Astrocyte dysfunction and neuronal network hyperactivity in a CRISPR engineered pluripotent stem cell model of frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most prevalent type of early-onset dementia and up to 40% of cases are familial forms. One of the genes mutated in patients is CHMP2B, which encodes a protein found in a complex important for maturation of late endosomes, an essential process for recycling membrane proteins through the endolysosomal system. Here, we have generated a CHMP2B-mutated human embryonic stem cell line using genome editing with the purpose to create a human in vitro FTD disease model. To date, most studies have focused on neuronal alterations; however, we present a new co-culture system in which neurons and astrocytes are independently generated from human embryonic stem cells and combined in co-cultures. With this approach, we have identified alterations in the endolysosomal system of FTD astrocytes, a higher capacity of astrocytes to uptake and respond to glutamate, and a neuronal network hyperactivity as well as excessive synchronization. Overall, our data indicates that astrocyte alterations precede neuronal impairments and could potentially trigger neuronal network changes, indicating the important and specific role of astrocytes in disease development.

Protocol for optical clearing and imaging of fluorescently labeled ex vivo rat brain slices.

Tissue clearing is commonly used for whole-brain imaging but seldom used for brain slices. Here, we present a simple protocol to slice, immunostain, and clear sections of adult rat brains for subsequent high-resolution confocal imaging. The protocol does not require toxic reagents or specialized equipment. We also provide instructions for culturing of rat brain slices free floating on permeable culture inserts, maintained in regular CO2 incubators, and handled only at media change.

Calcium-Permeable AMPA Receptors Mediate Timing-Dependent LTP Elicited by Low Repeat Coincident Pre- and Postsynaptic Activity at Schaffer Collateral-CA1 Synapses.

High-frequency stimulation induced long-term potentiation (LTP) and low-frequency stimulation induced LTD are considered as cellular models of memory formation. Interestingly, spike timing-dependent plasticity (STDP) can induce equally robust timing-dependent LTP (t-LTP) and t-LTD in response to low frequency repeats of coincident action potential (AP) firing in presynaptic and postsynaptic cells. Commonly, STDP paradigms relying on 25-100 repeats of coincident AP firing are used to elicit t-LTP or t-LTD, but the minimum number of repeats required for successful STDP is barely explored. However, systematic investigation of physiologically relevant low repeat STDP paradigms is of utmost importance to explain learning mechanisms in vivo. Here, we examined low repeat STDP at Schaffer collateral-CA1 synapses by pairing one presynaptic AP with either one postsynaptic AP (1:1 t-LTP), or a burst of 4 APs (1:4 t-LTP) and found 3-6 repeats to be sufficient to elicit t-LTP. 6× 1:1 t-LTP required postsynaptic Ca2+ influx via NMDARs and L-type VGCCs and was mediated by increased presynaptic glutamate release. In contrast, 1:4 t-LTP depended on postsynaptic metabotropic GluRs and ryanodine receptor signaling and was mediated by postsynaptic insertion of AMPA receptors. Unexpectedly, both 6× t-LTP variants were strictly dependent on activation of postsynaptic Ca2+-permeable AMPARs but were differentially regulated by dopamine receptor signaling. Our data show that synaptic changes induced by only 3-6 repeats of mild STDP stimulation occurring in ≤10 s can take place on time scales observed also during single trial learning.

Neuronal hyperactivity due to loss of inhibitory tone in APOE4 mice lacking Alzheimer's disease-like pathology.

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer's disease (AD). However, the reason APOE4 is associated with increased AD risk remains a source of debate. Neuronal hyperactivity is an early phenotype in both AD mouse models and in human AD, which may play a direct role in the pathogenesis of the disease. Here, we have identified an APOE4-associated hyperactivity phenotype in the brains of aged APOE mice using four complimentary techniques-fMRI, in vitro electrophysiology, in vivo electrophysiology, and metabolomics-with the most prominent hyperactivity occurring in the entorhinal cortex. Further analysis revealed that this neuronal hyperactivity is driven by decreased background inhibition caused by reduced responsiveness of excitatory neurons to GABAergic inhibitory inputs. Given the observations of neuronal hyperactivity in prodromal AD, we propose that this APOE4-driven hyperactivity may be a causative factor driving increased risk of AD among APOE4 carriers.

