On the Inadequacy of the Current Transgenic Animal Models of Alzheimer's Disease: The Path Forward.

For at least two reasons, the current transgenic animal models of Alzheimer's disease (AD) appear to be patently inadequate. They may be useful in many respects, the AD models; however, they are not. First, they are incapable of developing the full spectrum of the AD pathology. Second, they respond spectacularly well to drugs that are completely ineffective in the treatment of symptomatic AD. These observations indicate that both the transgenic animal models and the drugs faithfully reflect the theory that guided the design and development of both, the amyloid cascade hypothesis (ACH), and that both are inadequate because their underlying theory is. This conclusion necessitated the formulation of a new, all-encompassing theory of conventional AD-the ACH2.0. The two principal attributes of the ACH2.0 are the following. One, in conventional AD, the agent that causes the disease and drives its pathology is the intraneuronal amyloid-ß (iAß) produced in two distinctly different pathways. Two, following the commencement of AD, the bulk of Aß is generated independently of Aß protein precursor (AßPP) and is retained inside the neuron as iAß. Within the framework of the ACH2.0, AßPP-derived iAß accumulates physiologically in a lifelong process. It cannot reach levels required to support the progression of AD; it does, however, cause the disease. Indeed, conventional AD occurs if and when the levels of AßPP-derived iAß cross the critical threshold, elicit the neuronal integrated stress response (ISR), and trigger the activation of the AßPP-independent iAß generation pathway; the disease commences only when this pathway is operational. The iAß produced in this pathway reaches levels sufficient to drive the AD pathology; it also propagates its own production and thus sustains the activity of the pathway and perpetuates its operation. The present study analyzes the reason underlying the evident inadequacy of the current transgenic animal models of AD. It concludes that they model, in fact, not Alzheimer's disease but rather the effects of the neuronal ISR sustained by AßPP-derived iAß, that this is due to the lack of the operational AßPP-independent iAß production pathway, and that this mechanism must be incorporated into any successful AD model faithfully emulating the disease. The study dissects the plausible molecular mechanisms of the AßPP-independent iAß production and the pathways leading to their activation, and introduces the concept of conventional versus unconventional Alzheimer's disease. It also proposes the path forward, posits the principles of design of productive transgenic animal models of the disease, and describes the molecular details of their construction.

ACH2.0/E, the Consolidated Theory of Conventional and Unconventional Alzheimer's Disease: Origins, Progression, and Therapeutic Strategies.

The centrality of amyloid-beta (Aß) is an indisputable tenet of Alzheimer's disease (AD). It was initially indicated by the detection (1991) of a mutation within Aß protein precursor (AßPP) segregating with the disease, which served as a basis for the long-standing Amyloid Cascade Hypothesis (ACH) theory of AD. In the intervening three decades, this notion was affirmed and substantiated by the discovery of numerous AD-causing and AD-protective mutations with all, without an exception, affecting the structure, production, and intraneuronal degradation of Aß. The ACH postulated that the disease is caused and driven by extracellular Aß. When it became clear that this is not the case, and the ACH was largely discredited, a new theory of AD, dubbed ACH2.0 to re-emphasize the centrality of Aß, was formulated. In the ACH2.0, AD is caused by physiologically accumulated intraneuronal Aß (iAß) derived from AßPP. Upon reaching the critical threshold, it triggers activation of the autonomous AßPP-independent iAß generation pathway; its output is retained intraneuronally and drives the AD pathology. The bridge between iAß derived from AßPP and that generated independently of AßPP is the neuronal integrated stress response (ISR) elicited by the former. The ISR severely suppresses cellular protein synthesis; concurrently, it activates the production of a small subset of proteins, which apparently includes components necessary for operation of the AßPP-independent iAß generation pathway that are absent under regular circumstances. The above sequence of events defines "conventional" AD, which is both caused and driven by differentially derived iAß. Since the ISR can be elicited by a multitude of stressors, the logic of the ACH2.0 mandates that another class of AD, referred to as "unconventional", has to occur. Unconventional AD is defined as a disease where a stressor distinct from AßPP-derived iAß elicits the neuronal ISR. Thus, the essence of both, conventional and unconventional, forms of AD is one and the same, namely autonomous, self-sustainable, AßPP-independent production of iAß. What distinguishes them is the manner of activation of this pathway, i.e., the mode of causation of the disease. In unconventional AD, processes occurring at locations as distant from and seemingly as unrelated to the brain as, say, the knee can potentially trigger the disease. The present study asserts that these processes include traumatic brain injury (TBI), chronic traumatic encephalopathy, viral and bacterial infections, and a wide array of inflammatory conditions. It considers the pathways which are common to all these occurrences and culminate in the elicitation of the neuronal ISR, analyzes the dynamics of conventional versus unconventional AD, shows how the former can morph into the latter, explains how a single TBI can hasten the occurrence of AD and why it takes multiple TBIs to trigger the disease, and proposes the appropriate therapeutic strategies. It posits that yet another class of unconventional AD may occur where the autonomous AßPP-independent iAß production pathway is initiated by an ISR-unrelated activator, and consolidates the above notions in a theory of AD, designated ACH2.0/E (for expanded ACH2.0), which incorporates the ACH2.0 as its special case and retains the centrality of iAß produced independently of AßPP as the driving agent of the disease.

