Neuronal A2A receptor exacerbates synapse loss and memory deficits in APP/PS1 mice.

Early pathological upregulation of adenosine A2A receptors (A2ARs), one of the caffeine targets, by neurons is thought to be involved in the development of synaptic and memory deficits in Alzheimer's disease (AD) but mechanisms remain ill-defined. To tackle this question, we promoted a neuronal upregulation of A2AR in the hippocampus of APP/PS1 mice developing AD-like amyloidogenesis. Our findings revealed that the early upregulation of A2AR in the presence of an ongoing amyloid pathology exacerbates memory impairments of APP/PS1 mice. These behavioural changes were not linked to major change in the development of amyloid pathology but rather associated with increased phosphorylated tau at neuritic plaques. Moreover, proteomic and transcriptomic analyses coupled with quantitative immunofluorescence studies indicated that neuronal upregulation of the receptor promoted both neuronal and non-neuronal autonomous alterations, i.e. enhanced neuroinflammatory response but also loss of excitatory synapses and impaired neuronal mitochondrial function, presumably accounting for the detrimental effect on memory. Overall, our results provide compelling evidence that neuronal A2AR dysfunction, as seen in the brain of patients, contributes to amyloid-related pathogenesis and underscores the potential of A2AR as a relevant therapeutic target for mitigating cognitive impairments in this neurodegenerative disorder.

The Alzheimer's disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons.

Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

Integration of Microfluidic Devices with Microelectrode Arrays to Functionally Assay Amyloid-ß-Induced Synaptotoxicity.

Alzheimer's disease (AD) is a neurodegenerative disease and the most frequent cause of dementia. It is characterized by the accumulation in the brain of two pathological protein aggregates: amyloid-ß peptides (Aß) and abnormally phosphorylated tau. The progressive cognitive decline observed in patients strongly correlates with the synaptic loss. Many lines of evidence suggest that soluble forms of Aß accumulate into the brain where they cause synapse degeneration. Stopping their spreading and/or targeting the pathophysiological mechanisms leading to synaptic loss would logically be beneficial for the patients. However, we are still far from understanding these processes. Our objective was therefore to develop a versatile model to assay and study Aß-induced synaptotoxicity. We integrated a microfluidic device that physically isolates synapses from presynaptic and postsynaptic neurons with a microelectrode array. We seeded mouse primary cortical cells in the presynaptic and postsynaptic chambers. After functional synapses have formed in the synaptic chamber, we exposed them to concentrated conditioned media from cell lines overexpressing the wild-type or mutated amyloid precursor protein and thus secreting different levels of Aß. We recorded the neuronal activity before and after exposition to Aß and quantified Aß's effects on the connectivity between presynaptic and postsynaptic neurons. We observed that the application of Aß on the synapses for 48 h strongly decreased the interchamber connectivity without significantly affecting the neuronal activity in the presynaptic or postsynaptic chambers. Thus, through this model, we are able to functionally assay the impact of Aß peptides (or other molecules) on synaptic connectivity and to use the latter as a proxy to study Aß-induced synaptotoxicity. Moreover, since the presynaptic, postsynaptic, and synaptic chambers can be individually targeted, our assay provides a powerful tool to evaluate the involvement of candidate genes in synaptic vulnerability and/or test therapeutic strategies for AD.

High-Content Screening of Synaptic Density Modulators in Primary Neuronal Cultures.

The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high-content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real-world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384-well plate format. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of primary hippocampal rat neurons in 384-well plates Basic Protocol 2: Lentiviral shRNA transduction of primary neuronal culture in 384-well plates Basic Protocol 3: Immunostaining of the neuronal network and synaptic markers in 384-well plates Basic Protocol 4: Image acquisition using a high-throughput reader Basic Protocol 5: Image segmentation and analysis Basic Protocol 6: Synaptic density analysis.

Activity-Dependent Nr4a2 Induction Modulates Synaptic Expression of AMPA Receptors and Plasticity via a Ca2+/CRTC1/CREB Pathway.

Transcription factors have a pivotal role in synaptic plasticity and the associated modification of neuronal networks required for memory formation and consolidation. The nuclear receptors subfamily 4 group A (Nr4a) have emerged as possible modulators of hippocampal synaptic plasticity and cognitive functions. However, the molecular and cellular mechanisms underlying Nr4a2-mediated hippocampal synaptic plasticity are not completely known. Here, we report that neuronal activity enhances Nr4a2 expression and function in cultured mouse hippocampal neurons (both sexes) by an ionotropic glutamate receptor/Ca2+/cAMP response element-binding protein/CREB-regulated transcription factor 1 (iGluR/Ca2+/CREB/CRTC1) pathway. Nr4a2 activation mediates BDNF production and increases expression of iGluRs, thereby affecting LTD at CA3-CA1 synapses in acute mouse hippocampal slices (both sexes). Together, our results indicate that the iGluR/Ca2+/CREB/CRTC1 pathway mediates activity-dependent expression of Nr4a2, which is involved in glutamatergic synaptic plasticity by increasing BDNF and synaptic GluA1-AMPARs. Therefore, Nr4a2 activation could be a therapeutic approach for brain disorders associated with dysregulated synaptic plasticity.SIGNIFICANCE STATEMENT A major factor that regulates fast excitatory synaptic transmission and plasticity is the modulation of synaptic AMPARs. However, despite decades of research, the underlying mechanisms of this modulation remain poorly understood. Our study identified a molecular pathway that links neuronal activity with AMPAR modulation and hippocampal synaptic plasticity through the activation of Nr4a2, a member of the nuclear receptor subfamily 4. Since several compounds have been described to activate Nr4a2, our study not only provides mechanistic insights into the molecular pathways related to hippocampal synaptic plasticity and learning, but also identifies Nr4a2 as a potential therapeutic target for pathologic conditions associated with dysregulation of glutamatergic synaptic function.

