Characterization of Subcellular Organelles in Cortical Perisynaptic Astrocytes

Perisynaptic astrocytic processes (PAPs) carry out several different functions, from metabolite clearing to control of neuronal excitability and synaptic plasticity. All these functions are likely orchestrated by complex cellular machinery that resides within the PAPs and relies on a fine interplay between multiple subcellular components. However, traditional transmission electron microscopy (EM) studies have found that PAPs are remarkably poor of intracellular organelles, failing to explain how such a variety of PAP functions are achieved in the absence of a proportional complex network of intracellular structures. Here, we use serial block-face scanning EM to reconstruct and describe in three dimensions PAPs and their intracellular organelles in two different mouse cortical regions. We described five distinct organelles, which included empty and full endosomes, phagosomes, mitochondria, and endoplasmic reticulum (ER) cisternae, distributed within three PAPs categories (branches, branchlets, and leaflets). The majority of PAPs belonged to the leaflets category (~60%), with branchlets representing a minority (~37%). Branches were rarely in contact with synapses (<3%). Branches had a higher density of mitochondria and ER cisternae than branchlets and leaflets. Also, branches and branchlets displayed organelles more frequently than leaflets. Endosomes and phagosomes, which accounted for more than 60% of all the organelles detected, were often associated with the same PAP. Likewise, mitochondria and ER cisternae, representing ~40% of all organelles were usually associated. No differences were noted between the organelle distribution of the somatosensory and the anterior cingulate cortex. Finally, the organelle distribution in PAPs did not largely depend on the presence of a spine apparatus or a pre-synaptic mitochondrion in the synapse that PAPs were enwrapping, with some exceptions regarding the presence of phagosomes and ER cisternae, which were slightly more represented around synapses lacking a spine apparatus and a presynaptic mitochondrion, respectively. Thus, PAPs contain several subcellular organelles that could underlie the diverse astrocytic functions carried out at central synapses

Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation

Background: Prolonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER-stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss.Results: We used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD. Conclusions: We provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions

The impact of Sleep Deprivation in the brain: Endoplasmic Reticulum Stress and Myelin modification

