Alzheimers disease is a progressive neurodegenerative disorder affecting hippocampus and neocortical regions of the brain. Post mortem brain examination of severe AD patients reveals obvious reduction in brain size with atrophy particularly affecting the medial temporal lobes and the hippocampus, presenting as thinning of the cortical gyri, widening of the sulci, and enlargement of the lateral and third cerebral ventricles. (Dawbarn & Allen 2007). AD is characterised by the presence of two main pathological hallmarks in the striatum and neocortex of the Central Nervous System (CNS): extracellular senile plaques containing aggregated Î² amyloid (AÎ²) protein depositions surrounded by astrocytes, microglia and dystrophic neuritis; and intracellular neurofibrillary tangles (NFTs), consisting of fibrillar polymers of hyperphosphorylated tau protein. (Koechlig, 2010)
Since Alzheimer's disease was first described 1907 by Alois Alzheimer, significant progress has been made into understanding neuropathological mechanisms that drive neuronal failure. A large number of challenging hypotheses have been proposed to explain the pathogenesis of AD, however the mechanisms that drive neuronal failure have not yet been fully understood.
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For over two decades, the Amyloid Hypothesis dominated the stage of Alzheimer's disease research and provided the intellectual framework for therapeutic intervention. (Pimplikar, 2009). The amyloid hypothesis states that the AÎ² peptide is central to pathogenesis of AD, and is the proximal cause of multiple effects including neurofibrillary tangle formation, synaptic dysfunction and loss, and neuronal cell death. Although substantial genetic and biochemical data strongly support the amyloid hypothesis, there have been many recent contradictory findings, questioning and criticizing it.
Tau is another hypothesis proposed to explain the pathogenesis of AD. The hypothesis that tau is the primary causative factor has been grounded in the observation that deposition of amyloid plaques does not correlate well with neuron loss. (Schmitz et al. 2004). Tau is a microtubule associated protein, encoded by the tau gene on chromosome 17. It is mainly found in the neuronal cells, involved in microtubule polymerisation and stabilisation of neurons. Tau function is affected by its degree of phosphorylation in AD and hyperphosphorylation of tau depresses its binding to microtubules, inhibiting its ability to promote microtubule assembly leading to axonal transport impairment.
It is indisputable that APP is linked to the development of AD. APP is a transmembrane protein that plays several important cellular functions including synaptogenesis and synaptic plasticity. (Gralle, 2007) The gene encoding APP is located on chromosome 21. Patients with Down's syndrome (trisomy 21) develop early onset AD pathology which might be a result of increased AÎ² production due to the presence of an extra copy of APP. (Gralle, 2007) AÎ² and other products are formed when APP is cleaved by enzymatic activities known as Î²- secretase and Î³-secretase.
Different to the dogmatic hypothesis, more mechanistic kind of approach to understanding the disease process resulting in neuronal destruction has been proposed by Nicolaev et al (2010). He demonstrated that that an extracellular metabolite of APP (N-APP) activates the death cell receptor 6 (DR6) triggering neuronal degeneration via distinct capsases which may contribute to initiation or progression of AD. APP and DR6 are therefore the components of caspase dependant, self destruction program, that may in particular contribute to the initiation or progression of Azheimer's disease. This finding reveals yet another potential toxic mechanism that may contribute to Alzheimer's disease.
The study by Shankar et al (2008) is yet another important step towards establishing the cause of Alzheimer's Disease. The authors report that soluble AÎ² oligomers, specifically dimers, isolated from cerebral cortex of human subjects with AD are synaptotoxic. They inhibited long- term potentiation (LTP), enhanced long-term depression (LTD), and reduced dendritic spine density in rodent hippocampus. The extracted AÎ² oligomers from AD brain also impaired performance in memory of a learned behaviour in rats. Soluble dimers were shown to be the smallest synaptotoxic species, potently impairing synapse structure and function whilst insoluble amyloid plaques were noted to be inactive, supporting the hypothesis that plaques are not neurotoxic agents. These findings significantly strengthten the hypothesis that amyloid-beta oligomers are responsible for cognitive impairment in Alzheimer's disease.
Ittner et al. (2010) emerged critical finding that AmyloidÎ² (AÎ²) toxicity is tau dependant. Using tau mutant mice, it was found that tau has dendritic function in postsynaptic targeting of the tyrosine specific phospho-transferase Fyn. Fyn can phosphorylate the NMDA receptor subunit NR2B, promoting interaction with the scaffolding protein PSD95. This interaction appears important for AÎ²-induced neurotoxicity. In mutant mice with reduced dendritic Fyn expression there is uncoupling of the NR2B/PSD95 interaction and reduced AÎ² toxicity. Reduction of tau levels or targeting tau-dependant mechanisms are suitable strategies in treatment of AD, highlighting tau as an attractive drug target. These findings are a major step toward understanding neurodegeneration associated with Alzheimer's disease, sheding a new light on the role of tau, providing new insight into pathogenesis and opening up possibilities for new drug treatments.
