 Prejoutno, vseč, začela, da je zelo, da je hrabil na predstavno určite, in da je od vrčiteh pridost, jih je tukaj, je pričočen, da je zelo, da je zelo, da je odličen. Otevno vseč je izvečo, ovo 30 milijonih vrčitv, terje izvščenov, kaj je zelo, in nekako bolje se vrčite v pridosti, The clinical picture is heterogeneous, memory deficits are initial symptoms in most patients. However, there are non-amnestic presentation, including language dysfunction, visual spatial dysfunction, executive dysfunction, or other type of presentation, particularly in genetic forms. The molecular pathology of the disease is marked by misfolding and aggregation of amyloid beta in the extracellular space and protein tau in nerve cell bodies and processes. I have to underlined that in many instances other amyloid proteins are co-deposited in the brain of these people, including alpha-cinuclein, TDP-43, and C-statin C. The familial forms, which are rare forms, are linked to a mutation of the precursors of the amyloid beta protein, or pricine in one or pricine in two, which are the catalytic part of the gamma secretase, the TRIVS-APP, so the amyloid precursor protein, to generate a beta. Genetics plays a role also in sporadic forms of the disease in which the major risk factor is the ApoE-epsilon for allele. These mojiti bind to amyloid beta and affects A-beta clearance and aggregation. This is the picture in the end stage of Alzheimer's disease. As you see, all areas of cerebral cortex show a large amount of amyloid deposits that can even be seen and counted by eye, if you want. And there is accumulation in the same areas of hyperphosphorylation tau in the forms of neurofibrillary tangles in neural cell bodies and processes, particularly in this trophic neurase surrounding the amyloid plaques. The observation that mutation in amyloid precursor protein gene and pricine result in increased production and aggregation of A-beta led to the A-beta cascade hypothesis, which is the most widely accepted scenario. This hypothesis proposes that A-beta, which is a normal product of ATT processing, undergoes misfolding, or A-beta intermediates, particularly prone to aggregation, assembled into oligomers, high-order structure, so protofilaments, fibris, and finally in amyloid deposit, form of amyloid deposit that induz reaction of astrocyte, particularly of microgliaz, says that sustain a chronic neuroinflammation, and all these leads to tau pathology and nerve cell degeneration. There are many arguments in favor of this hypothesis I mentioned before, the linkage between genetic forms and mutation in APP and pricine in one gene, but also the trisomy of APP, such in down syndrome leads to full ad pathological pictures, also major risk factor for sporadike visapo E, which affects A-beta clearance. But there are also some factors against this hypothesis, including lack of correlation, close correlation between amyloid load and dementia, and the observation of frequent amyloid position without significant cognitive impairment among the elderly people. So most scientists think that additional events are required to cause neurodegeneration, and neuroinflammation can play a major role. In the last 25 years, many attempts have been done to modern disease using particular TG mice to investigate disease pathogenesis, identify therapeutic targets, develop disease-modifying treatments. Over 200 TG mouse lines, mouse models have been generated in the last years. These models are very far, actually, from the real world. So many models are generated with over expression of mutant APP, but many other models have double transgenic models with mutant APP and PS1. There are triple transgenic models with APP, PS1 and tau, and usually over expressed tau is a mutant tau, which is not found in Alzheimer's disease, but is linked to frontotemporal dementia. So as you see, the transitional value of this model is questionable, since they are highly manipulated, they do not reflect the real world. And indeed, many of these, or all of them do not recapitulate central aspects of disease, they can be useful to ask very specific questions, but not really to the pathogenesis. Concerning the A beta, so APP processing can follow two different pathways, non-amyloidogenic and the amyloidogenic pathway. In the amyloidogenic pathway, APP is cleared by beta in gamma secretase to generate A beta peptides. And the most studied peptides are A beta 40 and A beta 42. The 42 residue peptide is much more amyloidogenic than A beta 40. However, there are many other species that are generated in the APP processing that have been neglected for many, many years, but may play a role in this processor, we will see this in a minute. A beta produced by APP processing, A beta 42 particularly is highly amyloidogenic, and when you generate synthetic peptides of A beta 40 and 42, this peptide really assembles into oligomers and amyloid fibres in vitro, so these are oligomers and amyloid fibres. Solid state enema spectroscopy has been used to solve