Coexistence of Multiple Types of Synaptic Plasticity in Individual Hippocampal CA1 Pyramidal Neurons.

Understanding learning and memory mechanisms is an important goal in neuroscience. To gain insights into the underlying cellular mechanisms for memory formation, synaptic plasticity processes are studied with various techniques in different brain regions. A valid model to scrutinize different ways to enhance or decrease synaptic transmission is recording of long-term potentiation (LTP) or long-term depression (LTD). At the single cell level, spike timing-dependent plasticity (STDP) protocols have emerged as a powerful tool to investigate synaptic plasticity with stimulation paradigms that also likely occur during memory formation in vivo. Such kind of plasticity can be induced by different STDP paradigms with multiple repeat numbers and stimulation patterns. They subsequently recruit or activate different molecular pathways and neuromodulators for induction and expression of STDP. Dopamine (DA) and brain-derived neurotrophic factor (BDNF) have been recently shown to be important modulators for hippocampal STDP at Schaffer collateral (SC)-CA1 synapses and are activated exclusively by distinguishable STDP paradigms. Distinct types of parallel synaptic plasticity in a given neuron depend on specific subcellular molecular prerequisites. Since the basal and apical dendrites of CA1 pyramidal neurons are known to be heterogeneous, and distance-dependent dendritic gradients for specific receptors and ion channels are described, the dendrites might provide domain specific locations for multiple types of synaptic plasticity in the same neuron. In addition to the distinct signaling and expression mechanisms of various types of LTP and LTD, activation of these different types of plasticity might depend on background brain activity states. In this article, we will discuss some ideas why multiple forms of synaptic plasticity can simultaneously and independently coexist and can contribute so effectively to increasing the efficacy of memory storage and processing capacity of the brain. We hypothesize that resolving the subcellular location of t-LTP and t-LTD mechanisms that are regulated by distinct neuromodulator systems will be essential to reach a more cohesive understanding of synaptic plasticity in memory formation.

Theta Burst Firing Recruits BDNF Release and Signaling in Postsynaptic CA1 Neurons in Spike-Timing-Dependent LTP.

Timing-dependent LTP (t-LTP) is a physiologically relevant type of synaptic plasticity that results from repeated sequential firing of action potentials (APs) in pre- and postsynaptic neurons. t-LTP can be observed in vivo and is proposed to be a cellular correlate of memory formation. While brain-derived neurotrophic factor (BDNF) is essential to high-frequency stimulation-induced LTP in many brain areas, the role of BDNF in t-LTP is largely unknown. Here, we demonstrate a striking change in the expression mechanism of t-LTP in CA1 of the hippocampus following two distinct modes of synaptic activation. Single postsynaptic APs paired with presynaptic stimulation activated a BDNF-independent canonical t-LTP. In contrast, a theta burst of postsynaptic APs preceded by presynaptic stimulation elicited BDNF-dependent postsynaptic t-LTP that relied on postsynaptic BDNF secretion. This suggests that BDNF release during burst-like patterns of activity typically observed in vivo may play a crucial role during memory formation.

R6/2 Huntington's disease mice develop early and progressive abnormal brain metabolism and seizures.

A hallmark feature of Huntington's disease pathology is the atrophy of brain regions including, but not limited to, the striatum. Though MRI studies have identified structural CNS changes in several Huntington's disease (HD) mouse models, the functional consequences of HD pathology during the progression of the disease have yet to be investigated using in vivo functional MRI (fMRI). To address this issue, we first established the structural and functional MRI phenotype of juvenile HD mouse model R6/2 at early and advanced stages of disease. Significantly higher fMRI signals [relative cerebral blood volumes (rCBVs)] and atrophy were observed in both age groups in specific brain regions. Next, fMRI results were correlated with electrophysiological analysis, which showed abnormal increases in neuronal activity in affected brain regions, thus identifying a mechanism accounting for the abnormal fMRI findings. [(14)C] 2-deoxyglucose maps to investigate patterns of glucose utilization were also generated. An interesting mismatch between increases in rCBV and decreases in glucose uptake was observed. Finally, we evaluated the sensitivity of this mouse line to audiogenic seizures early in the disease course. We found that R6/2 mice had an increased susceptibility to develop seizures. Together, these findings identified seizure activity in R6/2 mice and show that neuroimaging measures sensitive to oxygen metabolism can be used as in vivo biomarkers, preceding the onset of an overt behavioral phenotype. Since fMRI-rCBV can also be obtained in patients, we propose that it may serve as a translational tool to evaluate therapeutic responses in humans and HD mouse models.