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Neither of the dogmatic hypothesis so far could sufficiently explain the pathogenesis of AD or provide neurodegeneration cure. However, none of the currently perceived weaknesses of the dogmatic hypothesis provide a compelling reason to abandon them, although together they certainly point to important gaps in our understanding of AD. Recent studies seem to be receding from the dogmatic hypothesis, focusing on mechanistic understanding of the disease process which might be capable of filling in the gaps generated by the dogmatic hypothesis.
So what are the Alzheimer's molecular players? Is Alzheimer's caused by plaques and tangles in the brain? Which of the molecular mechanisms correlates best with neuropathological features seen in AD?
The aim of this dissertation is to explore and evaluate the molecular mechanisms of neuronal failure in Alzheimer's Disease. Although a large number of hypotheses have been proposed to explain the mechanisms that result in neuronal degeneration in AD, this still remains poorly understood. Development of a comprehensive therapeutic treatment AD is also limited by our understanding of the disease process. There is an ongoing controversy over the molecular mechanisms that result in the symptoms seen in AD, that this dissertation will aim to resolve. It is well established that both AÎ² and tau proteins are somehow involved in AD pathogenesis but what is the connection between them and which protein acts upstream of the other?
The gap is going to be closed with the use of recent study materials undertaking more mechanistic rather than dogmatic approach, by focusing on molecular mechanisms in studying the disease process. The work by Nicolaev et al. (2010), Shankar et al. (2008), Ittner et al. (2010) is going to be evaluated in terms of their relevance to initiation and development of AD and the possibility of discovering effective drug therapies that would tackle the initial pathways of the disease process.
The amyloid hypothesis
For over two decades, Amyloid hypothesis governed the stage of Alzheimer's disease research and provided the intellectual foundation for therapeutic intervention. The amyloid hypothesis states that the AÎ² peptide is fundamental to pathogenesis of AD, and is the proximal cause of multiple effects including neurofibrillary tangle formation, dysfunction and of synapses, as well as neuronal cell death. For long it has been reckoned that the AÎ² peptides derived from proteolytic cleavage of APP to be cardinal toxic species in Alzheimer's Disease pathogenesis. However, so far the amyloid hypothesis has failed to explain its pathogenesis or provide neurodegeneration cure. There are substantial genetic and biochemical data strongly supporting the amyloid hypothesis, however, more recent articles seem to question as well as criticize the amyloid hypothesis, calling for its reassessment.
Amyloid cascade hypothesis
APP cleavage and amyloid cascade pictures.
Describe what is happening in terms of the amyloid cascade hypothesis.
Figure 1. The amyloid cascade hypothesis
Structure and function of the Amyloid Precursor Protein
Despite sometimes contradictory findings with regard to the amyloid hypothesis, the indisputable genetic evidence that APP is linked to the development of AD is difficult to counter. Therefore continuing rational efforts aimed at understanding the biology and molecular neuropathology of APP will lead to further insights which will better elucidate the role of APP and AÎ² in the development of AD.
Amyloid Precursor Protein (APP) is a type I transmembrane glycoprotein with a relatively long N-terminal extracellular domain and short C-terminal cytoplasmic domain. (Dawbarn & Allen 2007). This type of structure led its discoverers to suggest that APP might function as a receptor (Kang et al., 1987). Although functions, ligands and intracellular pathways of many cellular receptors have been established in vitro, the functional importance of APP in nervous tissue has been slow to emerge.
APP consists of up to 770 amino acids (in its longest isoform). So far it has not been crystalised satisfactory. The APP gene has 19 exons (Yoshikai, 1991). Several isoforms of APP are generated by alternative splicing of exons 7, 8 and 15, all of them coding for domains localized in the extracellular portion of the molecule. (Gralle, 2007)
It is known that the amyloid precursor protein (APP) is a transmembrane protein that plays major roles in the regulation of several important cellular functions, especially in the nervous system, where it is involved in synaptogenesis and synaptic plasticity. (Gralle, 2007)
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APP is subject to endoproteolytic cleavage by several proteases, named in order of their historical discovery, Î±-secretase, Î²-secretase and Î³-secretase. (Dawbarn and Allen 2007).