the structure of this A beta 42 fibres. The core of the fibres consists of a dimer of A beta 42 molecules per fibre layer, with residues 15 to 42, so that C terminal 2-3, which forms a double horseshoe-like cross-betasheet entity, while residues 1 to 14 are partially ordered in the beta strand from strand conformation. Concerning the second layer of AD, so microtubula-associated protein tau, these protein exist in six different isoforms that can be grouped into major families with three or four microtubule binding domain. Binding of tau to microtubules depends on its phosphorylation state. The tau has a large number of serine and trionine that can be phosphorylated, so in this state tau undergoes hyperphosphorylation, misfolding and aggregation into perylical filaments and straight filaments, which disrupt the cytoskeleton. The structure of tau filaments has been investigated by CryoEM by the Cambridge group and both pHF and straight filaments are composed by two identical protofilament with C-shaped subunits, which adopt a combined cross-beta-betaelic structure. The core of both protofilaments is composed only by a small part of tau, in particular the microtubule binding domain 3 and 4 and 10 amino acid C terminal to the repeats, while the N terminal is the terminal termini of tau form a fascicult. I have to underline that this flanking region may play different roles also in the process. They can capture for instance monomers for elongation, mediate chaperone binding, mediate membrane binding, toxic assembly, so these flanking parts are very important, although they are not fully investigated. The difference between perylical filaments and straight filaments is due to differences in their interprotofilament packing, which is symmetrical in pHF and is asymmetrical in straight filaments, and the ordered core defines the seed of aggregation of tau in Alzheimer's disease. A central issue to be addressed is the high phenotypic heterogeneity of AD. Alzheimer's disease is often seen as a single homogenous pathological entity, indeed the disease is highly heterogeneous, and its complexity is a key issue to be addressed, since it has important implications, particularly for the development of specific therapeutic agents. The clinical heterogeneity of sporadic forms of Alzheimer's disease is incorporated into the diagnostic criteria and reflects different brain regional distribution of the pathological process. In most instances the presentation is amnestic. I say so in 15-20% of patients is non-amnestic, but this heterogeneity is even more pronounced, is remarkable, really, in genetic form associated with presenium mutation, which may present with cerebellar attacks, for instance, or displace plastic paraparesis or Parkinsonian syndrome. As you see, the clinical picture of Alzheimer's forms linked to presenium mutation is highly, highly variable. This is an example of a young patient carrying the presenium mutation who had an initial ataxia syndrome and behavioral disturbances, and only in less stages of the disease appear at the men's condition. The neuropathology show a high involvement, a profound involvement of the cerebellum by a beta deposition, which is not a common feature of the disease. Also heterogeneity is associated with APP mutation, which can be linked to classical AD phenotype, in most instances, but can result in amyloid congophilic angiopathy with strokes, for instance, or a combination of the two. The clinical heterogeneity is parallel by striking variation in a beta-related neuropathological profiles as shown by this panel, which highlights great differences in density, type, and distribution of a beta deposition in the cerebral cortex, for instance. So you can see, in some instances, a very important congophilic angiopathy, in other index, almost half sense of congophilic angiopathy, and this cotton-hole plaque instead of dense plaque and so on. So there is a huge variation in neuropathology. The understanding of the molecular basis of a scientific diversity has an important implication for the development of treatment strategies. In the last part of my talk, I will focus on heterogeneity of a beta pathology and neuroinflammation. So I will make a case of a beta pathology in neuroinflammation. Robert Tico and co-worker Satena Ege investigated possible correlation between Alzheimer's phenotype and structural variation of the beta fibers. They use extracts of cerebral cortex from three distinct subtypes of Alzheimer's disease, a typical form with long-duration, a posterior cortical atrophy variant, a rapid progressive form. The samples were used as a seed, and a beta 40 and 42 as substrate, and the fibers obtained by seed growth were analyzed by transmission electron microscopy and solistate NMR spectroscopy. Typical AD and also the posterior cortical atrophy variant show a specific predominant a beta fiber structure, while rapid progressive AD show a high proportion of additional a beta fiber structure. And the authors conclude that