Early intraneuronal amyloid triggers neuron-derived inflammatory signaling in APP transgenic rats and human brain.

Chronic inflammation during Alzheimer's disease (AD) is most often attributed to sustained microglial activation in response to amyloid-ß (Aß) plaque deposits and cell death. However, cytokine release and microgliosis are consistently observed in AD transgenic animal models devoid of such pathologies, bringing into question the underlying processes that may be at play during the earliest AD-related immune response. We propose that this plaque-independent inflammatory reaction originates from neurons burdened with increasing levels of soluble and oligomeric Aß, which are known to be the most toxic amyloid species within the brain. Laser microdissected neurons extracted from preplaque amyloid precursor protein (APP) transgenic rats were found to produce a variety of potent immune factors, both at the transcript and protein levels. Neuron-derived cytokines correlated with the extent of microglial activation and mobilization, even in the absence of extracellular plaques and cell death. Importantly, we identified an inflammatory profile unique to Aß-burdened neurons, since neighboring glial cells did not express similar molecules. Moreover, we demonstrate within disease-vulnerable regions of the human brain that a neuron-specific inflammatory response may precede insoluble Aß plaque and tau tangle formation. Thus, we reveal the Aß-burdened neuron as a primary proinflammatory agent, implicating the intraneuronal accumulation of Aß as a significant immunological component in the AD pathogenesis.

The Amyloid Cascade Hypothesis 2.0 for Alzheimer's Disease and Aging-Associated Cognitive Decline: From Molecular Basis to Effective Therapy.

With the long-standing amyloid cascade hypothesis (ACH) largely discredited, there is an acute need for a new all-encompassing interpretation of Alzheimer's disease (AD). Whereas such a recently proposed theory of AD is designated ACH2.0, its commonality with the ACH is limited to the recognition of the centrality of amyloid-ß (Aß) in the disease, necessitated by the observation that all AD-causing mutations affect, in one way or another, Aß. Yet, even this narrow commonality is superficial since AD-causing Aß of the ACH differs distinctly from that specified in the ACH2.0: Whereas in the former, the disease is caused by secreted extracellular Aß, in the latter, it is triggered by Aß-protein-precursor (AßPP)-derived intraneuronal Aß (iAß) and driven by iAß generated independently of AßPP. The ACH2.0 envisions AD as a two-stage disorder. The first, asymptomatic stage is a decades-long accumulation of AßPP-derived iAß, which occurs via internalization of secreted Aß and through intracellular retention of a fraction of Aß produced by AßPP proteolysis. When AßPP-derived iAß reaches critical levels, it activates a self-perpetuating AßPP-independent production of iAß that drives the second, devastating AD stage, a cascade that includes tau pathology and culminates in neuronal loss. The present study analyzes the dynamics of iAß accumulation in health and disease and concludes that it is the prime factor driving both AD and aging-associated cognitive decline (AACD). It discusses mechanisms potentially involved in AßPP-independent generation of iAß, provides mechanistic interpretations for all principal aspects of AD and AACD including the protective effect of the Icelandic AßPP mutation, the early onset of FAD and the sequential manifestation of AD pathology in defined regions of the affected brain, and explains why current mouse AD models are neither adequate nor suitable. It posits that while drugs affecting the accumulation of AßPP-derived iAß can be effective only protectively for AD, the targeted degradation of iAß is the best therapeutic strategy for both prevention and effective treatment of AD and AACD. It also proposes potential iAß-degrading drugs.

