Search

Abstract Synaptic dysfunction is an early feature in Alzheimer’s disease (AD) pathogenesis and a major morphological correlate of memory deficits. Given the main synaptic location of N-methyl-D-aspartate receptors (NMDARs), their dysregulation has been implicated in these pathological effects. Here, to detect possible alterations in the expression and synaptic localisation of the GluN1 subunit in the brain of amyloidogenic APP/PS1 mice, we employed histoblot and SDS-digested freeze-fracture replica labelling (SDS-FRL) techniques. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus in a layer-dependent manner, in the cortex and the caudate putamen of APP/PS1 transgenic mice at 12 months of age but was unaltered at 1 and 6 months. Using quantitative SDS-FRL, we unravelled the molecular organisation of GluN1 in seven excitatory synapse populations at a high spatial resolution in the CA1 and CA3 fields and the DG of the hippocampus in 12-month-old APP/PS1 mice. In the CA1 field, the labelling density for GluN1 in the excitatory synapses established on spines and interneurons, was significantly reduced in APP/PS1 mice compared to age-matched wild-type mice in the stratum lacunosum-moleculare but unaltered in the stratum radiatum. In the CA3 field, synaptic GluN1 was reduced in mossy fibre-CA3 pyramidal cell synapses but unaltered in the A/C-CA3 pyramidal cell synapses. In the DG, the density of GluN1 in granule cell-perforant pathway synapses was reduced in APP/PS1 mice. Altogether, our findings provide evidence of specific alterations of synaptic GluN1 in the trisynaptic circuit of the hippocampus in Aβ pathology. This differential vulnerability in the disruption of NMDARs may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit and cognitive impairment characteristic of APP/PS1 mice.

[INLINE:1] Plaques of amyloid-β (Aβ) and neurofibrillary tangles are the main pathological characteristics of Alzheimer's disease (AD). However, some older adult people with AD pathological hallmarks can retain cognitive function. Unraveling the factors that lead to this cognitive resilience to AD offers promising prospects for identifying new therapeutic targets. Our hypothesis focuses on the contribution of resilience to changes in excitatory synapses at the structural and molecular levels, which may underlie healthy cognitive performance in aged AD animals. Utilizing the Morris Water Maze test, we selected resilient (asymptomatic) and cognitively impaired aged Tg2576 mice. While the enzyme-linked immunosorbent assay showed similar levels of Aβ42 in both experimental groups, western blot analysis revealed differences in tau pathology in the pre-synaptic supernatant fraction. To further investigate the density of synapses in the hippocampus of 16–18 month-old Tg2576 mice, we employed stereological and electron microscopic methods. Our findings indicated a decrease in the density of excitatory synapses in the stratum radiatum of the hippocampal CA1 in cognitively impaired Tg2576 mice compared with age-matched resilient Tg2576 and non-transgenic controls. Intriguingly, through quantitative immunoelectron microscopy in the hippocampus of impaired and resilient Tg2576 transgenic AD mice, we uncovered differences in the subcellular localization of glutamate receptors. Specifically, the density of GluA1, GluA2/3, and mGlu5 in spines and dendritic shafts of CA1 pyramidal cells in impaired Tg2576 mice was significantly reduced compared with age-matched resilient Tg2576 and non-transgenic controls. Notably, the density of GluA2/3 in resilient Tg2576 mice was significantly increased in spines but not in dendritic shafts compared with impaired Tg2576 and non-transgenic mice. These subcellular findings strongly support the hypothesis that dendritic spine plasticity and synaptic machinery in the hippocampus play crucial roles in the mechanisms of cognitive resilience in Tg2576 mice.

Abstract Alzheimer’s disease (AD) is characterized by a reorganization of brain activity determining network hyperexcitability and loss of synaptic plasticity. Precisely, a dysfunction in metabotropic GABAB receptor signalling through G protein-gated inwardly rectifying K+ (GIRK or Kir3) channels on the hippocampus has been postulated. Thus, we determined the impact of amyloid-β (Aβ) pathology in GIRK channel density, subcellular distribution, and its association with GABAB receptors in hippocampal CA1 pyramidal neurons from the APP/PS1 mouse model using quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL) and proximity ligation in situ assay (P-LISA). In wild type mice, single SDS-FRL detection revealed a similar dendritic gradient for GIRK1 and GIRK2 in CA1 pyramidal cells, with higher densities in spines, and GIRK3 showed a lower and uniform distribution. Double SDS-FRL showed a co-clustering of GIRK2 and GIRK1 in post- and presynaptic compartments, but not for GIRK2 and GIRK3. Likewise, double GABAB1 and GIRK2 SDS-FRL detection displayed a high degree of co-clustering in nanodomains (40–50 nm) mostly in spines and axon terminals. In APP/PS1 mice, the density of GIRK2 and GIRK1, but not for GIRK3, was significantly reduced along the neuronal surface of CA1 pyramidal cells and in axon terminals contacting them. Importantly, GABAB1 and GIRK2 co-clustering was not present in APP/PS1 mice. Similarly, P-LISA experiments revealed a significant reduction in GABAB1 and GIRK2 interaction on the hippocampus of this animal model. Overall, our results provide compelling evidence showing a significant reduction on the cell surface density of pre- and postsynaptic GIRK1 and GIRK2, but not GIRK3, and a decline in GABAB receptors and GIRK2 channels co-clustering in hippocampal pyramidal neurons from APP/PS1 mice, thus suggesting that a disruption in the GABAB receptor–GIRK channel membrane assembly causes dysregulation in the GABAB signalling via GIRK channels in this AD animal model.

Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41–360 to 105–800 μg/g DW, respectively, with crocetin, crocin 1 [(crocetin-(β-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(β-D-glucosyl)-(β-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value.

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.

Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transduce signals from G protein-coupled receptors (GPCRs) to effector ion channels and enzymes Gαo, a member of the pertussis toxin-sensitive Gi/o family, is widely expressed in the brain, although its role within a neuronal context remains largely unknown. Using immunohistochemical and quantitative immunoelectron microscopy techniques, we have investigated the expression, cellular and subcellular localization of Gαo in the cerebellar cortex. Histoblot revealed that Gαo is expressed in many brain regions, including the cerebellum. At the cellular level, Gαo protein was distributed in Purkinje cells, basket cells, stellate cells, granule cells and Golgi cells. At the subcellular level, pre-embedding immunoelectron microscopy revealed mainly a postsynaptic localization of Gαo along the extrasynaptic plasma membrane of Purkinje cell dendritic shafts and spines, and dendrites of basket, stellate and granule cells. To a lesser extent, immunolabeling for Gαo was localized in different types of axon terminals establishing excitatory synapses. Moreover, post-embedding immunoelectron microscopy revealed the synaptic localization of Gαo on PSDs of glutamatergic synapses between Purkinje cell spines and parallel fiber terminals and its co-localization with GABAB1 in the same spines. Quantitative analysis of Gαo immunoparticles revealed they preferentially localized on the cytoplasmic face of the plasma membrane. Furthermore, the analysis revealed a high concentration of Gαo around excitatory synapses on Purkinje cell dendritic spines, but a uniform distribution in granule cell dendrites. These molecular-anatomical findings suggest that Gαo is a major signal transducer of specific GPCRs in different neuronal populations in the cerebellum.

Synapse loss occurs early in Alzheimer’s disease (AD) patients and animal models. Alterations at synaptic level are a major morphological correlate of the memory deficits and related symptoms of AD. Given the predominant roles of synaptic AMPA receptors (AMPARs) in excitatory synaptic transmission in the brain, changes in their dynamic regulation are also implicated in the pathophysiology of AD. Here, we used immunolocalization techniques to analyze the expression and subcellular distribution of AMPARs in the hippocampal region of APP/PS1 mouse model of AD. Immunoblots and histoblots revealed that the total amount of AMPARs and their regional expression pattern in the hippocampus was similar in APP/PS1 mice and in age-matched wild type mice. At the ultrastructural level, two synapse populations were examined using SDS-digested freeze-fracture replica labeling in the stratum radiatum in mice: (i) on spines of CA1 pyramidal cells; and (ii) on randomly found dendritic shafts of CA1 interneurons. While 1- and 6-months-old APP/PS1 mice exhibited no change, we observed a significant reduction at 12 months in AMPAR density at synapses in both pyramidal cells and interneurons, compared to wild-type. This reduction of AMPARs in dendritic spines was accompanied by a significant increase in AMPAR subunit proteins identified in intracellular compartments. Our data demonstrate an age-dependent reduction of synaptic AMPARs in APP/PS1 mice, which may contribute to impaired learning and memory at later stages of AD.

G protein-gated inwardly rectifying K+ (GIRK) channels are the main targets controlling excitability and synaptic plasticity on hippocampal neurons. Consequently, dysfunction of GIRK-mediated signalling has been implicated in the pathophysiology of Alzheimer´s disease (AD). Here, we provide a quantitative description on the expression and localisation patterns of GIRK2 in two transgenic mice models of AD (P301S and APP/PS1 mice), combining histoblots and immunoelectron microscopic approaches. The histoblot technique revealed differences in the expression of GIRK2 in the two transgenic mice models. The expression of GIRK2 was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered in APP/PS1 mice at 12 months compared to age-matched wild type mice. Ultrastructural approaches using the pre-embedding immunogold technique, demonstrated that the subcellular localisation of GIRK2 was significantly reduced along the neuronal surface of CA1 pyramidal cells, but increased in its frequency at cytoplasmic sites, in both P301S and APP/PS1 mice. We also found a decrease in plasma membrane GIRK2 channels in axon terminals contacting dendritic spines of CA1 pyramidal cells in P301S and APP/PS1 mice. These data demonstrate for the first time a redistribution of GIRK channels from the plasma membrane to intracellular sites in different compartments of CA1 pyramidal cells. Altogether, the pre- and post-synaptic reduction of GIRK2 channels suggest that GIRK-mediated alteration of the excitability in pyramidal cells could contribute to the cognitive dysfunctions as described in the two AD animal models.

