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Numerous Aß proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated Aß proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent Aß-amyloidosis. Though the brain-derived Aß proteoform landscape is largely occupied by Aß1-42, 25 different forms of Aß with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble Aß, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.
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Enfermedad de Alzheimer , Amiloidosis , Ratones , Humanos , Animales , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Espectrometría de MasasRESUMEN
RATIONALE: Extracellular proteolytic activity plays an important role in memory formation and the preservation of cognitive function. Previous studies have shown increased levels of plasminogen activator inhibitor-1 (PAI-1) in the brain of mouse models of Alzheimer's disease (AD) and plasma of AD patients, associated with memory and cognitive decline; however, the exact function of PAI-1 in AD onset and progression is largely unclear. OBJECTIVE: In this study, we evaluated a novel PAI-1 inhibitor, TM5A15, on its ability to prevent or reverse memory deficits and decrease Aß levels and plaque deposition in APP/PS1 mice. METHODS: We administered TM5A15 mixed in a chow diet to 3-month and 9-month-old APP/PS1 mice before and after neuropathological changes were distinguishable. We then evaluated the effects of TM5A15 on memory function and neuropathology at 9 months and 18 months of age. RESULTS: In the younger mice, 6 months of TM5A15 treatment protected against recognition and short-term working memory impairment. TM5A15 also decreased oligomer levels and amyloid plaques, and increased mBDNF expression in APP/PS1 mice at 9 months of age. In aged mice, 9 months of TM5A15 treatment did not significantly improve memory function nor decrease amyloid plaques. However, TM5A15 treatment showed a trend in decreasing oligomer levels in APP/PS1 mice at 18 months of age. CONCLUSION: Our results suggest that PAI-1 inhibition could improve memory function and reduce the accumulation of amyloid levels in APP/PS1 mice. Such effects are more prominent when TM5A15 is administered before advanced AD pathology and memory deficits occur.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Ratones , Humanos , Animales , Lactante , Péptidos beta-Amiloides/metabolismo , Ratones Transgénicos , Placa Amiloide/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/uso terapéutico , Enfermedad de Alzheimer/metabolismo , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/prevención & control , Trastornos de la Memoria/complicaciones , Modelos Animales de Enfermedad , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/genéticaRESUMEN
The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.
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Enfermedad de Alzheimer , Anticuerpos de Cadena Única , Ratones , Ratas , Humanos , Animales , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/terapia , Enfermedad de Alzheimer/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Sinapsis/metabolismo , Neuronas/metabolismo , Trastornos de la Memoria/genética , Trastornos de la Memoria/terapiaRESUMEN
AIMS: An obstacle to developing new treatment strategies for Alzheimer's disease (AD) has been the inadequate translation of findings in current AD transgenic rodent models to the prediction of clinical outcomes. By contrast, nonhuman primates (NHPs) share a close neurobiology with humans in virtually all aspects relevant to developing a translational AD model. The present investigation used African green monkeys (AGMs) to refine an inducible NHP model of AD based on the administration of amyloid-beta oligomers (AßOs), a key upstream initiator of AD pathology. METHODS: AßOs or vehicle were repeatedly delivered over 4 weeks to age-matched young adult AGMs by intracerebroventricular (ICV) or intrathecal (IT) injections. Induction of AD-like pathology was assessed in subregions of the medial temporal lobe (MTL) by quantitative immunohistochemistry (IHC) using the AT8 antibody to detect hyperphosphorylated tau. Hippocampal volume was measured by magnetic resonance imaging (MRI) scans prior to, and after, intrathecal injections. RESULTS: IT administration of AßOs in young adult AGMs revealed an elevation of tau phosphorylation in the MTL cortical memory circuit compared with controls. The largest increases were detected in the entorhinal cortex that persisted for at least 12 weeks after dosing. MRI scans showed a reduction in hippocampal volume following AßO injections. CONCLUSIONS: Repeated IT delivery of AßOs in young adult AGMs led to an accelerated AD-like neuropathology in MTL, similar to human AD, supporting the value of this translational model to de-risk the clinical trial of diagnostic and therapeutic strategies.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Chlorocebus aethiops , Fosforilación , Primates/metabolismo , Lóbulo Temporal/patología , Proteínas tau/metabolismoRESUMEN