Next Generation Therapeutic Strategy for Treatment and Prevention of Alzheimer's Disease and Aging-Associated Cognitive Decline: Transient, Once-in-a-Lifetime-Only Depletion of Intraneuronal Aß (iAß) by Its Targeted Degradation via Augmentation of Intra-iAß-Cleaving Activities of BACE1 and/or BACE2.

Although the long-standing Amyloid Cascade Hypothesis (ACH) has been largely discredited, its main attribute, the centrality of amyloid-beta (Aß) in Alzheimer's disease (AD), remains the cornerstone of any potential interpretation of the disease: All known AD-causing mutations, without a single exception, affect, in one way or another, Aß. The ACH2.0, a recently introduced theory of AD, preserves this attribute but otherwise differs fundamentally from the ACH. It posits that AD is a two-stage disorder where both stages are driven by intraneuronal (rather than extracellular) Aß (iAß) albeit of two distinctly different origins. The first asymptomatic stage is the decades-long accumulation of Aß protein precursor (AßPP)-derived iAß to the critical threshold. This triggers the activation of the self-sustaining AßPP-independent iAß production pathway and the commencement of the second, symptomatic AD stage. Importantly, Aß produced independently of AßPP is retained intraneuronally. It drives the AD pathology and perpetuates the operation of the pathway; continuous cycles of the iAß-stimulated propagation of its own AßPP-independent production constitute an engine that drives AD, the AD Engine. It appears that the dynamics of AßPP-derived iAß accumulation is the determining factor that either drives Aging-Associated Cognitive Decline (AACD) and triggers AD or confers the resistance to both. Within the ACH2.0 framework, the ACH-based drugs, designed to lower levels of extracellular Aß, could be applicable in the prevention of AD and treatment of AACD because they reduce the rate of accumulation of AßPP-derived iAß. The present study analyzes their utility and concludes that it is severely limited. Indeed, their short-term employment is ineffective, their long-term engagement is highly problematic, their implementation at the symptomatic stages of AD is futile, and their evaluation in conventional clinical trials for the prevention of AD is impractical at best, impossible at worst, and misleading in between. In contrast, the ACH2.0-guided Next Generation Therapeutic Strategy for the treatment and prevention of both AD and AACD, namely the depletion of iAß via its transient, short-duration, targeted degradation by the novel ACH2.0-based drugs, has none of the shortcomings of the ACH-based drugs. It is potentially highly effective, easily evaluable in clinical trials, and opens up the possibility of once-in-a-lifetime-only therapeutic intervention for prevention and treatment of both conditions. It also identifies two plausible ACH2.0-based drugs: activators of physiologically occurring intra-iAß-cleaving capabilities of BACE1 and/or BACE2.

Neuron Loss in Alzheimer's Disease: Translation in Transgenic Mouse Models.

Transgenic mouse models represent an essential tool for the exploration of Alzheimer's disease (AD) pathological mechanisms and the development of novel treatments, which at present provide only symptomatic and transient effects. While a variety of mouse models successfully reflects the main neuropathological hallmarks of AD, such as extracellular amyloid-ß (Aß) deposits, intracellular accumulation of Tau protein, the development of micro- and astrogliosis, as well as behavioral deficits, substantial neuron loss, as a key feature of the disease, seems to be more difficult to achieve. In this review, we summarize information on classic and more recent transgenic mouse models for AD, focusing in particular on loss of pyramidal, inter-, and cholinergic neurons. Although the cause of neuron loss in AD is still a matter of scientific debate, it seems to be linked to intraneuronal Aß accumulation in several transgenic mouse models, especially in pyramidal neurons.