While the trafficking of transmembrane APP still remains incompletely understood, the biological effects of the cleaved and secreted form of APP (known as sAPP, for secreted APP) have been thoroughly investivated. The Î±-secretase cleaves the APP 12 amino acid residues upstream from the extracellular face of the membrane, releasing the extracellular domain sAPPÎ±, known to have many functions. However, the most important roles of sAPPÎ± is modulation of transmission at the synapse and neuroprotection against ischemic and excitotoxic injury. (Dawbarn and Allen, 2007)
Alternative proteolytic processing of APP releases potentially neurotoxic species, including the amyloid-Î² (AÎ²) peptide that is centrally implicated in the pathogenesis of Alzheimer's disease (AD). (Gralle, 2007).
More studies need to be done in relation to the structure and function of APP as it is certain that this molecule is involved in AD. Better understanding of the molecule will enhance development of a successful AD therapy.
4. The amyloid-Î² peptide
APP was initially discovered because of the characteristic deposition of plaques in demented patients, containing the amyloid Î² peptide, which is a proteolytic fragment derived from APP. The AÎ² is derived from the cleavage of APP by Î²-secretase. The amyloid aggregates that accumulate in vivo in the brains of AD patients have long been thought to be potent neurotoxins. Given that AÎ² amyloid plaques are found extracellularly, much focus has been placed on the role of extracellular conformers of AÎ². However, since the intracellular pools of AÎ² have been detected in 1993, they were postulated to influence the development of AD. The research has then headed away from that theory, with neuropathological observations suggesting that the species responsible for synaptic dysfunction in AD patients may be soluble oligomers of AÎ² which interfere with synaptic plasticity rather than the fibrillar amyloid aggregates that were initially considered to be neurotoxic species. (Gralle, 2007)
The Tau protein and Tau hypothesis
Tau is one of the series of microtubule associated proteins (MAPs) which maintain the microtubule network in neurons by facilitating assembly and stabilization of microtubules. Tau gene is located on chromosome 17. Tau function is affected by its degree of phosphorylation and abnormally phosphorylated tau is accumulated as interneuronal tangles of pared helical filaments, twisted ribbons, and/or straight filaments. (Dawbarn and Allen, 2007). Results from various studies suggest that the abnormal hyperhosphorylation of tau precedes its accumulation into tangles.
The Tau hypothesis stating that tau is the primary causative factor originates from the observation that deposition of AP's does not correlate well with neuron loss.
Tau protein pathology, seen as neurofibrillary degeneration, is a hallmark of Alzheimer's Disease. To date, the most established and the most compelling cause of dysfunctional tau in AD and related tauopathies is the abnormal hyperphosporylation of tau. The abnormal hyperphosphorylation results not only in the loss of tau function of promoting assembly and stabilizing microtubules but also in gain of a toxic function whereby the pathological tau sequesters not only normal tau, but also other two neuronal microtubule-associated proteins (MAPs) and causes inhibition and distruption to microtubules. (Dawbarn and Allen, 2007).
Support for tau hypothesis is also derived from other tauopathies, in which the same protein is misfolded. However, a majority of researches support the hypothesis that not tau but the amyloid is the primary causative agent.
Mechanistic approaches aimed at understanding neurodegenerative processes in AD
New molecular model for AD proposed by Nicolaev et. al.- Activation of DR6 by N-APP triggers neuronal degeneration via distinct caspases.
It is well known that APP is the bad actor in the brain in Alzheimer's Disease. Knowing that APP is a large protein that sits in the cell membrane, for many years research in field of AD had focused on the part of the protein called AÎ². However, the AÎ² toxicity and its contribution to the degeneration in AD has been argued.
Nicolaev et al. have undertaken a completely different approach to previous studies of APP involvement in neurodegenerative processes. The authors found that not the AÎ² as previously thought but different part of APP, called the N-APP may be involved in the process, triggering neuron death and degeneration. Nicolaev et al. described a new novel role for APP in neurodegeneration that appears completely independent of AÎ² toxicity, which could finally explain why only certain neurons are affected in AD pathology. They reported that the extracellular domain of Amyloid Precursor Protein (N-APP) binds Death Receptor 6 (DR6), activating a widespread caspase-dependent self-destruction program that targets both axons and cell bodies.
During nervous system development, initial formative phase of nervous system involving generation of neurons and extension of axons is followed by a regressive phase where inappropriate axonal branches are pruned to refine connections and many neurons are culled to match the numbers of neurons and targeted cells. Loss of neurons and branches also underlies the pathophysiology of many neurodegenerative diseases. The mechanisms of events that happen during regressive phase are not fully understood, however, its known that degeneration can occur both "passively" from loss of support from trophic factors like Nerve Growth Factor (NGF) and "actively" from mechanism where extrinsic signals trigger degeneration via proapototic receptors, including some members of the Tumor Necrosis Factor (TNF) receptor superfamily. However, the full complement of degeneration triggers together with intracellular mechanisms of neuronal dismantling remains incompletely understood.