variation in a beta fiber structure may correlate with variation in AD phenotype in analogy to distinct prion strains that are related to different conformance of PRP scrappy. And indeed prion disease are a prime example of how different conformance and assembly states of the same protein are associated with profoundly different clinical pathological phenotype. As you can see here, a classical form of crotseljakov disease with a high involvement of the cerebral cortex and relatively sparing of the cerebellum in form with a high involvement of the cerebellum and these forms are associated with two different conformance of PRP scrappy. My group investigated this issue with a different, more direct approach. Brain samples were collected from 24 patient with genetic sporadic forms of Alzheimer's disease and biochemical profiling of a beta species in affected brain regions and purified amelod fraction was performed using cell detox mass spectrometry and in addition amelod fraction were injected into transenic mice to analyze the propagation pattern of amelod pathology. As mentioned earlier so a large number of a beta peptide in addition to a beta 1442 are generated in APP processing including a variety of N and C terminal tronkated fragments. Cell detox mass spectrometry show that amelod fractions from different patient contain different A beta species and these fingerprint profiles could be clustered into three main groups, profile 1, 2 and 3. The profile 1 was found in about 70% of sporadic Alzheimer's patient and in patient with presinil mutation where the predominant species appear to be A beta 42 or N terminal tronkated A beta 42 or modified A beta 42 species. Profile 2 consisted in a combination of A beta 40 and 42 and was found in about 30% of patient with sporadic AD. Profile 3 was found in familiar AD linked to APP mutation and was characterized by A beta 40 peptide and A beta 138. In addition to this there were final species as you can see here. Now it is well known that accumulation of A beta in the brain is parallel by a reduction of A beta peptide in CSF in the cerebrospinal fluid. As a control we then compare the A beta profile in brain amelod fraction and in the cerebrospinal fluid and found exactly a mirror image supporting our result of the characterization of amelod fraction. These four, three different A beta profile show this in chemical physical properties in term of resistance to K-digestion as you can see here aggregation kinetics and also the different A beta profile show different propagation pattern in transgenomic mice expressing the Swedish mutation is a double mutation just upstream the end terminus of A beta. These mice generate high amount of white A beta and when injected with the different amelod fraction belonging to different profiles there was a propagation pattern which was different with major involvement of hippocampus and thalamus in some instances only thalamus in others only hippocampus in others and also the topology of the amelod position in cell walls versus neuropil was different and so on. Our findings are consistent with the result of Matthias Juker group that used luminescent conugate oligotiofenis to investigate the molecular architecture of amelod deposits in brain tissue Alzheimer patient and found differences among genetic sporadic subtype in between typical and sporadic AD and posterior cortical atrophy and these differences quite interesting were maintained for transmission to transgenic mice. Recently we investigated the potential contribution of neuroinflammation to phenotypic diversity of AD to this end we carried out the pathological and biochemical study on brain tissue from 24 patients with sporadic and genetic forms of Alzheimer and investigated the correlation between the neuroinflammatory molecules and the clinical pathological feature of the patient these slides show the neuro pathological assessments of micro glial cells were differently represented in familial versus sporadic AD patients with regard to morphology, distribution and density we then investigated whether these differences could be associated with differences in production of inflammatory factors to study show high levels of these factors in AD sample respect to control and a predominance of anti-inflammatory pro-inflammatory cytokines in AD brains Analyzate were assigned to for biological classes cytokines chemokines matrix metalloproteinase in innate immunity factors as you can see the most represented classes where matrix metalloproteins and innate immunity factors were suggesting a major contribution to neuroinflammation Finally, we analyzed the association weight of the for cytokines families with available clinical, neuropatological biochemical data and based on hierarchical cluster analysis of our AD court the court was grouping three neuroinflammatory cluster, cluster one characterized by patients with early stage atones and death and intense microglare reaction, cluster two patient with the fastest disease progression and