TOM1 Regulates Neuronal Accumulation of Amyloid-ß Oligomers by FcγRIIb2 Variant in Alzheimer's Disease.

Emerging evidences suggest that intraneuronal Aß correlates with the onset of Alzheimer's disease (AD) and highly contributes to neurodegeneration. However, critical mediator responsible for Aß uptake in AD pathology needs to be clarified. Here, we report that FcγRIIb2, a variant of Fcγ-receptor IIb (FcγRIIb), functions in neuronal uptake of pathogenic Aß. Cellular accumulation of oligomeric Aß1-42, not monomeric Aß1-42 or oligomeric Aß1-40, was blocked by Fcgr2b knock-out in neurons and partially in astrocytes. Aß1-42 internalization was FcγRIIb2 di-leucine motif-dependent and attenuated by TOM1, a FcγRIIb2-binding protein that repressed the receptor recycling. TOM1 expression was downregulated in the hippocampus of male 3xTg-AD mice and AD patients, and regulated by miR-126-3p in neuronal cells after exposure to Aß1-42 In addition, memory impairments in male 3xTg-AD mice were rescued by the lentiviral administration of TOM1 gene. Augmented Aß uptake into lysosome caused its accumulation in cytoplasm and mitochondria. Moreover, neuronal accumulation of Aß in both sexes of 3xTg-AD mice and memory deficits in male 3xTg-AD mice were ameliorated by forebrain-specific expression of Aß-uptake-defective Fcgr2b mutant. Our findings suggest that FcγRIIb2 is essential for neuropathic uptake of Aß in AD.SIGNIFICANCE STATEMENT Accumulating evidences suggest that intraneuronal Aß is found in the early step of AD brain and is implicated in the pathogenesis of AD. However, the critical mediator involved in these processes is uncertain. Here, we describe that the FcγRIIb2 variant is responsible for both neuronal uptake and intraneuronal distribution of pathogenic Aß linked to memory deficits in AD mice, showing a pathologic significance of the internalized Aß. Further, Aß internalization is attenuated by TOM1, a novel FcγRIIb2-binding protein. Together, we provide a molecular mechanism responsible for neuronal uptake of pathogenic Aß found in AD.

Evidence of intraneuronal Aß accumulation preceding tau pathology in the entorhinal cortex.

Growing evidence gathered from transgenic animal models of Alzheimer's disease (AD) indicates that the intraneuronal accumulation of amyloid-ß (Aß) peptides is an early event in the AD pathogenesis, producing cognitive deficits before the deposition of insoluble plaques. Levels of soluble Aß are also a strong indicator of synaptic deficits and concurrent AD neuropathologies in post-mortem AD brain; however, it remains poorly understood how this soluble amyloid pool builds within the brain in the decades leading up to diagnosis, when a patient is likely most amenable to early therapeutic interventions. Indeed, characterizing early intracellular Aß accumulation in humans has been hampered by the lack of Aß-specific antibodies, variability in the quality of available human brain tissue and the limitations of conventional microscopy. We therefore sought to investigate the development of the intraneuronal Aß pathology using extremely high-quality post-mortem brain material obtained from a cohort of non-demented subjects with short post-mortem intervals and processed by perfusion-fixation. Using well-characterized monoclonal antibodies, we demonstrate that the age-dependent intraneuronal accumulation of soluble Aß is pervasive throughout the entorhinal cortex and hippocampus, and that this phase of the amyloid pathology becomes established within AD-vulnerable regions before the deposition of Aß plaques and the formation of tau neurofibrillary tangles. We also show for the first time in post-mortem human brain that Aß oligomers do in fact accumulate intraneuronally, before the formation of extracellular plaques. Finally, we validated the origin of the Aß-immunopositive pool by resolving Aß- and APP/CTF-immunoreactive sites using super resolution structured illumination microscopy. Together, these findings indicate that the lifelong accrual of intraneuronal Aß may be a potential trigger for downstream AD-related pathogenic events in early disease stages.

A Bifunctional Anti-Amyloid Blocks Oxidative Stress and the Accumulation of Intraneuronal Amyloid-Beta.

There is growing recognition regarding the role of intracellular amyloid beta (Aß) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aß likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aß and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aß accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aß. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aß. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aß-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aß targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aß, its promotion of ROS, and Aß metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aß oligomer formation.