Decrease of Tau hyperphosphorylation by 17ß estradiol requires sphingosine kinase in a glutamate toxicity model.

Several studies have linked estrogens with sphingosine kinase (SphK) activity, enzyme responsible of sphingosine-1-phosphate synthesis (S-1P), however their possible interaction in the nervous system is not documented yet. In the present study, we developed a glutamate toxicity model in SH-SY5Y cells to evaluate the possible effect of the inhibition of SphK activity on the protective capability of 17ß-estradiol (E2). Glutamate induced cytoskeletal actin changes associated to cytotoxic stress, significant increase of apoptotic-like nuclear fragmentation, Tau hyperphosphorylation and increase of p25/p35 cleavage. These effects were prevented by E2 pre-treatment during 24 h. Although the inhibition of SphK did not block this protective effect, significantly increased Tau hyperphosphorylation by glutamate, in a way that was not reverted by E2. Our results suggest that the decrease of glutamate-induced Tau hyperphosphorylation by 17ß-estradiol requires SphK.

El 17β - estradiol disminuye la expresión y asociación de quinasas responsables de la hiperfosforilación de Tau / 17 β - estradiol decreases the expression and association of kinases responsible of Tau hyperphosphorylation

Introducción: Un componente molecular predominante en el estudio de las enfermedades neurodegenerativas es la presencia del complejo Tau-GSK3β y su asociación con agregados proteicos al interior de la célula. Evidencias considerables muestran que GSK3β es el principal causante de la hiperfosforilación de Tau. Sin embargo, son poco claros los eventos moleculares que gobiernan este complejo. Objetivo: Determinar el efecto del 17 β-estradiol en la expresión y asociación de las quinasas responsables de la hiperfosforilación de Tau. Métodos: Se realizaron tratamientos con 17 β-estradiol en hipocampo de rata Wistar adulta ovariectomizada y en cultivos primarios de hipocampo de rata tratados con b-amiloide. Se evaluó la asociación de complejos proteicos por co-inmunoprecipitación, ensayo de toxicidad por liberación LDH y cambios morfológicos celulares por microscopía confocal. Resultados: Este estudio mostró evidencias de que el estradiol disocia complejos macromoleculares como Tau/GSK3β, Tau/GluR2/3, Tau/FAK, Tau/Fyn en hipocampo de rata adulta. Ademßs, disminuyó la expresión de GSK3β-ptyr por el tratamiento hormonal y éste reguló la defosforilación de Tau en un modelo de excitoxicidad poráβ-amiloide. Conclusiones: Lo anterior sugiere, nuevos blancos que contribuyen al estudio de la neuroprotección y plasticidad neuronal mediada por el estrógeno.

Introduction: A predominant molecular component analyzed in the study of neurodegenerative diseases is the presence of the Tau-GSK3β complex and its association with protein aggregation into the cell. Several evidences show that GSK3β has an important role in abnormal pattern of the phosphorylation of Tau. However, the molecular events that are governing this complex are unknown. Aim: To determine the effect of 17 β-estradiol treatment on the expression and association of Tau hyperphosphorylation responsible kinases. Methods: 17 β-estradiol treatments were realized in the hippocampus of ovariectomized adult wistar rats and in hippocampal primary cultures treated with β-amiloid. Protein complex association was assessed by co-immunoprecipitation, toxicity assay by LDH release and cell morphologic changes by confocal microscopy. Results: Our results show that 17β-estradiol produced dissociation of macromolecular complexes like Tau/GSK3β, Tau /GluR2/3, Tau/FAK, and Tau/Fyn in hippocampus of adult rat. In addition the expression of GSK3β-ptyr was decreased by the hormonal treatment and this one regulated the defosforilation of Tau in an excitotoxicity model by β-amiloid. Conclusions: It suggests new targets that will contribute to neuroprotection and neuronal plasticity studies mediated by the estrogen.