RESUMO
Several mutations conferring protection against Alzheimer's disease (AD) have been described, none as profound as the A673T mutation, where carriers are four times less likely to get AD compared to noncarriers. This mutation results in reduced amyloid beta (Aß) protein production in vitro and lower lifetime Aß concentration in carriers. Better understanding of the protective mechanisms of the mutation may provide important insights into AD pathophysiology and identify productive therapeutic intervention strategies for disease modification. Aß(1-42) protein forms oligomers that bind saturably to a single receptor site on neuronal synapses, initiating the downstream toxicities observed in AD. Decreased formation, toxicity, or stability of soluble Aß oligomers, or reduction of synaptic binding of these oligomers, may combine with overall lower Aß concentration to underlie A673T's disease protecting mechanism. To investigate these possibilities, we compared the formation rate of soluble oligomers made from Icelandic A673T mutant and wild type (wt) Aß(1-42) synthetic protein, the amount and intensity of oligomer bound to mature primary rat hippocampal/cortical neuronal synapses, and the potency of bound oligomers to impact trafficking rate in neurons in vitro using a physiologically relevant oligomer preparation method. At equal protein concentrations, mutant protein forms approximately 50% or fewer oligomers of high molecular weight (>50 kDa) compared to wt protein. Mutant oligomers are twice as potent at altering the cellular vesicle trafficking rate as wt at equivalent concentrations, however, mutant oligomers have a >4-fold lower binding affinity to synaptic receptors (Kd = 1,950 vs. 442 nM). The net effect of these differences is a lower overall toxicity at a given concentration. This study demonstrates for the first time that mutant A673T Aß oligomers prepared with this method have fundamentally different assembly characteristics and biological impact from wt protein and indicates that its disease protecting mechanism may result primarily from the mutant protein's much lower binding affinity to synaptic receptors. This suggests that therapeutics that effectively reduce oligomer binding to synapses in the brain may be beneficial in AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Neurônios/metabolismo , Animais , Humanos , Ligação Proteica , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
α-Synuclein oligomers are thought to have a pivotal role in sporadic and familial Parkinson's disease (PD) and related α-synucleinopathies, causing dysregulation of protein trafficking, autophagy/lysosomal function, and protein clearance, as well as synaptic function impairment underlying motor and cognitive symptoms of PD. Moreover, trans-synaptic spread of α-synuclein oligomers is hypothesized to mediate disease progression. Therapeutic approaches that effectively block α-synuclein oligomer-induced pathogenesis are urgently needed. Here, we show for the first time that α-synuclein species isolated from human PD patient brain and recombinant α-synuclein oligomers caused similar deficits in lipid vesicle trafficking rates in cultured rat neurons and glia, while α-synuclein species isolated from non-PD human control brain samples did not. Recombinant α-synuclein oligomers also increased neuronal expression of lysosomal-associated membrane protein-2A (LAMP-2A), the lysosomal receptor that has a critical role in chaperone-mediated autophagy. Unbiased screening of several small molecule libraries (including the NIH Clinical Collection) identified sigma-2 receptor antagonists as the most effective at blocking α-synuclein oligomer-induced trafficking deficits and LAMP-2A upregulation in a dose-dependent manner. These results indicate that antagonists of the sigma-2 receptor complex may alleviate α-synuclein oligomer-induced neurotoxicity and are a novel therapeutic approach for disease modification in PD and related α-synucleinopathies.