INTRODUCTION: Although mouse models of Alzheimer's disease (AD) have increased our understanding of the molecular basis of the disease, none of those models represent late-onset Alzheimer's Disease which accounts for >90% of AD cases, and no therapeutics developed in the mouse (with the possible exceptions of aduhelm/aducanumab and gantenerumab) have succeeded in preventing or reversing the disease. This technology has allowed much progress in understanding the molecular basis of AD. To further enhance our understanding, we used wild-type rabbit (with a nearly identical amino acid sequence for amyloid as in humans) to model LOAD by stressing risk factors including age, hypercholesterolemia, and elevated blood glucose levels (BGLs), upon an ε3-like isoform of apolipoprotein. We report a combined behavioral, imaging, and metabolic study using rabbit as a non-transgenic model to examine effects of AD-related risk factors on cognition, intrinsic functional connectivity, and magnetic resonance-based biomarkers of neuropathology. METHODS: Aging rabbits were fed a diet enriched with either 2% cholesterol or 10% fat/30% fructose. Monthly tests of novel object recognition (NOR) and object location memory (OLM) were administered to track cognitive impairment. Trace eyeblink conditioning (EBC) was administered as a final test of cognitive impairment. Magnetic resonance imaging (MRI) was used to obtain resting state connectivity and quantitative parametric data (R2*). RESULTS: Experimental diets induced hypercholesterolemia or elevated BGL. Both experimental diets induced statistically significant impairment of OLM (but not NOR) and altered intrinsic functional connectivity. EBC was more impaired by fat/fructose diet than by cholesterol. Whole brain and regional R2* MRI values were elevated in both experimental diet groups relative to rabbits on the control diet. DISCUSSION: We propose that mechanisms underlying LOAD can be assessed by stressing risk factors for inducing AD and that dietary manipulations can be used to assess etiological differences in the pathologies and effectiveness of potential therapeutics against LOAD. In addition, non-invasive MRI in awake, non-anesthetized rabbits further increases the translational value of this non-transgenic model to study AD.
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Human amyloid beta peptide (Aß) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AßOs), which are known to accumulate in Alzheimer's disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AßO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aß as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AßOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AßO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AßOs, are linked to Alzheimer's disease (AD). To test whether the AßOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AßO-selective antibody to prematurely lower the levels of AßOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AßOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Retina/metabolismo , Animales , Encéfalo/metabolismo , Pollos/metabolismo , Espacio Extracelular/metabolismo , Sinapsis/metabolismoRESUMEN
Abnormalities in brain glucose metabolism and accumulation of abnormal protein deposits called plaques and tangles are neuropathological hallmarks of Alzheimer's disease (AD), but their relationship to disease pathogenesis and to each other remains unclear. Here we show that succinylation, a metabolism-associated post-translational protein modification (PTM), provides a potential link between abnormal metabolism and AD pathology. We quantified the lysine succinylomes and proteomes from brains of individuals with AD, and healthy controls. In AD, succinylation of multiple mitochondrial proteins declined, and succinylation of small number of cytosolic proteins increased. The largest increases occurred at critical sites of amyloid precursor protein (APP) and microtubule-associated tau. We show that in vitro, succinylation of APP disrupted its normal proteolytic processing thereby promoting Aß accumulation and plaque formation and that succinylation of tau promoted its aggregation to tangles and impaired microtubule assembly. In transgenic mouse models of AD, elevated succinylation associated with soluble and insoluble APP derivatives and tau. These findings indicate that a metabolism-linked PTM may be associated with AD.
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Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Placa Amiloide/metabolismo , Procesamiento Proteico-Postraduccional , Ácido Succínico/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Animales , Autopsia , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Placa Amiloide/genética , Placa Amiloide/patología , Agregado de Proteínas , Proteolisis , Proteoma/genética , Proteoma/metabolismo , Proteínas tau/genéticaRESUMEN
Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.
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Reactores Biológicos , Encéfalo/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Organoides/citología , HumanosRESUMEN
Improvements have been made in the diagnosis of Alzheimer's disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by magnetic resonance imaging (MRI) and positron emission tomography (PET) scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques-species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid ß oligomers (AßOs), rather, are now widely accepted as the Aß species most germane to AD onset and progression. Here we report evidence further supporting the role of AßOs as pathological instigators of AD and introduce promising anti-AßO diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, Aß oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of AßOs is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these AßOs on memory is in harmony with findings that intraventricular injection of synthetic AßOs into wild type mice induced hippocampal dependent memory dysfunction within 24 h. Compelling support for the conclusion that endogenous AßOs cause memory loss was found in experiments showing that intranasal inoculation of AßO-selective antibodies into 5xFAD mice completely restored memory function, measured 30-40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of AßOs in instigating memory loss and salient AD neuropathology, and they demonstrate that AßO selective antibodies have potential both for therapeutics and for diagnostics.