microRNAs as Early Biomarkers of Alzheimer's Disease: A Synaptic Perspective.

Pathogenic processes underlying Alzheimer's disease (AD) affect synaptic function from initial asymptomatic stages, long time before the onset of cognitive decline and neurodegeneration. Therefore, reliable biomarkers enabling early AD diagnosis and prognosis are needed to maximize the time window for therapeutic interventions. MicroRNAs (miRNAs) have recently emerged as promising cost-effective and non-invasive biomarkers for AD, since they can be readily detected in different biofluids, including cerebrospinal fluid (CSF) and blood. Moreover, a growing body of evidence indicates that miRNAs regulate synaptic homeostasis and plasticity processes, suggesting that they may be involved in early synaptic dysfunction during AD. Here, we review the current literature supporting a role of miRNAs during early synaptic deficits in AD, including recent studies evaluating their potential as AD biomarkers. Besides targeting genes related to Aß and tau metabolism, several miRNAs also regulate synaptic-related proteins and transcription factors implicated in early synaptic deficits during AD. Furthermore, individual miRNAs and molecular signatures have been found to distinguish between prodromal AD and healthy controls. Overall, these studies highlight the relevance of considering synaptic-related miRNAs as potential biomarkers of early AD stages. However, further validation studies in large cohorts, including longitudinal studies, as well as implementation of standardized protocols, are needed to establish miRNA-based biomarkers as reliable diagnostic and prognostic tools.

Proteasomal-Mediated Degradation of AKAP150 Accompanies AMPAR Endocytosis during cLTD.

The number and function of synaptic AMPA receptors (AMPARs) tightly regulates excitatory synaptic transmission. Current evidence suggests that AMPARs are inserted into the postsynaptic membrane during long-term potentiation (LTP) and are removed from the membrane during long-term depression (LTD). Dephosphorylation of GluA1 at Ser-845 and enhanced endocytosis are critical events in the modulation of LTD. Moreover, changes in scaffold proteins from the postsynaptic density (PSD) could be also related to AMPAR regulation in LTD. In the present study we analyzed the effect of chemical LTD (cLTD) on A-kinase anchoring protein (AKAP)150 and AMPARs levels in mouse-cultured neurons. We show that cLTD induces AKAP150 protein degradation via proteasome, coinciding with GluA1 dephosphorylation at Ser-845 and endocytosis of GluA1-containing AMPARs. Pharmacological inhibition of proteasome activity, but not phosphatase calcineurin (CaN), reverted cLTD-induced AKAP150 protein degradation. Importantly, AKAP150 silencing induced dephosphorylation of GluA1 Ser-845 and GluA1-AMPARs endocytosis while AKAP150 overexpression blocked cLTD-mediated GluA1-AMPARs endocytosis. Our results provide direct evidence that cLTD-induced AKAP150 degradation by the proteasome contributes to synaptic AMPARs endocytosis.

Altered microRNAs related to synaptic function as potential plasma biomarkers for Alzheimer's disease.

BACKGROUND: Several evidences suggest that failure of synaptic function occurs at preclinical stages of Alzheimer's disease (AD) preceding neuronal loss and the classical AD pathological hallmarks. Nowadays, there is an urgent need to identify reliable biomarkers that could be obtained with non-invasive methods to improve AD diagnosis at early stages. Here, we have examined plasma levels of a group of miRNAs related to synaptic proteins in a cohort composed of cognitive healthy controls (HC), mild cognitive impairment (MCI) and AD subjects. METHODS: Plasma and brain levels of miRNAs were analysed in two different cohorts including 38 HC, 26 MCI, 56 AD dementia patients and 27 frontotemporal dementia (FTD) patients. D'Agostino and Pearson and Shapiro-Wilk tests were used to evaluate data normality. miRNA levels between groups were compared using a two-sided nonparametric Mann-Whitney test and sensitivity and specificity was determined by receiver operating characteristic curve analysis. RESULTS: Significant upregulation of miR-92a-3p, miR-181c-5p and miR-210-3p was found in the plasma of both MCI and AD subjects. MCI patients that progress to AD showed higher plasma levels of these miRNAs. By contrast, no changes in miR-92a-3p, miR-181c-5p or miR-210-3p levels were observed in plasma obtained from a cohort of FTD. CONCLUSION: Our study shows that plasma miR-92a-3p, miR-181c-5p and miR-210-3p constitute a specific molecular signature potentially useful as a potential biomarker for AD.