Senescence-related impairment of autophagy induces toxic intraneuronal amyloid-ß accumulation in a mouse model of amyloid pathology.

Aging is the main risk factor for Alzheimer's disease (AD) and other neurodegenerative pathologies, but the molecular and cellular changes underlying pathological aging of the nervous system are poorly understood. AD pathology seems to correlate with the appearance of cells that become senescent due to the progressive accumulation of cellular insults causing DNA damage. Senescence has also been shown to reduce the autophagic flux, a mechanism involved in clearing damaged proteins from the cell, and such impairment has been linked to AD pathogenesis. In this study, we investigated the role of cellular senescence on AD pathology by crossing a mouse model of AD-like amyloid-ß (Aß) pathology (5xFAD) with a mouse model of senescence that is genetically deficient for the RNA component of the telomerase (Terc-/-). We studied changes in amyloid pathology, neurodegeneration, and the autophagy process in brain tissue samples and primary cultures derived from these mice by complementary biochemical and immunostaining approaches. Postmortem human brain samples were also processed to evaluate autophagy defects in AD patients. Our results show that accelerated senescence produces an early accumulation of intraneuronal Aß in the subiculum and cortical layer V of 5xFAD mice. This correlates with a reduction in amyloid plaques and Aß levels in connecting brain regions at a later disease stage. Neuronal loss was specifically observed in brain regions presenting intraneuronal Aß and was linked to telomere attrition. Our results indicate that senescence affects intraneuronal Aß accumulation by impairing autophagy function and that early autophagy defects can be found in the brains of AD patients. Together, these findings demonstrate the instrumental role of senescence in intraneuronal Aß accumulation, which represents a key event in AD pathophysiology, and emphasize the correlation between the initial stages of amyloid pathology and defects in the autophagy flux.

Effect of Lecanemab in Early Alzheimer's Disease: Mechanistic Interpretation in the Amyloid Cascade Hypothesis 2.0 Perspective.

In clinical trials, lecanemab and donanemab showed statistically significant yet marginal slowdown of Alzheimer's disease (AD)-associated cognitive decline. This could be due to their sub-optimal design and/or deployment; alternatively, their limited efficiency could be intrinsic. Distinguishing between the two is of great importance considering the acute need of efficient AD therapy and tremendous resources being invested in its pursuit. The present study analyzes the mode of operation of lecanemab and donanemab within the framework of recently proposed Amyloid Cascade Hypothesis 2.0 and concludes that the second possibility is correct. It suggests that substantial improvement of the efficiency of these drugs in symptomatic AD is unlikely and proposes the alternative therapeutic strategy.

Principles of Design of Clinical Trials for Prevention and Treatment of Alzheimer's Disease and Aging-Associated Cognitive Decline in the ACH2.0 Perspective: Potential Outcomes, Challenges, and Solutions.

With the Amyloid Cascade Hypothesis (ACH) largely discredited, the ACH2.0 theory of Alzheimer's disease (AD) has been recently introduced. Within the framework of the ACH2.0, AD is triggered by amyloid-ß protein precursor (AßPP)-derived intraneuronal Aß (iAß) and is driven by iAß produced in the AßPP-independent pathway and retained intraneuronally. In this paradigm, the depletion of extracellular Aß or suppression of Aß production by AßPP proteolysis, the two sources of AßPP-derived iAß, would be futile in symptomatic AD, due to its reliance on iAß generated independently of AßPP, but effective in preventing AD and treating Aging-Associated Cognitive Decline (AACD) driven, in the ACH2.0 framework, by AßPP-derived iAß. The observed effect of lecanemab and donanemab, interpreted in the ACH2.0 perspective, supports this notion and mandates AD-preventive clinical trials. Such trials are currently in progress. They are likely, however, to fail or to yield deceptive results if conducted conventionally. The present study considers concepts of design of clinical trials of lecanemab, donanemab, or any other drug, targeting the influx of AßPP-derived iAß, in prevention of AD and treatment of AACD. It analyzes possible outcomes and explains why selection of high-risk asymptomatic participants seems reasonable but is not. It argues that outcomes of such AD preventive trials could be grossly misleading, discusses inevitable potential problems, and proposes feasible solutions. It advocates the initial evaluation of this type of drugs in clinical trials for treatment of AACD. Whereas AD protective trials of these drugs are potentially of an impractical length, AACD clinical trials are expected to yield unequivocal results within a relatively short duration. Moreover, success of the latter, in addition to its intrinsic value, would constitute a proof of concept for the former. Furthermore, this study introduces concepts of the active versus passive iAß depletion, contends that targeted degradation of iAß is the best therapeutic strategy for both prevention and treatment of AD and AACD, proposes potential iAß-degrading drugs, and describes their feasible and unambiguous evaluation in clinical trials.