Nicolaev et al. studied the expression of all TNF receptor superfamily (members of which are well known for their roles in apoptotic signaling) in neuronal development. The experiment was performed on mice embryos in their midgestation period with the use of in situ hybridization technique. Nicolaev et. al identified DR6 as a candidate, as it was expressed at low levels in neuronal progenitors in the spinal cord but at high levels in differentiating neurons. Also because DR6-expressing neurons are known to be apoptotic, the authors examined whether DR6 regulates neuronal death following trophic factor deprivation in vitro, focusing on three sets of spinal neurons: commissural, motor and sensory.
They found that DR6 receptor is widely expressed by neurons as they differentiate and become pro-apoptotic, linking both passive and active degeneration mechanisms. Following trophic deprivation, DR6 triggers neuronal cell body and axon degeneration, requiring both Bax (an effector in the intrinsic apoptotic pathway) and caspase-3. DR6 is activated by a prodegenerative ligand(s) that is surface-tethered but released in active form upon trophic deprivation.
The authors discovered that DR6 regulates both axonal pruning and neuronal death both in vitro and in vivo. They performed small interfering RNA (siRNA) knock-down of DR6 and discovered that it protected commissural neurons from degeneration. They also screened monoclonal antibodies to DR6 to check their ability to mimic the protection. By selecting anti-DR6 they discovered that it inhibited the degeneration, mimicking DR6 knock-down. Trophic factor deprivation resulted in massive cell death and axonal degeneration which were inhibited with antiDR6, with this antibody being function-blocking. They also examined DR6 function in cell death in vivo and found that antagonizing DR6 delays death of multiple neuronal populations both in vitro and in vivo.
They speculated that because DR6 is expressed not only by cell bodies but also axons, the protection of axons by inhibition of DR6 might reflect a direct role for DR6 in axons. By performing both in vitro and in vivo experiments they discovered DR6 regulates axonal pruning in both and blocking DR6 function delays pruning. Because DR6 regulates cell body apoptosis together with axonal degeneration, the authors have also wondered whether there is an apoptotic pathway involvement in axons and they found that Bax is required in axons as local sensory axon degeneration was blocked by genetic deletion of Bax or by local addition of Bax inhibitor.
They found that caspase-3 mediates the cell body but not axon degeneration. Whilst pro-caspase-3 was highly enriched in cell bodies, pro-capase-6 was present in both cell bodies and axons. When they caspase-6 inhibitor blocked degeneration of sensory, motor and commissural axons they have decided to confirm their predictions with RNA interference in sensory and commissural neurons and found that caspase-3 knock-down protected cell bodies significantly but had only a minor protective effect on axons, whilst caspase-6 knock down protected axons significantly with minor effect on cell bodies. They therefore came to the conclusion that distinct caspases mediate both cell body and axon degeneration.
Because DR6 is a receptor like protein, the authors have wondered whether its activation is ligand dependent. Although they were not sure if a ligand was necessary for this process, they speculated that if this was the case, the DR6 ectodomain could be capable of binding the ligand and blocking its action. They incubated neurons with soluble DR6 ectodomain construct and noticed that it prevented degeneration following trophic factor deprivation, suggesting that a ligand was necessary for the process. The authors considered the APP might activate DR6 because AD is marked by neuron and axon degeneration and they had previously found APP to be highly expressed by developing neurons and especially axons. Other APP properties which made it a candidate for a ligand were the fact that its ectodomain can be shed in a regulated fashion and APP is tied to degeneration through its links to Alzheimer' disease. To search for DR6 binding sites on axons and in conditioned medium they fused DR6 ectodomain with alkaline phosphatase which was visualized by AP biochemistry. The authors have shown that the extacellular fragment of APP is indeed a ligand for DR6 and DR6 ligand(s) on neurons is shed in response to trophic deprivation.
They also found that N-APP is not only necessary but also sufficient for degeneration with the use of loss of function studies. They found that the degeneration of sensory and commissural neurons by trophic factor deprivation was inhibited by anti-N-APP(poly) which also inhibited death of sensory cell bodies without affecting loss of surface APP following trophic deprivation. They also found that trophic factor deprivation triggers shedding of surface APP in a beta-secretase (BACE)-dependent manner. BACE inhibitors impaired degeneration of sensory axons, cell bodies and commissural axons following trophic deprivation. Trophic factors inhibit signaling downstream of DR6.
The authors have also noticed that physiological degeneration does not appear to involve AÎ² toxicity. Knowing that BACE cleavage of APP is followed by gamma secretase cleavage, yelding AÎ² peptides and that AÎ² peptides can be neurotoxic, they examined whether they contribute to degeneration and found that synthetic AÎ²1-42 trigerred degeneration.