highest amount of cytokines in the beta species cluster three patient with longest disease duration and low neuroinflammatory profile in beta levels so cluster two was the most aggressive clinical phenotype, cluster three, the lesser the and this is the last slide to summarize our views of the phenotypic heterogeneity of AD is associated with differences in biochemical composition, chemical physical properties, higher order aggregation pattern and targeting of the beta species differential involvement of microglare resulting in different neuroinflammatory profiles these variables along with other factors contributed by medical complexity of Alzheimer's disease and must be taken into account for patient certification, the existence of molecular subgroups of AD can account for responders and non-responders to specific treatments and has important implication for the development of disease modifying therapies. Thank you for your attention. Ane, do we have questions? I think there was something on the whole stuff. Good so I don't see any questions in the chat right now. I have one question if it's possible, so you mentioned on your last slide molecular subgroups is it could you specify what is is it pure assembly of a beta monomers Or it is more complex subgroups? To, in me, this is simplification. So I was referring to the A beta species involved in amylo deposition. We don't know yet which of these species reared to be the amylo itself, but I'm sure that there are assemblies of also in to je na ono illuminadnje, katericina skupov, ko je zvečno��, zato je ještje, še nekaj ležiti in zaznakati nekaj ležiti, nekaj k過來, nekaj prav, nekaj nešte kodi, nekaj nešte kodi, nekaj kodi, če zelo. terapije naredne in immunoterapije z antivodijem v spesivnih spesivnih in spesivnih asemlih, oligomir, sofibris in zelo. In tudi je to nekaj semplifikacija, ker je v mnohem tudi tukaj izgleda. In tudi je tudi tukaj izgleda, kaj je tukaj izgleda izgleda. In soothing needs may be related toakat that Alzheimer's diseases are not in single conditions, but they are in multiple conditions with different polycolysis substrate. So there is just a question from Christian Korp, do you know if co-aggregation, co-aggregating proteins might contribute to clustering? So I guess it is similar, is it single proteins or is it co-aggregation here? There is a co-aggregation of some of these peptides. For instance, in mutant, in genetic forms with mutation in APPs, there is a co-aggregation of A beta 38 and A beta 40, for instance. But, I mean, also this is still a preliminary picture, I think. So the things are much more complex. So I have a question my own as well, because I am curious on this take with the different polymorphic forms of the vipals as well and the indications of relation to the phenotypes as well. So I think it is more clear for the mutations than for the actual different vipal forms in my understanding. Or what should I take on this? Yes, I mean, in brain tissue of patient with mutation, this is more striking, but also sporadic AD is heterogeneous. So there are forms where amyl protein contains, the aggregates contain basically only 142 peptides, which can be full length or truncated at n-terminus, for instance 342 or 1142 with pyroglutamate, position 3 or 11, while other types of sporadic AD had just A beta 40 peptides, or a combination of A beta 40 and 42, so which, I mean, result in a very complex picture so that has to be still defined in detail. May I ask? Yeah. Final question and then we move. There are some claims about some involvement of the heart in Alzheimer's disease, some damage of A beta to cardiomyocyte and because you and Periot are both present in the audience to be nice to have your comment about this, but there is no evidence of involvement of the heart. There are some claims, some paper reporting, some damage and some systemic effect of A beta deposition, both in the brain and in the heart, which is your impression with your patients so you don't see any really connection between the two diseases, the brain disease and the heart. In Alzheimer's disease, I don't see a connection, actually. And not just Peri, yeah, go ahead. Yeah, sorry, my video is disabled, but I don't see any connection. And I think unfortunately there's a lot of confounders because you're dealing with an elderly population that are likely to have both diseases, actually. And you certainly see progressive cognitive decline in patients with cardiac amyloidosis, but I'm not sure that's directly related to... But my question is, this progressive cognitive decline is associated with A beta pathology in the brain? No, I don't think we have good evidence for that at all. I think it's more to do with a general disability. And frailty? Also probably the cardiac problem can affect, I mean, the brain function. The basis of this was discovery of A beta aggregating, the cardiomyoside in the heart. So I don't know if it's just a molecular observation or some clinical implication. That's the point.