The Amyloid Cascade Hypothesis 2.0: On the Possibility of Once-in-a-Lifetime-Only Treatment for Prevention of Alzheimer's Disease and for Its Potential Cure at Symptomatic Stages.

We posit that Alzheimer's disease (AD) is driven by amyloid-ß (Aß) generated in the amyloid-ß protein precursor (AßPP) independent pathway activated by AßPP-derived Aß accumulated intraneuronally in a life-long process. This interpretation constitutes the Amyloid Cascade Hypothesis 2.0 (ACH2.0). It defines a tandem intraneuronal-Aß (iAß)-anchored cascade occurrence: intraneuronally-accumulated, AßPP-derived iAß triggers relatively benign cascade that activates the AßPP-independent iAß-generating pathway, which, in turn, initiates the second, devastating cascade that includes tau pathology and leads to neuronal loss. The entire output of the AßPP-independent iAß-generating pathway is retained intraneuronally and perpetuates the pathway's operation. This process constitutes a self-propagating, autonomous engine that drives AD and ultimately kills its host cells. Once activated, the AD Engine is self-reliant and independent from Aß production in the AßPP proteolytic pathway; operation of the former renders the latter irrelevant to the progression of AD by relegating its iAß contribution to insignificant, and brands its manipulation for therapeutic purposes, such as BACE (beta-site AßPP-cleaving enzyme) inhibition, as futile. In the proposed AD paradigm, the only valid direct therapeutic strategy is targeting the engine's components, and the most effective feasible approach appears to be the activation of BACE1 and/or of its homolog BACE2, with the aim of exploiting their Aß-cleaving activities. Such treatment would collapse the iAß population and 'reset' its levels below those required for the operation of the AD Engine. Any sufficiently selective iAß-depleting treatment would be equally effective. Remarkably, this approach opens the possibility of a short-duration, once-in-a-lifetime-only or very infrequent, preventive or curative therapy for AD; this therapy would be also effective for prevention and treatment of the 'common' pervasive aging-associated cognitive decline. The ACH2.0 clarifies all ACH-unresolved inconsistencies, explains the widespread 'resilience to AD' phenomenon, predicts occurrences of a category of AD-afflicted individuals without excessive Aß plaque load and of a novel type of familial insusceptibility to AD; it also predicts the lifespan-dependent inevitability of AD in humans if untreated preventively. The article details strategy and methodology to generate an adequate AD model and validate the hypothesis; the proposed AD model may also serve as a research and drug development platform.

Differential Effects of Alzheimer's Disease Aß40 and 42 on Endocytosis and Intraneuronal Trafficking.

Anomalous neuronal accumulation of Aß peptides was shown to affect synaptic transmission and contribute to neurodegeneration in Alzheimer's disease (AD) brain. Neuronal cells internalize amyloid beta (Aß) peptides from the brain extracellular space even under normal physiological conditions, and these endocytotic pathways go awry during AD progression. We hypothesized that exposure to toxic Aß species accumulating in AD brain contributes to perturbations in neuronal endocytosis. We have shown substantial down-regulation of KEGG endocytotic pathway genes in AD patient brain regions that accumulate Aß compared to those in non-demented individuals. While both Aß40 and Aß42 perturbed endocytosis and intracellular trafficking in neuronal cells, Aß40 had a greater effect than Aß42. Moreover, Aß40 decreased the neuronal uptake and lysosomal accumulation of Aß42, which tends to oligomerize at low lysosomal pH. Hence, Aß40 may reduce the prevalence of stable Aß42 oligomers that are closely associated with neurodegeneration and are intercellularly propagated across the vulnerable brain regions to eventually nucleate as amyloid plaques. In conclusion, elevated brain Aß levels and Aß42:40 ratio apparent in the early stages of AD could perturb intraneuronal trafficking, augment the anomalous accumulation of amyloid peptides in AD brain, and drive AD pathogenesis.