Nicolaev et al. findings showed that extracellular fragment of APP is indeed a ligand for DR6 and it triggers a widespread self destruction program that relies on caspases and that while caspase-3 is involved in cell death of the neuron body, its caspase-6 that is required for axon degeneration and that this is triggered when relatively unknown part of APP (N-APP) binds to DR6. They proposed that the embryonic pruning mechanism might be abnormally activated in AD, believing that this could be involved in either initiating or helping the progression of this illness either alone or with other mechanisms such as amyloid-beta toxicity. This mechanistic approach provides new evidence into APP involvement in neurodegeneration. This discovery also raises the possibility that similar events occur in mature neurons in the brain. Because the APP is involved in the above mentioned self-destruction mechanism, perhaps it could contribute to AD. This is a new discovery, shedding light on yet another possible mechanism involved in the pathology of AD and opens new ways to interpret AD pathogenesis at the molecular level.
The authors showed that DR6 regulates neuronal death and axonal pruning not only in vitro but also in vivo embryonic mouse models. They showed that DR6 plays a key role in the process of activating an apoptotic pathway in commissural, motor and sensory neurons, dependent on activation of caspase 6 in axons and caspase 3 in cell bodies. The article provides compelling evidence that DR6 mediates axonal pruning and degeneration induced by trophic-factor withdrawal in developing neurons, and that the extracellular sequences of APP bind to DR6 with high affinity and specificity and that the activation of DR6 by N-APP triggers neuronal degeneration via distinct caspases.
Although the experiment seems very convincing and there is a lot of complementary, biochemical and cell biological data relevant for APP biology, it seems too early to consider this theory to be equivalent to amyloid hypothesis as there is no human or clinical data available yet with the experiment only performed on mice. Although the relevance of the experiment to Alzheimer's dementia is suspected, this has not yet been sufficiently proven. It is known that during embryo development many more neurons are formed in the brain than needed and those that not make proper connections are removed by a process involving axonal pruning and cell death. The article provides a significant scientific contribution into neuroscience, however, the physiological significance of the findings by Nicolaev et. al needs to be further established.
The discovery could potentially explain why only specific neurons are targeted in the disease despite widespread expression of the APP in the Central Nervous System. The authors have shown that DR6 expression in the mature brain is enriched in hippocampus and forebrain cholinergic neurons, sites of known AD pathology. This suggests that under certain conditions, shedding of the APP ectodomain might trigger a self-desctruction pathway, contributing to neurodegeneration in these neurons.
Although the study relied on in vitro (lab cultures) and in vivo (using mutant mouse models) tests only with no human clinical applications, this discovery could potentially offer new targets for therapy and development, perhaps by trying to prevent the initiation or progression of the disease by blocking the portion of APP molecule or others downstream signaling mechanisms that has been proposed.
The findings that APP/DR6 activation requires BACE but not Î±-secretase cleavage, that BACE processing is followed by other cleavage events, and that this pathway is AÎ² independent are interesting and leave many open questions. Although the study addresses a developmental function, it could offer novel insights into pathogenesis and therapeutic intervention as APP/DR6/caspase 6 are expressed in both developing neurons and adult brains.
However, a question remains here on how this discovery relates to tau hypothesis, how this model would lead to tau hyperphosphorylation, NFT formation and the disease considered by many researchers as tauopathy. However, it was suggested by the scientists that activation of the caspase-6 occuring downstream of N-APP/DR6 binding might tie APP to the pathology of tau.
Although this discovery provides a good foundation to N-APP involvement in neurodegeneration, much more work is to be done to corroborate these results and explore the mechanisms in greater detail and how this is applicable to AD development in human AD brains. Perhaps mutations that might be associated with those proposed new players and its association with risk factors in AD might need consideration. Also trying to develop potential therapeutic candidates to interfere with the steps of proposed pathways should be sought.
Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory (Shankar et al. 2008). Another step towards establishing the cause of AD.
AÎ² in culture cells and mouse brains has been shown to be neurotoxic. However, the results of experiments from the in vitro-culture cells and in vivo-animal models cannot be directly compared to human AD cases and events occurring in human brains can only be predicted but not confirmed. So, how about the human brain? Are AÎ² peptides indeed toxic in human AD brains as predicted?
