Reply to: Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years.
These observations enables a viable interpretation of the results in this fied of AD research. Each of these stages can be compared with the control group without any AD-type changes
Reply to: Neuroinflammation in Alzheimer's disease and mild cognitive impairment: a field in its infancy.
This paper shows the importance to consider the inflammatory nature of brain damage in AD.
Reply to: A mutation in APP protects against Alzheimer's disease and age-related cognitive decline.
These observations fit well with the recent findings that beta amyloid belongs to the family of antimictrobial peptides
Reply to: Alzheimer’s Disease and Spirochetosis: A Causal Relationship
Herbert B. Allen, MD and Erum Ilyas MD, Drexel University College of Medicine, Department of Dermatology
Dr. McCully mentions that homocysteine is a risk factor for Alzheimer’s disease (AD); and, further, it has been shown to be a risk factor for arteriosclerotic heart disease. Homocysteine has been observed to encourage organisms to “make” biofilms . Inasmuch as we have found biofilms in the plaques of arteriosclerosis , and, we and others have also found them in AD, it seems plausible to implicate the biofilm-forming effect of homocysteine in both the worsening of AD and in arteriosclerosis. This is similar to the effect of diabetes on AD where the hyperosmolality in diabetes leads to the creation of more biofilms, and that leads to deterioration in AD .
Drs. Singhrao, Balin, and Poster mention other organisms (in addition to spirochetes) in AD. Again, considered in the light of microbes making biofilms, it has been well documented that biofilms of one species have receptors for organisms of other species (and vice versa). This allows many different organisms to take up residence in the biofilm . The biofilm, in this instance, is more comparable to a “hotel” than a “single family home”.
 Belval SC, Gal L, Margiewes S, Garmyn D, Piveteau P, Guzzo J (2006) Assessment of the roles of LuxS, S-Ribosyl Homocysteine, and Autoinducer 2 in cell attachment during biofilm formation by Listeria monocytogenes EGF-e. Appl Environ Microbiol 72, 2644-2650.
 Allen HB, Boles J, Morales D, Ballal S, Joshi SG (2016) Arteriosclerosis: the novel finding of biofilms and innate immune system activity within the plaques. J Med Surg Pathol 1, 135.
 Allen HB, Husienzad L Joshi SG (2016) Letter-to-the-Editor: The impact of diabetes on Alzheimer’s disease. J Alzheimers Dis, http://www.j-alz.com/content/impact-diabetes-alzheimers-disease.
 Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS (2003) Bacterial coaggregation: an integral process in the development of multi-species biofilms. Trends Microbiol 11, 94-100.
Reply to: What is wrong with Alzheimer’s disease clinical research?
Comment by Sally Hunter and Carol Brayne
Given the recent failure of the solanezumab clinical trials, it is understandable that discussions relating to the current state of Alzheimer disease (AD) research focus on the failure of the amyloid cascade hypothesis (ACH) and to a lesser extent the presenilin (PS) hypothesis (PSH). For decades the ACH and to a lesser extent the PSH have driven research with the amyloid beta protein (Aβ), proposed as neurotoxic and causal, as the measure of outcome for experimental design. However, there is a third relevant yet almost completely neglected hypothesis, the amyloid precursor protein (APP) matrix approach (AMA) [1-5] that takes a systems biology approach. This hypothesis suggests an opposing view to the ACH, is compatible with a modified PSH, and addresses long standing concerns relating to complexity within the APP proteolytic system.
The AMA starts with the suggestion that APP is a hub  and describes how the balance of flow through the various cleavage pathways represents a dynamic and iterative system that receives biological information representing the current state of the cell via regulation of the various cleavages and outputs the integrated biological information via the ratios of its proteolytic fragments. This system can respond to wider changes in the cell by changing the relative proportions of the proteolytic fragments released. Several features support this proposal, APP is constantly expressed, has a short half-life of between 1-4 hours, APP concentration is rate limiting and as a result its multiple cleavages compete. This view predicts that as flow through the β pathway increases, flow through the α pathway (and others) is reduced. Expanding this view, we see that any change in flow through the APP proteolytic system involves both gain and loss in equal measure. Therefore we can never be sure that the physiological changes that we see in experimental systems where Aβ expression is increased are due solely to Aβ – loss of full length APP, loss of the N-terminal sAPPα and P3 from reduced α-cleavage or the loss of sAPPβ’ and Aβ’ from β’ cleavage confounds this interpretation.