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AIMS: Amyloid ß-oligomers (AßO) are potent modulators of Alzheimer's pathology, yet their impact on one of the earliest brain regions to exhibit signs of the condition, the locus coeruleus (LC), remains to be determined. Of particular importance is whether AßO impact the spontaneous excitability of LC neurons. This parameter determines brain-wide noradrenaline (NA) release, and thus NA-mediated brain functions, including cognition, emotion and immune function, which are all compromised in Alzheimer's patients. Therefore, the aim of the study was to determine the expression profile of AßO in the LC of Alzheimer's patients and to probe their potential impact on the molecular and functional correlates of LC excitability, using a mouse model of increased Aß production (APP-PSEN1). METHODS AND RESULTS: Immunohistochemistry and confocal microscopy, using AßO-specific antibodies, confirmed LC AßO expression both intraneuronally and extracellularly in both Alzheimer's and APP-PSEN1 samples. Patch clamp electrophysiology recordings revealed that APP-PSEN1 LC neuronal hyperexcitability accompanied this AßO expression profile, arising from a diminished inhibitory effect of GABA due to impaired expression and function of the GABA-A receptor (GABAA R) α3 subunit. This altered LC α3-GABAA R expression profile overlapped with AßO expression in samples from both APP-PSEN1 mice and Alzheimer's patients. Finally, strychnine-sensitive glycine receptors (GlyRs) remained resilient to Aß-induced changes and their activation reversed LC hyperexcitability. CONCLUSIONS: The data suggest a close association between AßO and α3-GABAA Rs in the LC of Alzheimer's patients, and their potential to dysregulate LC activity, thereby contributing to the spectrum of pathology of the LC-NA system in this condition.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Locus Coeruleus/patología , Neuronas/patología , Enfermedad de Alzheimer/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Locus Coeruleus/metabolismo , Locus Coeruleus/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/fisiologíaRESUMEN
The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.
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Amiloide/inmunología , Amiloidosis/diagnóstico , Síndromes de Neurotoxicidad/diagnóstico , Agregación Patológica de Proteínas/diagnóstico , Amiloide/antagonistas & inhibidores , Amiloidosis/inmunología , Amiloidosis/patología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Síndromes de Neurotoxicidad/inmunología , Síndromes de Neurotoxicidad/patología , Fragmentos de Péptidos/inmunología , Agregación Patológica de Proteínas/inmunología , Anticuerpos de Dominio Único , Relación Estructura-ActividadRESUMEN
INTRODUCTION: Amyloid-beta oligomers (AßOs) accumulate in Alzheimer's disease and may instigate neuronal pathology and cognitive impairment. We examined the ability of a new probe for molecular magnetic resonance imaging (MRI) to detect AßOs in vivo, and we tested the behavioral impact of AßOs injected in rabbits, a species with an amino acid sequence that is nearly identical to the human sequence. METHODS: Intracerebroventricular (ICV) injection with stabilized AßOs was performed. Rabbits were probed for AßO accumulation using ACUMNS (an AßO-selective antibody [ACU193] coupled to magnetic nanostructures). Immunohistochemistry was used to verify AßO presence. Cognitive impairment was evaluated using object location and object recognition memory tests and trace eyeblink conditioning. RESULTS: AßOs in the entorhinal cortex of ICV-injected animals were detected by MRI and confirmed by immunohistochemistry. Injections of AßOs also impaired hippocampal-dependent, but not hippocampal-independent, tasks and the area fraction of bound ACUMNs correlated with the behavioral impairment. DISCUSSION: Accumulation of AßOs can be visualized in vivo by MRI of ACUMNS and the cognitive impairment induced by the AßOs can be followed longitudinally with the novel location memory test.