Synergistic Effect on Neurodegeneration by N-Truncated Aß4-42 and Pyroglutamate Aß3-42 in a Mouse Model of Alzheimer's Disease.

The N-terminally truncated pyroglutamate Aß3-42 (AßpE3-42) and Aß4-42 peptides are known to be highly abundant in the brain of Alzheimer's disease (AD) patients. Both peptides show enhanced aggregation and neurotoxicity in comparison to full-length Aß, suggesting that these amyloid peptides may play an important role in the pathogenesis of AD. The aim of the present work was to study the direct effect of the combination of AßpE3-42 and Aß4-42 on ongoing AD-related neuron loss, pathology, and neurological deficits in transgenic mice. Bigenic mice were generated by crossing the established TBA42 and Tg4-42 mouse models expressing the N-truncated Aß peptides AßpE3-42 and Aß4-42, respectively. After generation of the bigenic mice, detailed phenotypical characterization was performed using either immunostainings to evaluate amyloid pathology or quantification of neuron numbers using design-based stereology. The elevated plus maze was used to study anxiety levels. In order to evaluate sensori-motor deficits, the inverted grid, the balance beam and the string suspension tasks were applied. We could demonstrate that co-expression of AßpE3-42 and Aß4-42 accelerates neuron loss in the CA1 pyramidal layer of young bigenic mice as seen by reduced neuron numbers in comparison to single transgenic homozygous mice expressing either AßpE3-42 or Aß4-42. This observation coincides with the robust intraneuronal Aß accumulation observed in the bigenic mice. In addition, loss of anxiety and motor deficits were enhanced in an age-dependent manner. The sensori-motor deficits correlate with the abundant spinal cord pathology, as demonstrated by robust intracellular Aß accumulation within motor neurons and extracellular Aß deposition. Our observations demonstrate that a combination of AßpE3-42 and Aß4-42 has a stronger effect on ongoing AD pathology than the peptides alone. Therefore, AßpE3-42 and Aß4-42 might represent excellent potential therapeutic targets and diagnostic markers for AD.

Synaptophysin depletion and intraneuronal Aß in organotypic hippocampal slice cultures from huAPP transgenic mice.

BACKGROUND: To date, there are no effective disease-modifying treatments for Alzheimer's disease (AD). In order to develop new therapeutics for stages where they are most likely to be effective, it is important to identify the first pathological alterations in the disease cascade. Changes in Aß concentration have long been reported as one of the first steps, but understanding the source, and earliest consequences, of pathology requires a model system that represents all major CNS cell types, is amenable to repeated observation and sampling, and can be readily manipulated. In this regard, long term organotypic hippocampal slice cultures (OHSCs) from neonatal amyloid mice offer an excellent compromise between in vivo and primary culture studies, largely retaining the cellular composition and neuronal architecture of the in vivo hippocampus, but with the in vitro advantages of accessibility to live imaging, sampling and intervention. RESULTS: Here, we report the development and characterisation of progressive pathological changes in an organotypic model from TgCRND8 mice. Aß1-40 and Aß1-42 rise progressively in transgenic slice culture medium and stabilise when regular feeding balances continued production. In contrast, intraneuronal Aß continues to accumulate in close correlation with a specific decline in presynaptic proteins and puncta. Plaque pathology is not evident even when Aß1-42 is increased by pharmacological manipulation (using calpain inhibitor 1), indicating that soluble Aß species, or other APP processing products, are sufficient to cause the initial synaptic changes. CONCLUSIONS: Organotypic brain slices from TgCRND8 mice represent an important new system for understanding mechanisms of Aß generation, release and progressive toxicity. The pathology observed in these cultures will allow for rapid assessment of disease modifying compounds in a system amenable to manipulation and observation.