Metabotropic glutamate receptor subtype 5 (mGlu5) is implicated in the pathophysiology of Alzheimer’s disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu5 protein in the hippocampus, in addition to downstream molecules, i.e., Gq/11 and PLCβ1, was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu5-associated molecules and downstream signalling pathways in the progression of the pathology.

The K+ channel interacting proteins (KChIPs) are a family of cytosolic proteins that interact with Kv4 channels, leading to higher current density, modulation of channel inactivation and faster recovery from inactivation. Using immunohistochemical techniques at the light and electron microscopic level combined with quantitative analysis, we investigated the cellular and subcellular localisation of KChIP3 and KChIP4 to compare their distribution patterns with those for Kv4.2 and Kv4.3 in the cerebellar cortex. Immunohistochemistry at the light microscopic level demonstrated that KChIP3, KChIP4, Kv4.2 and Kv4.3 proteins were widely expressed in the cerebellum, with mostly overlapping patterns. Immunoelectron microscopic techniques showed that KChIP3, KChIP4, Kv4.2 and Kv4.3 shared virtually the same somato-dendritic domains of Purkinje cells and granule cells. Application of quantitative approaches showed that KChIP3 and KChIP4 were mainly membrane-associated, but also present at cytoplasmic sites close to the plasma membrane, in dendritic spines and shafts of Purkinje cells (PCs) and dendrites of granule cells (GCs). Similarly, immunoparticles for Kv4.2 and Kv4.3 were observed along the plasma membrane and at intracellular sites in the same neuron populations. In addition to the preferential postsynaptic distribution, KChIPs and Kv4 were also distributed presynaptically in parallel fibres and mossy fibres. Immunoparticles for KChIP3, KChIP4 and Kv4.3 were detected in parallel fibres, and KChIP3, KChIP4, Kv4.2 and Kv4.3 were found in parallel fibres, indicating that composition of KChIP and Kv4 seems to be input-dependent. Together, our findings unravelled previously uncharacterised KChIP and Kv4 subcellular localisation patterns in neurons, revealed that KChIP have additional Kv4-unrelated functions in the cerebellum and support the formation of macromolecular complexes between KChIP3 and KChIP4 with heterotetrameric Kv4.2/Kv4.3 channels.

Metabotropic γ-aminobutyric acid (GABAB) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) has been implicated in Alzheimer´s disease (AD). Given the involvement of GABAB receptors in AD, to determine their subcellular localisation and possible alteration in granule cells of the DG in a mouse model of AD at 12 months of age, we used high-resolution immunoelectron microscopic analysis. Immunohistochemistry at the light microscopic level showed that the regional and cellular expression pattern of GABAB1 was similar in an AD model mouse expressing mutated human amyloid precursor protein and presenilin1 (APP/PS1) and in age-matched wild type mice. High-resolution immunoelectron microscopy revealed a distance-dependent gradient of immunolabelling for GABAB receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABAB receptors at the neuronal surface of these postsynaptic compartments of granule cells was significantly reduced in APP/PS1 mice. Parallel to this reduction in surface receptors, we found a significant increase in GABAB1 at cytoplasmic sites. GABAB receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABAB receptors in axon terminals contacting dendritic spines of granule cells, which was more pronounced in the outer than in the inner molecular layer. Altogether, our data showing post- and presynaptic reduction in surface GABAB receptors in the DG suggest the alteration of the GABAB-mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of AD.

The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.

The Kv4 family of voltage-gated K+ channels underlie the fast transient (A-type) outward K+ current. Although A-type currents are critical to determine somato-dendritic integration in central neurons, relatively little is known about the precise subcellular localisation of the underlying channels in hippocampal circuits. Using histoblot and immunoelectron microscopic techniques, we investigated the expression, regional distribution and subcellular localisation of Kv4.2 and Kv4.3 in the adult brain, as well as the ontogeny of their expression during postnatal development. Histoblot demonstrated that Kv4.2 and Kv4.3 proteins were widely expressed in the brain, with mostly non-overlapping patterns. During development, levels of Kv4.2 and Kv4.3 increased with age but showed marked region- and developmental stage-specific differences. Immunoelectron microscopy showed that labelling for Kv4.2 and Kv4.3 was differentially present in somato-dendritic domains of hippocampal principal cells and interneurons, including the synaptic specialisation. Quantitative analyses indicated that most immunoparticles for Kv4.2 and Kv4.3 were associated with the plasma membrane in dendritic spines and shafts, and that the two channels showed very similar distribution patterns in spines of principal cells and along the surface of granule cells. Our data shed new light on the subcellular localisation of Kv4 channels and provide evidence for their non-uniform distribution over the plasma membrane of hippocampal neurons.