In contrast to both the ACH and PSH, which aim to explain the available evidence in relation to the roles of Aβ in AD, the AMA aims to explain the evidence in relation to the behaviour of the entire proteolytic system in both normal and disease states and requires that all the proteolytic fragments are measured. The AMA predicts that the relative ratios of all the fragments are required to describe the state of the APP proteolytic system. This is a major departure from current experimental designs where no study to date has systematically measured these fragments. Exploring this interpretation leads to the conclusion that the confounding effects of all the other proteolytic fragments potentially undermine all current research. An examination of the cross-reactivities of commonly used antibodies illustrates this view . The updated neuropathological diagnostic protocols have suggested that since the monoclonal antibody 4G8 reveals more Aβ pathology it should be the standard antibody used in routine neuropathological diagnosis. However it is not clear where this additional sensitivity comes from or what it represents. This increased reactivity may be due to cross reactivity with P3, the fragment arising from sequential α and γ cleavages, and also cross-reactivity with various catabolic fragments. Further confounding is introduced by the complex molecular behaviours of Aβ and P3 - there is no panel of antibodies that can distinguish between P3 and Aβ across all aggregations states, (monomers, dimers, oligomers and fibrils), across all sequence variations and across solubilities. How then do we best interpret any signal from this or any antibody?
Unlike the ACH or PSH which focus narrowly on evidence relating to β and γ cleavages and interprets evidence from other cellular systems as secondary or as a consequence of these cleavages, the AMA allows evidence from wider cellular systems to drive APP cleavage. Therefore there is no overarching “causal feature” and various factors such as synaptic activity, immune signalling, metabolism cholesterol etc. can be both drivers of and be driven by APP cleavage through complex regulatory feedback. The coherent behaviour of the APP proteolytic system and its regulatory factors can be understood as contributing to a homeostatic point that allows proper cellular function and in the case of neurons, supports appropriate synaptic plasticity. With ageing, the AMA suggests that the APP system becomes increasingly decoherent so that changes in, say, immune signalling or energy metabolism etc. can stress the homeostatic point beyond that which allows full integration of the biological information. Ageing and cellular senescence programs may alter the homeostatic point to which cellular systems cohere . This re-programming of the homeostatic point has relevance to therapeutic approaches that aim to reduce Aβ. According to the AMA, loss of physiologically relevant Aβ may increase flow through β pathways to replace the Aβ removed so that no long term change would be expected and further, flow through the other APP cleavages would be reduced while this adjustment is in progress with further loss of function from other cleavage fragments.
The AMA predicts that each mutation in APP and PS can be understood as altering the homeostatic point or the behaviour of the APP proteolytic system as a whole, allowing changes that happen with e.g. age to impact at an earlier age/stage. Each mutation has the potential to alter the homeostatic balance in this system in subtly different ways. If we take this interpretation further, the AMA predicts that multiple disease pathways are possible, each with different drivers and that those arising from mutations do not necessarily relate to those arising through ageing. Further, the AMA suggests that a detailed investigation of each mutation in relation to neuropathology and cognitive status from a natural history perspective will help clarify how the entire system works – an approach that has not yet been acknowledged as useful.
Because the AMA predicts multiple disease pathways, it also suggests that, unlike familial AD where the presence of a disease associated mutation is a qualitative diagnostic feature, defining sporadic AD as a single disease process in the older population is not possible. Therefore from the perspective of the AMA, there is no way to accurately select sporadic AD cases and controls in randomised controlled trials (RCTs) and any attempt to do so will result in confounding and/or bias.
It is understandable that the wider research community has not fully engaged with the AMA. The AMA was first described in 2005 in a Master’s thesis for the Open University, UK. It was first submitted to a journal in 2006 where it was rejected without peer review. There followed a cycle of re-write, update, resubmit and rejection without peer review until 2012 – journal editors thus denied the AD research community an alternative framework for many years. When work based on the AMA actually gets to peer review, the perspective of the dominant ACH leads invariably to rejection – again blocking access for the wider research community to this alternative perspective. It is possible that other approaches have similarly had major challenges in being visible to the scientific community. There is a reluctance to engage in a constructive way with avenues outside the role of Aβ, including hypotheses relating to the roles of vascular change, the immune system, metabolic regulation, metal homeostasis and oxidative stress etc. in the evolution of dementia.
In terms of strategy in the immediate future, the AMA is clear.
1) In order to fully account for and understand the potentially confounded nature of AD research, we need to accurately describe the APP proteolytic system as a synergistic whole in humans. This will allow us to characterise experimental models in relation to the human so that we can better assess which experimental models of disease are appropriate. Currently this approach is entirely missing from AD research strategy.
2) Experimental techniques to accurately and reliably measure all the proteolytic fragments need to be developed and validated – not a straightforward task given the complexity of the APP system.
3) Since the AMA predicts that rate and timing of APP cleavage is as important as any cleavage products, experimental techniques to accurately and reliably measure protein-protein interactions, affinities and rates of reaction need to be developed, standardised and validated.
4) We need population approaches to generate reliable evidence that describes the APP proteolytic system in ‘normal’ and ‘disease’ states and how this system interacts with wider cellular systems with a view to better defining possible disease pathways in the older population. We may then be able to better select cases/controls for RCTs that reduce confounding and bias.