Gene Dosage Dependent Aggravation of the Neurological Phenotype in the 5XFAD Mouse Model of Alzheimer's Disease.

In the present report, we extend previous findings in the 5XFAD mouse model with regard to a characterization of behavioral deficits and neuropathological alterations. We demonstrate that these mice develop a robust age-dependent motor phenotype and spatial reference memory deficits when bred to homozygosity, leading to a strongly reduced age of onset of behavioral symptoms. At postnatal day sixteen, abundant AßPP was detected in subiculum and cortical pyramidal neurons. From six weeks on, intraneuronal Aß could be detected which was much more abundant in homozygous mice. The same gene-dosage effect was seen on memory and motor deficits. While at 2 months of age neither heterozygous nor homozygous 5XFAD mice show any neurological phenotype except for alterations in anxiety behavior, at 5 months they were clearly evident. Interestingly, despite abundant motor deficiencies, homozygous 5XFAD mice were able to perform the acquisition training of the Morris water maze task with no difference in the swimming performance between the groups. Therefore the aggravated spatial memory and spatial reference memory deficits of the homozygous mice correlated with the elevated soluble and insoluble Aß levels. Homozygous 5XFAD mice represent a model with several advantages in comparison to the heterozygous mice, developing amyloid pathology much more rapidly together with a neurological phenotype. These advantages allow reducing the number of animals for Alzheimer's disease research.

Neprilysin deficiency alters the neuropathological and behavioral phenotype in the 5XFAD mouse model of Alzheimer's disease.

The deposition of amyloid-ß (Aß) is one of the major neuropathological hallmarks of Alzheimer's disease (AD). In the case of sporadic AD, an imbalance in Aß in production and clearance seems to be the reason for an enhanced Aß accumulation. Besides a systematic clearance through the blood-brain barrier, Aß is cleared from the brain by Aß-degrading enzymes. The metalloprotease neprilysin (NEP) is an important Aß-degrading enzyme as shown by numerous in vitro, in vivo and reverse genetics studies. 5XFAD mice represent an early-onset AD mouse model which develops plaque pathology starting with 2 months of age in addition to robust behavioral deficits at later time points. By crossing 5XFAD mice with homozygous NEP-knock-out mice (NEP-/-), we show that hemizygous NEP deficiency aggravates the behavioral and neuropathological phenotype of 5XFAD mice. We found that 5XFAD mice per se showed strongly decreased NEP expression levels compared to wildtype mice, which was aggravated by NEP reduction. 5XFAD/NEP+/- mice demonstrated impairment in spatial working memory and increased astrocytosis in all studied brain areas, in addition to an overall increased level of soluble Aß42 as well as region-specific increases in extracellular Aß deposition. Surprisingly, in young mice, a more abundant cortical Aß plaque pathology was observed in 5XFAD compared to 5XFAD/NEP+/- mice. Additionally, young 5XFAD/NEP+/- as well as hemi- and homozygous NEP knockout mice showed elevated levels of endothelin-converting enzyme 1 (ECE1), suggesting a mutual regulation of ECE1 and NEP at young ages. The present data indicate that NEP mainly degrades soluble Aß peptides, which confirms previous observations. Increased ECE1 levels correlated well with the strongly reduced extracellular plaque load in young 5XFAD/NEP+/- mice and might suggest a reciprocal effect between ECE and NEP activities in Aß degradation.

Intracellular accumulation of amyloid-Beta - a predictor for synaptic dysfunction and neuron loss in Alzheimer's disease.

Despite of long-standing evidence that beta-amyloid (Abeta) peptides have detrimental effects on synaptic function, the relationship between Abeta, synaptic and neuron loss is largely unclear. During the last years there is growing evidence that early intraneuronal accumulation of Abeta peptides is one of the key events leading to synaptic and neuronal dysfunction. Many studies have been carried out using transgenic mouse models of Alzheimer's disease (AD) which have been proven to be valuable model systems in modern AD research. The present review discusses the impact of intraneuronal Abeta accumulation on synaptic impairment and neuron loss and provides an overview of currently available AD mouse models showing these pathological alterations.