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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.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patología , Inflamación/patología , Neuronas/inmunología , Placa Amiloide/patología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Amiloidosis , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/inmunología , Placa Amiloide/metabolismo , Ratas , Ratas TransgénicasRESUMEN
Introduction: ß-Amyloid protein (Aß) putatively plays a seminal role in synaptic loss in Alzheimer's disease (AD). While there is no consensus regarding the synaptic-relevant species of Aß, it is known that Aß oligomers (AßOs) are noticeably increased in the early stages of AD, localizing at or within the synapse. In cell and animal models, AßOs have been shown to attach to synapses and instigate synapse dysfunction and deterioration. To establish the pathological mechanism of synaptic loss in AD, it will be important to identify the synaptic targets to which AßOs attach. Methods: An unbiased approach using far western ligand blots has identified three synaptic proteins to which AßOs specifically attach. These proteins (p100, p140, and p260) were subsequently enriched by detergent extraction, ultracentrifugation, and CHT-HPLC column separation, and sequenced by LC-MS/MS. P100, p140, and p260 were identified. These levels of AßOs targets in human AD and aging frontal cortexes were analyzed by quantitative proteomics and western-blot. The polyclonal antibody to AßOs was developed and used to block the toxicity of AßOs. The data were analyzed with one-way analysis of variance. Results: AßOs binding proteins p100, p140, and p260 were identified as Na/K-ATPase, synGap, and Shank3, respectively. α3-Na/K-ATPase, synGap, and Shank3 proteins showed loss in the postsynaptic density (PSD) of human AD frontal cortex. In short term experiments, oligomers of Aß inhibited Na/K-ATPase at the synapse. Na/K-ATPase activity was restored by an antibody specific for soluble forms of Aß. α3-Na/K-ATPase protein and synaptic ß-amyloid peptides were pulled down from human AD synapses by co-immunoprecipitation. Results suggest synaptic dysfunction in early stages of AD may stem from inhibition of Na/K-ATPase activity by Aß oligomers, while later stages could hypothetically result from disrupted synapse structure involving the PSD proteins synGap and Shank3. Conclusion: We identified three AßO binding proteins as α3-Na/K-ATPase, synGap, and Shank3. Soluble Aß oligomers appear capable of attacking neurons via specific extracellular as well as intracellular synaptic proteins. Impact on these proteins hypothetically could lead to synaptic dysfunction and loss, and could serve as novel therapeutic targets for AD treatment by antibodies or other agents.
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Synapse degeneration and dendritic spine dysgenesis are believed to be crucial early steps in Alzheimer's disease (AD), and correlate with cognitive deficits in AD patients. Soluble amyloid beta (Aß)-derived oligomers, also termed Aß-derived diffusible ligands (ADDLs), accumulate in the brain of AD patients and play a crucial role in AD pathogenesis. ADDLs bind to mature hippocampal neurons, induce structural changes in dendritic spines and contribute to neuronal death. However, mechanisms underlying structural and toxic effects are not fully understood. Here, we report that ADDLs bind to cultured mature cortical pyramidal neurons and induce spine dysgenesis. ADDL treatment induced the rapid depletion of kalirin-7, a brain-specific guanine-nucleotide exchange factor for the small GTPase Rac1, from spines. Kalirin-7 is a key regulator of dendritic spine morphogenesis and maintenance in forebrain pyramidal neurons and here we show that overexpression of kalirin-7 prevents ADDL-induced spine degeneration. Taken together, our results suggest that kalirin-7 may play a role in the early events leading to synapse degeneration, and its pharmacological activation may prevent or delay synapse pathology in AD.