Assuntos
Doença de Parkinson/metabolismo , Receptores sigma/antagonistas & inibidores , Receptores sigma/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagia/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Metabolismo dos Lipídeos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Doença de Parkinson/patologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas de Transporte Vesicular/metabolismo , alfa-Sinucleína/farmacologiaRESUMO
INTRODUCTION: Amyloid beta (Aß) oligomers are one of the most toxic structural forms of the Aß protein and are hypothesized to cause synaptotoxicity and memory failure as they build up in Alzheimer's disease (AD) patients' brain tissue. We previously demonstrated that antagonists of the sigma-2 receptor complex effectively block Aß oligomer toxicity. CT1812 is an orally bioavailable, brain penetrant small molecule antagonist of the sigma-2 receptor complex that appears safe and well tolerated in healthy elderly volunteers. We tested CT1812's effect on Aß oligomer pathobiology in preclinical AD models and evaluated CT1812's impact on cerebrospinal fluid (CSF) protein biomarkers in mild to moderate AD patients in a clinical trial (ClinicalTrials.gov NCT02907567). METHODS: Experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer binding to synapses in vitro, to human AD patient post mortem brain tissue ex vivo, and in living APPSwe /PS1dE9 transgenic mice in vivo. Additional experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer-induced deficits in membrane trafficking rate, synapse number, and protein expression in mature hippocampal/cortical neurons in vitro. The impact of CT1812 on cognitive function was measured in transgenic Thy1 huAPPSwe/Lnd+ and wild-type littermates. A multicenter, double-blind, placebo-controlled parallel group trial was performed to evaluate the safety, tolerability, and impact on protein biomarker expression of CT1812 or placebo given once daily for 28 days to AD patients (Mini-Mental State Examination 18-26). CSF protein expression was measured by liquid chromatography with tandem mass spectrometry or enzyme-linked immunosorbent assay in samples drawn prior to dosing (Day 0) and at end of dosing (Day 28) and compared within each patient and between pooled treated versus placebo-treated dosing groups. RESULTS: CT1812 significantly and dose-dependently displaced Aß oligomers bound to synaptic receptors in three independent preclinical models of AD, facilitated oligomer clearance into the CSF, increased synaptic number and protein expression in neurons, and improved cognitive performance in transgenic mice. CT1812 significantly increased CSF concentrations of Aß oligomers in AD patient CSF, reduced concentrations of synaptic proteins and phosphorylated tau fragments, and reversed expression of many AD-related proteins dysregulated in CSF. DISCUSSION: These preclinical studies demonstrate the novel disease-modifying mechanism of action of CT1812 against AD and Aß oligomers. The clinical results are consistent with preclinical data and provide evidence of target engagement and impact on fundamental disease-related signaling pathways in AD patients, supporting further development of CT1812.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Cognição/efeitos dos fármacos , Camundongos Transgênicos , Receptores sigma/antagonistas & inibidores , Idoso , Animais , Encéfalo/metabolismo , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Sinapses/metabolismoRESUMO
BACKGROUND: Mature primary neuronal cultures are an important model of the nervous system, but limited scalability has been a major challenge in their use for drug discovery of neurodegenerative diseases. This work describes a method for improving scalability through the use of larger format microtiter plates while preserving culture quality. NEW METHOD: Here we describe a method and quality control procedures for growing embryonic day 18 rat hippocampal/cortical neuronal cultures in 384-well microtiter plates for three weeks in vitro. RESULTS: We use these cultures in two assays measuring intracellular lipid vesicle trafficking and synapse density for routine screening of small molecule libraries. Together this culture system and screening platform have successfully identified therapeutics capable of improving cognitive function in transgenic models of Alzheimer's disease that have advanced to clinical trials, validating their translational applicability. COMPARISON WITH EXISTING METHODS: Our method enables the growth of healthy, mature neurons in larger format microtiter plates than in traditional primary neuronal culturing protocols, making it ideal for drug screening and mechanism of action studies. CONCLUSION: The predictive capacity of this culture system and screening platform provides a method for rapidly identifying novel disease-modifying neurodegenerative therapeutics.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Animais , Descoberta de Drogas , Doenças Neurodegenerativas/tratamento farmacológico , Neurônios , RatosRESUMO
An unbiased phenotypic neuronal assay was developed to measure the synaptotoxic effects of soluble Aß oligomers. A collection of CNS druglike small molecules prepared by conditioned extraction was screened. Compounds that prevented and reversed synaptotoxic effects of Aß oligomers in neurons were discovered to bind to the sigma-2 receptor complex. Select development compounds displaced receptor-bound Aß oligomers, rescued synapses, and restored cognitive function in transgenic hAPP Swe/Ldn mice. Our first-in-class orally administered small molecule investigational drug 7 (CT1812) has been advanced to Phase II clinical studies for Alzheimer's disease.
RESUMO
Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Sinapses/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Química Farmacêutica , Cognição/efeitos dos fármacos , Transtornos Cognitivos/tratamento farmacológico , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Transgênicos , Neuroglia/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismoRESUMO
Amyloid beta (Abeta) 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.