5) We need to investigate in detail how wider cellular systems interact with the regulation of APP cleavage so that contributions from important hypotheses that have been relatively ignored relating to cellular systems such as energy and metabolism, oxidative stress, immune signalling, calcium regulation etc. can be understood in both familial and sporadic disease.
6) Evidence relating to familial AD arising from mutations requires re-interpretation. Subtle differences arising from each mutation have the potential to increase our understanding of the APP proteolytic system as a whole and crucially, this evidence can be reliably interpreted as relating to an AD pathway since the presence of a fully penetrant mutation is a qualitative diagnostic feature.
Simultaneously following the above recommendations will enable us to better understand the complex problems presented by AD and move towards a rational identification of therapeutic targets. From the perspective of the AMA, we haven’t even started to describe the problem, never mind developing therapeutic solutions.
 Hunter S, Arendt T, Brayne C. The Senescence Hypothesis of Disease Progression in Alzheimer Disease: an Integrated Matrix of Disease Pathways for FAD and SAD. Mol Neurobiol 2013.
 Hunter S, Brayne C. Relationships between the amyloid precursor protein and its various proteolytic fragments and neuronal systems. Alzheimers Res Ther 2012;4:10.
 Hunter S, Friedland RP, Brayne C. Time for a change in the research paradigm for Alzheimer's disease: the value of a chaotic matrix modeling approach. CNS Neurosci Ther 2010;16:254-62.
 Hunter S, Martin S, Brayne C. The APP Proteolytic System and Its Interactions with Dynamic Networks in Alzheimer's Disease. Methods Mol Biol 2016;1303:71-99.
 Hunter S, Brayne C. Integrating Data for Modeling Biological Complexity. In: Kasabov N, editor. Springer Handbook of Bio-/Neuro-informatics. Berlin Heidelberg: Springer-Verlag, 2014. p. 921-40.
 Turner PR, O'Connor K, Tate WP, Abraham WC. Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog Neurobiol 2003;70:1-32.
 Hunter S, Brayne C. Do anti-amyloid beta protein antibody cross reactivities confound Alzheimer disease research? Journal of negative results in biomedicine 2017;16:1.
Reply to: What is wrong with Alzheimer’s disease clinical research?
Jack de la Torre's has written an insightful article on the inability, or unwillingness, of researchers in the field of Alzheimer's disease to determine if a target of interest is truly causative of the disease. Specifically as this relates to the beta amyloid hypothesis. Unfortunately, I think this inability to test one's target-hypothesis can be generalized across the CNS neurodegenerative research space, where we presently have only symptomatic therapeutic relief for any of the chronic neurodegenerative indications. What if, in fact, our early conclusion, "that either we find a means to curb further neurodegeneration by means of a neuroprotective agent, or that we must stop the disease prior to disease onset" is omitting another therapeutic strategy that could, for the first time, reverse disease? We have known for over a decade that the adult brain can indeed produce new neurons and seemingly initiate this process in response to neurodegeneration. So the third option would take advantage of the ability by the brain to self-regenerate, however insufficient under chronic neurodegenerative conditions, and push this process in a (i) disease, and (ii) region-specific and (iii) temporal way.
Indeed, this has been Neuronascent's view point all along, in that we take advantage of the highly regulated process of new neuron formation, in select regions of the adult brain, to replace dying and lost neurons in aging and neurodegenerative disorders, i.e. endogeneous regeneration. We are not suggesting returning to the idea of growth factor injections, with the many systemic issues that this often causes. The ideal process-stimulation point should be downstream of growth factors, hormones and other initiators of cellular events. We are also not suggesting administration of general mitogens, that would not qualify as either region-specific or working in a temporal way (i.e. timed to the occurrence of neuronal loss). Instead, the aim should be toward selective therapeutics, that first push neuronal progenitors to not only proliferate (i.e. neurogenesis), but to secondly push the migration and differentiation and thirdly the maturation of neurons -- leading to an associated reversal of behavioral impairment in chronic neurodegenerative disorders. It is time to test a completely new hypothesis, not one based on a single target for neuroprotection in these very complex neuron-dying diseases, like Alzheimer's and Parkinson's disease. The hypothesis to be tested looks at the possibility to pharmacologically push the brain in a safe and non-invasive manner to regenerate itself. This hypothesis could even bring back the possibility of a disease-modifying therapy for Alzheimer's disease patients. It is time to invest in such a paradigm-shift. This is not an issue of a "potential" therapeutic discovery program, years away from human testing. Neuronascent, Inc. already has a patented, neuron regenerative NCE, manufactured for the clinic, that has shown to be safe and which could potentially enter the clinic in 2017.
Judith Kelleher-Andersson, Ph.D., is President and CEO of Neuronascent, Inc. and is the inventor on all of Neuronascent’s technologies.