In 2008 a group of scientists took a critical step towards establishing the cause of Alzheimer's Disease. Shankar and colleagues have isolated soluble AÎ² oligomers from cerebral cortex brain tissue of human subjects at autopsy with clinical and neuropathological diagnosis of AD. Aqueously soluble (Tris-buffered saline (TBS)), detergent-soluble and "insoluble" extracts were prepared by sequential centrifugation of brain homogenates from humans with neuropathologically confirmed dementia. Sensitive immunoprecipitation/Western blotting revealed AÎ² monomers and lithium dodecylsulfate (LDS)-stable dimmers and trimers in all three extracts, whilst extracts from some non-AD subject showed models levels of AÎ² in the insoluble extracts but little/none in the soluble (TBS) extracts in comparison to AD cases. The authors were astonished by the discovery of AÎ² in the insoluble but not soluble fraction of a particular subject with AD histopathology but no clinical features of AD.
Although the AÎ² was detectable an all three extracts, the researchers have focused on the TBS-soluble fraction due to AD clinical features strongly correlating with soluble levels of AÎ², with a particular attention on the earliest AÎ² assemblies: soluble oligomers that form initially from monomers.
The scientists started with extracts of soluble AÎ² isolated from mouse hippocampus and questioned if they altered long term potentiation (LTP). They found that the control and cortex AD extracts (TBS) did not alter basal synaptic transmission or paired-pulse ratio, indicating that neurotransmitter release was unaffected. They found that the AD TBS inhibited LTP, whilst immunodepletion of AD TBS with AÎ² antiserum prevented LTP inhibition. The results suggest that the soluble AÎ² is the necessary player in inhibition of the long term potentiation.
LTD of hyppocampal synapses is known to be induced by repetitive stimulation. Authors induced LTD with AD TBS and found that it was NMDAR-independent. mGluR activation was necessary for the LTD facilitation by soluble AÎ², whilst SIB1757 did not prevent AD TBS-mediated LTP inhibition.
In human moloclonal AÎ² antibody was passively administered and found that it was able to block LTD. Antibodies to the free N-terminus of AÎ² almost completely precipitated soluble AÎ² from AD TBS and also prevented the LTD facilitation, whilst antibodies to the AÎ² C-termini weakly precipitated AÎ² and did not block the LTD effect. AÎ² mid region antibodies of the AD TBS only partially blocked the effect. Also the N-terminal but not C-terminal antibodies neutralized the LTP deficit.
The scientist then moved on to assessments of the effect of soluble AD cortical extracts directly on memory function. They used rats in this part of the study and trained them on a step-through passive avoidance task. When they injected AD TBS/immunodepleted AD TBS (AD TBS-ID) into the lateral ventricle, they found that AD TBS administered 3 hours post-training significantly impaired the animal's recall of the learned behaviour 48 hours later.
Decreased synapse density is the strongest neuropathological correlate of the degree of dementia in AD (Terry et al. 1991) To determine whether soluble AÎ² in AD brain contributes directly to synapse loss, the authors quantified dendritic spine density in GFP-transfected pyramidal cells in organotypic rat hippocampal slices. In order to reconstitute brain extract in slice culture medium, TBS extracts underwent non-denaturating size exclusion chromatography (SEC). Pyramidal neurons in slices were cultured for 10 days with plain medium or medium reconstituted with lyophilized SEC fractions of Control TBS and SEC fractions from AD TBS and found that only AD TBS caused a significant decrease in spinal density almost by a half in comparison to control. Whilst metabolic glutamate receptor antagonist did not prevent the loss of spines in AD TBS fraction, the NMDAR antagonist prevented the decrease in spine density in AD TBS fraction. These findings support prior evidence that NMDAR activation is necessary for AÎ² spine loss.
The authors have wondered which soluble AÎ² species was responsible for the synapse AD physiology. With the use of LDS-PAGE gels, mass spectrophotometry and IP and found that AÎ² dimers were responsible for the effect on LTP. The also aimed to established which soluble AÎ² was responsible for the impaired synaptic plasticity and found hat soluble AÎ² dimmers inhibited LTP.
They have also wondered whether amyloid cores isolated from AD cortex can inhibit hippocampal LTP. They isolated fibrillar AÎ² from neuritic plaques and found that in physiologic buffer amyloid cores did not acutely release soluble AÎ² dimers to alter synaptic plasticity and that highly insoluble AÎ² aggregates such as amyloid plaque cores represent dimer-rich structures that do not readily associate.
The authors effectively showed that soluble AÎ² isolated directly from AD brains is able to decrease dendritic spine density, inhibit LTP and facilitate LTD in hippocampus and interfere with the memory of a learnt behaviour in rats. The authors have found that neither AÎ² monomers nor insoluble amyloid plaque cores significantly altered synaptic plasticity opposing to AÎ² dimers which were shown to contribute directly to synapse disfunction in AD patients. Soluble AÎ² dimers from AD subjects were shown to induce their effects by perturbing glutameric synaptic transmission.
