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1.
Acta Neuropathol ; 147(1): 32, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38319380

RESUMO

Synapse loss correlates with cognitive decline in Alzheimer's disease, and soluble oligomeric amyloid beta (Aß) is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how Aß leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds Aß and mediates toxicity. While many candidates have been observed in model systems, their relevance to human AD brain remains unknown. This is in part due to methodological limitations preventing visualization of Aß binding at individual synapses. To overcome this limitation, we combined two high resolution microscopy techniques: array tomography and Förster resonance energy transfer (FRET) to image over 1 million individual synaptic terminals in temporal cortex from AD (n = 11) and control cases (n = 9). Within presynapses and post-synaptic densities, oligomeric Aß generates a FRET signal with transmembrane protein 97. Further, Aß generates a FRET signal with cellular prion protein, and post-synaptic density 95 within post synapses. Transmembrane protein 97 is also present in a higher proportion of post synapses in Alzheimer's brain compared to controls. We inhibited Aß/transmembrane protein 97 interaction in a mouse model of amyloidopathy by treating with the allosteric modulator CT1812. CT1812 drug concentration correlated negatively with synaptic FRET signal between transmembrane protein 97 and Aß. In human-induced pluripotent stem cell derived neurons, transmembrane protein 97 is present in synapses and colocalizes with Aß when neurons are challenged with human Alzheimer's brain homogenate. Transcriptional changes are induced by Aß including changes in genes involved in neurodegeneration and neuroinflammation. CT1812 treatment of these neurons caused changes in gene sets involved in synaptic function. These data support a role for transmembrane protein 97 in the synaptic binding of Aß in human Alzheimer's disease brain where it may mediate synaptotoxicity.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Proteínas de Membrana , Animais , Humanos , Camundongos , Peptídeos beta-Amiloides , Encéfalo , Sinapses , Proteínas de Membrana/metabolismo
3.
J Neurosci Methods ; 358: 109180, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33836174

RESUMO

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 , Ratos
4.
Alzheimers Dement ; 17(8): 1365-1382, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33559354

RESUMO

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/metabolismo
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