY-1 and IDENTITY-2 with 3000 pts total). Both trials were to run until 2012, but were halted in 2011 because both increased risk of skin cancer and infections and showed lack of efficacy. Notably, both cognition and function only did not improve but worsened in all treatment groups. Has been terminated.
Adams et al 2018
phase III trial has recently been published by Adams et al (2018) using patisiran, an RNA-interfering agent that specifically inhibits hepatic synthesis of transthyretin, that was shown to improve multiple clinical manifestations of hereditary transthyretin amyloidosis.