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Péptidos beta-Amiloides/toxicidad , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Animales , Células Cultivadas , Degeneración Nerviosa , Células Piramidales/metabolismo , Células Piramidales/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Amyloid ß oligomers (AßOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AßO-targeting diagnostics and therapeutics, the AßO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AßOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aß in a soluble oligomeric conformation. With DFDNB, solutions of Aß that would otherwise convert to large aggregates instead yield solutions of stable AßOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AßOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AßO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AßOs in structure-function studies.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Reactivos de Enlaces Cruzados/farmacología , Neuronas/patología , Animales , Humanos , Ratones , RatasRESUMEN
BACKGROUND: Slice cultures have been prepared from several organs. With respect to the brain, advantages of slice cultures over dissociated cell cultures include maintenance of the cytoarchitecture and neuronal connectivity. Slice cultures from adult human brain have been reported and constitute a promising method to study neurological diseases. Despite this potential, few studies have characterized in detail cell survival and function along time in short-term, free-floating cultures. NEW METHOD: We used tissue from adult human brain cortex from patients undergoing temporal lobectomy to prepare 200 µm-thick slices. Along the period in culture, we evaluated neuronal survival, histological modifications, and neurotransmitter release. The toxicity of Alzheimer's-associated Aß oligomers (AßOs) to cultured slices was also analyzed. RESULTS: Neurons in human brain slices remain viable and neurochemically active for at least four days in vitro, which allowed detection of binding of AßOs. We further found that slices exposed to AßOs presented elevated levels of hyperphosphorylated Tau, a hallmark of Alzheimer's disease. COMPARISON WITH EXISTING METHOD(S): Although slice cultures from adult human brain have been previously prepared, this is the first report to analyze cell viability and neuronal activity in short-term free-floating cultures as a function of days in vitro. CONCLUSIONS: Once surgical tissue is available, the current protocol is easy to perform and produces functional slices from adult human brain. These slice cultures may represent a preferred model for translational studies of neurodegenerative disorders when long term culturing in not required, as in investigations on AßO neurotoxicity.
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Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Adulto , Análisis de Varianza , Epilepsia del Lóbulo Temporal/patología , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Fosfopiruvato Hidratasa/metabolismo , Cloruro de Potasio/farmacología , Proteínas tau/metabolismoRESUMEN
The amyloid-ß oligomer (AßO) hypothesis was introduced in 1998. It proposed that the brain damage leading to Alzheimer's disease (AD) was instigated by soluble, ligand-like AßOs. This hypothesis was based on the discovery that fibril-free synthetic preparations of AßOs were potent CNS neurotoxins that rapidly inhibited long-term potentiation and, with time, caused selective nerve cell death (Lambert et al., 1998). The mechanism was attributed to disrupted signaling involving the tyrosine-protein kinase Fyn, mediated by an unknown toxin receptor. Over 4,000 articles concerning AßOs have been published since then, including more than 400 reviews. AßOs have been shown to accumulate in an AD-dependent manner in human and animal model brain tissue and, experimentally, to impair learning and memory and instigate major facets of AD neuropathology, including tau pathology, synapse deterioration and loss, inflammation, and oxidative damage. As reviewed by Hayden and Teplow in 2013, the AßO hypothesis "has all but supplanted the amyloid cascade." Despite the emerging understanding of the role played by AßOs in AD pathogenesis, AßOs have not yet received the clinical attention given to amyloid plaques, which have been at the core of major attempts at therapeutics and diagnostics but are no longer regarded as the most pathogenic form of Aß. However, if the momentum of AßO research continues, particularly efforts to elucidate key aspects of structure, a clear path to a successful disease modifying therapy can be envisioned. Ensuring that lessons learned from recent, late-stage clinical failures are applied appropriately throughout therapeutic development will further enable the likelihood of a successful therapy in the near-term.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/terapia , Animales , Humanos , Modelos NeurológicosRESUMEN
Alzheimer's disease (AD) is a devastating neurological disorder that still lacks an effective treatment, and this has stimulated an intense pursuit of disease-modifying therapeutics. Given the increasingly recognized link between AD and defective brain insulin signaling, we investigated the actions of liraglutide, a glucagon-like peptide-1 (GLP-1) analog marketed for treatment of type 2 diabetes, in experimental models of AD. Insulin receptor pathology is an important feature of AD brains that impairs the neuroprotective actions of central insulin signaling. Here, we show that liraglutide prevented the loss of brain insulin receptors and synapses, and reversed memory impairment induced by AD-linked amyloid-ß oligomers (AßOs) in mice. Using hippocampal neuronal cultures, we determined that the mechanism of neuroprotection by liraglutide involves activation of the PKA signaling pathway. Infusion of AßOs into the lateral cerebral ventricle of non-human primates (NHPs) led to marked loss of insulin receptors and synapses in brain regions related to memory. Systemic treatment of NHPs with liraglutide provided partial protection, decreasing AD-related insulin receptor, synaptic, and tau pathology in specific brain regions. Synapse damage and elimination are amongst the earliest known pathological changes and the best correlates of memory impairment in AD. The results illuminate mechanisms of neuroprotection by liraglutide, and indicate that GLP-1 receptor activation may be harnessed to protect brain insulin receptors and synapses in AD. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.