These various effects could particularly be attributed to AÎ² dimmers. Metabotropic glutamate receptors (mGluR) were found to be required for LTD enhancement whilst the NMDA receptors (NMDAR) for spine loss, however these receptors are unlikely to be the sole effector targets of soluble AÎ² oligomers. Antibodies co-administration to the AÎ² N-terminus prevented the LTP and LTD deficits, whereas antibodies to the mid-region or C-terminus were less effective. Insoluble amyloid plaque cores from AD cortex did not impair LTP unless they were first solubilized to release AÎ² dimers, suggesting that plaque cores are largely inactive but sequester AÎ² dimers that are synaptotoxic.
Soluble dimers were shown to be the smallest synaptotoxic species, potently impairing synapse structure and function whilst insoluble amyloid plaques were noted to be inactive, supporting the hypothesis that plaques are not neurotoxic agents. The authors have tried to minimize the bias of their experiment, however they could not rule out the possibility that a small molecule might be attached to the dimer and be responsible for the toxicity. However, its worth noticing they have shown that synthetic dimers (made by oxidizing monomers with cysteine substitutions for serine at position 26) were similarly toxic, showing that dimers alone could impair synaptic function. The work by Shankar and colleagues significantly strengthen the hypothesis that amyloid-beta oligomers are responsible for cognitive impairment in Alzheimer's disease being able to distrupt synaptic function.
Shankar et al. is another important step towards understanding the neuropathology of AÎ². Scientists observed that AÎ² oligomers were neurotoxic not in cell culture, test tube or animal modes but indeed in human subjects. Although the specific molecular initiators of AD process still remain unknown, biochemical studies indicate that the severity of cognitive impairment in AD correlates strongly with the cortical levels of soluble AÎ² rather than insoluble amyloid plaques. Understanding how AÎ² impairs hyppocampal synaptic function at the molecular level could enable the development of specific neuroprotective therapies for AD.
Mechanistic approach of neuronal degeneration proposed by Ittner et al. Dendritic Function of Tau Mediates Amyloid-beta toxicity in Alzheimer's Disease Mouse Models.
Although it is known that both proteins are involved in AD, the connection between the two respective proteins AÎ² and tau has remained mysterious over years. The study by Ittner et al. reveals yet another molecular mechanism significantly contributing to understanding the pathology of AD. In this study, critical finding of molecular mechanism that links tau to the AÎ² toxicity at the synapse have been emerged. The results of the study suggest that the mechanism of AÎ² toxicity is tau dependant. This might be the discovery of the missing link between these two proteins that has puzzled the scientists over years. The researches show the previously unknown function of tau in the dendrite.
Within the last decade, researches have shown that AÎ² can worsen tau pathology, therefore concluded that it must act upstream of tau. (Gotz, 2001 and Lewis, 2001). In both people, and animal modes AÎ² is known to have excitotoxic effects. However, it was not clear how tau mediated excitotoxicity.
Ittner et. al. based their experiments on previous tau related discoveries and the fact that tau protein contains binding site for kinase Fyn, interacting via its aminoterminal projection domain,Lee etÂ al., 1998 G. Lee, S.T. Newman, D.L. Gard, H. Band and G. Panchamoorthy, Tau interacts with src-family non-receptor tyrosine kinases, J. Cell Sci. 111 (1998), pp. 3167-3177. View Record in Scopus Cited By in Scopus (14 phosporylating NR subunit 2 (NR2) to facilitate interaction of the NR complex at the postsynaptic density protein 95 (PSD-95) linking NR to synaptic excitotoxic downstream signaling. Reduction of Fyn in APP transgenic mice prevents AÎ² toxicity, while overexpression enhances it.
Ittner et al in its study address how tau confers AÎ² toxicity. Researchers have generated transgenic mouse models (Î”tau74) expressing only the amino-terminal projection domain (PD) of tau and crossed them with AÎ²-forming APP23 and tauâˆ’/âˆ’ mice.
Ittner et al. emerged critical finding that AÎ² toxicity is tau dependant. Using tau mutant mice, it was found that tau has dendritic function in postsynaptic targeting of the tyrosine specific phospho-transferase Fyn. Fyn can phosphorylate the NMDA receptor subunit NR2B, promoting interaction with the scaffolding protein PSD95. This interaction appears important for AÎ²-induced neurotoxicity. In mutant mice with reduced dendritic Fyn expression there is uncoupling of the NR2B/PSD95 interaction and reduced AÎ² toxicity.
When tau is deleted or mistargeted in AD mouse model, survival and memory improve to those of wild-type levels, although plaque burden of AÎ² levels do not change. The authors have shown that dendritic localization of Fyn is Tau-dependent. They have shown that Tau associated with PSD complex by using coIP, PSD purification and immunochemistry with enhanced antigen retrieval. Scientists showed that the additional role of tau in dendrites becomes pivotal in AD, particularly in mediating early AÎ² toxicity.
Using tau mutant mice, it was found that tau has dendritic function in postsynaptic targeting of the tyrosine specific phospho-transferase Fyn. Fyn can phosphorylate the NMDA receptor subunit NR2B, promoting interaction with the scaffolding protein PSD95. This interaction appears important for AÎ²-induced neurotoxicity. In mutant mice with reduced dendritic Fyn expression there is uncoupling of the NR2B/PSD95 interaction and reduced AÎ² toxicity.
The dendritic role of tau that confers AÎ² toxicity at the postsynapse might have direct implications for pathogenesis and treatment of AD, suggesting several therapeutic possibilities and tau becoming an attractive drug target for AD therapy.
Ittner et al showed that reduction of tau levels can improve symptoms in AD mouse models, indicating a possibility of it being beneficial in human. Distruption in the NR2b-PSF-95 interaction or with tau projection domain should be considered when looking at possible therapies because it was shown that these are able to weaken excitotoxicity without interfering with synaptic transmission in mouse models. Scientists therefore propose that reduction of tau levels or targeting tau-dependant mechanisms would be the suitable strategies in treatment of AD.
However, again so far this discovery has been achieved with the use of mouse models and not human subjects, suggesting that there is a possibility however no guarantee that exactly the same process occurs in human brain.
It is crucial to note that the specific connection between AÎ² and tau revealed by this and previous studies significantly contribute to the progress in neuroscience and therefore deserves and should be explored further.
In the last two decades, a vast amount of research has focused on the structure and metabolism of AÎ² with much less attention being directed into investigation of the structure and function of APP and complexity of the interactions between the molecule and others including Tau.
The scientists are now certain that APP is involved in development of AD, therefore in my opinion more studies should be aimed at understanding the molecular mechanisms in which APP plays a part. Further studies into the APP molecule structure and function need to be done in order to understand the initiation and progression of AD as well as to develop effective drug therapies. Understanding the molecular pathways of Tau involvement however cannot be abandoned, particularly in view of the recent discoveries pointing at tau and the reduction of its levels being able to improve the symptoms seen in AD mouse models.
In my opinion the study by Shankar et al. deserves a particular attention. The group of scientists have made a significant contribution into understanding the Alzheimer's Disease process by studying AÎ² involvement its development not just in a cell culture or with the use of animal models but on human subjects. This study strengthens the hypothesis of amyloid-beta oligomers being responsible for cognitive impairment in AD, able to disrupt synaptic function.
Nicolaev et al. is an excellent study, explaining the novel role for APP in neurodegeneration. The group of scientists have revealed that signaling of the APP through DR6 is central to the developmental selection process of the nervous system, where excess neurons and axons are eliminated to refine neuronal connections, with the finding having a potential relevance to neurodegenerative disease. However, although the study is very convincing, there is still no human data available and the study has been done with the use of mice in their midgestation period where many neurons undergo a selection process where neurons and axons are known to be eliminated to refine neuronal connections. It is possible that this mechanism is hijacked in AD, however, more studies need to be done to ascertain the relevance of this discovery to the development of the disease.
The study by Ittner et al. reveals although previously suspected, however, not fully ascertained connection between AÎ² and tau that definitely deserves an appreciation.
Although they are different discoveries in relation to Alzheimer's Disease have been made by three different groups of scientists, all of them deserve a praise. It is difficult to ascertain whether any of those newly invented pathways are right or wrong. However, I all studies have been well designed and possible bias eliminated. There is still a possibility that all the discovered pathways are the pieces of puzzle and combined together could explain the neurodegenerative process of AD. Nicolaev et al. proposed that the embryonic pruning mechanism involving N-APP binding to DR6, might be abnormally activated in AD and contribute to its initiation or progression, either alone or together with other mechanisms such as AÎ² toxicity. This suggestion links Nicolaev et al. study to Shankar et al. and the finding of the soluble AÎ² neurotoxic oligomers being molecular players in the disease. It is also possible to link the study by Ittner et al. to both discoveries. The authors revealed what have appeared to be the missing link between the two mysterious players and tied tau to the AÎ² toxicity with AÎ² toxicity being tau dependant.
I believe that each of above mentioned studies bring us a step closer in understanding the process of this devastating disease. They all in their own way contribute to a progress in neuroscience. Although more experimental work need to be done in relation to the newly discovered pathways, they all provide a significant foundation and point at possible therapeutic targets that deserve and should be explored further.