La privazione del sonno (SD) è un enorme problema per la società moderna. Studi clinici e preclinici hanno dimostrato che la SD è in grado di indurre cambiamenti molecolari, fisiologici e biochimici a livello del sistema nervoso centrale. Studi precedenti hanno mostrato che la privazione del sonno porta a stress del reticolo endoplasmatico (ER) nei neuroni, con un numero di proteine specifiche dell'ER che vengono regolate al rialzo per mantenere una proteostasi cellulare ottimale. Inoltre, utilizzando la microscopia elettronica e confocale, è stato recentemente dimostrato che la restrizione prolungata del sonno (~5 giorni) riduce lo spessore della mielina e aumenta la lunghezza del nodo di Ranvier senza influire sulla lunghezza internodale, suggerendo così che la perdita di sonno può portare a una ristrutturazione plastica della mielina. Attraverso un approccio multidisciplinare, abbiamo esplorato meglio i cambiamenti che la SD induce nella rete ER e nella ristrutturazione della mielina. Qui abbiamo verificato che la SD promuove cambiamenti nella rete ER. I neuroni della corteccia frontale (strato IIIII) di topi in stato di dormienza (S, n = 4, 127 neuroni) e di topi senza sonno per 6 ore (SD, n = 4, 132 neuroni) sono stati analizzati con la microscopia elettronica. Si è scoperto che nei topi SD, i contatti tra ER e mitocondri (mitocondri associati alle membrane, MAM) erano più numerosi, che i mitocondri tendevano a formare cluster e la forma del ER era più compatta. I contatti tra ER e membrana plasmatica (MAP) non sono cambiati. Interessantemente, l'analisi trascrittomico di un database di espressione genica precedente (GSE48369, Bellesi et al. 2013) ha rivelato che vdac1, un trascritto chiave di MAMs, è stato regolato al rialzo dopo SD. Inoltre, è stato valutato se la SD è in grado di influire sulla fluidità della membrana della mielina mediante l'utilizzo della spettroscopia di fluorescenza a stato stazionario. In particolare, sono stati utilizzati due fluorofori, il 2-dimetilamino-(lauroil)-naftalene (Laurdan), situato all'interfaccia idrofobica-idrofila della membrana e l'1,6-difenil-1,3,5-esatriene, incorporato nella regione lipidica idrofobica. È stata trovata un'aumento significativo della fluidità di membrana nel nucleo della membrana della mielina in SD rispetto a S (anisotropia DPH: S [0,231 ± 0,008]; SD [0,222 ± 0,005], p <0,00019), mentre non sono state rilevate differenze a livello di gruppi testa polari. Così, un aumento della fluidità della regione interna della membrana mielinica potrebbe contribuire alle modifiche morfologiche della mielina indotte dalla privazione del sonno. Inoltre, utilizzando gli stessi micrografi utilizzati per la rete ER, è stata effettuata un'ispezione visiva di centinaia di PAP segmentate manualmente intorno alle sinapsi eccitatorie del cortex somatosensoriale e cingolato. Sono state rilevate diverse organelle distinte all'interno dei PAP, tra cui endosomi vuoti e riempiti, fagosomi, mitocondri e cisterne del reticolo endoplasmatico, distribuiti all'interno di tre categorie di PAP (rami, rametti e foglie). La maggior parte dei PAP apparteneva alla categoria delle foglie (~ 60%), mentre i rametti rappresentavano una minoranza (~ 37%). I rami erano raramente a contatto con le sinapsi (<3%). I rami avevano una maggiore densità di mitocondri e cisterne rispetto al ER nei rametti e nelle foglie. Inoltre, i rami e i rametti mostravano organelle più frequenti rispetto alle foglie. Endosomi e fagosomi, che rappresentavano oltre il 60% di tutte le organelle rilevate, erano spesso associati allo stesso PAP. Allo stesso modo, i mitocondri e le cisterne del ER, che rappresentano circa il 40% di tutte le organelle, erano associati ad essi. Non sono state riscontrate differenze tra la distribuzione delle organelle nel cortex somatosensoriale e nel cortex cingolato anteriore. Infine, la distribuzione delle organelle nei PAP non dipendeva dalla presenza di un'apparato spinale o di un mitocondrio presinaptico nell'avvolgimento sinaptico dei PAP, con alcune eccezioni riguardanti la presenza di fagosomi e cisterne del ER, leggermente più rappresentati intorno alle sinapsi che mancano rispettivamente di un apparato spinale e di un mitocondrio presinaptico. Pertanto, i PAP contengono diverse organelle subcellulari che potrebbero sottostare alle diverse funzioni astrocitiche svolte centralmente.Sleep deprivation (SD) is a huge problem for modern society. Clinical and preclinical studies have shown that SD is capable of inducing molecular, physiological, and biochemical changes at the central nervous system level. Previous molecular work has shown that sleep deprivation leads to endoplasmic reticulum (ER) stress in neurons, with a number of ER-specific proteins upregulated to maintain optimal cellular proteostasis. Furthermore, using electron and confocal microscopy, it has recently been shown that prolonged sleep restriction (~ 5 days) reduces myelin thickness and increases Ranvier node length without affecting internodal length, thus suggesting that sleep loss can lead to plastic remodelling of myelin. Through a multidisciplinary approach we have better explored the changes that SD induces in the ER network and at remodelling myelin. Here we have verified that SD promotes changes in the ER network. Frontal cortex (layer IIIII) neurons from dormant (S, n = 4, 127 neurons) and sleepless 6 hours (SD, n = 4, 132 neurons) mice were analysed with electron microscopy. It was found that in SD mice, the contacts between ER and mitochondria (mitochondria associated with membranes, MAM) were more numerous, that mitochondria tend to form clusters and the shape of the ER is more compact. The contacts between ER and plasma membrane (MAP) did not change. Interestingly, transcriptomic analysis of a previous gene expression database (GSE48369, Bellesi et al. 2013) revealed that vdac1, a key transcript of MAMs was upregulated after SD. Furthermore, it was evaluated whether SD is able to influence the fluidity of the myelin membrane through the use of steady-state fluorescence spectroscopy. In particular, two fluorophores were used, 2- dimethylamino- (lauroyl) -naphthalene (Laurdan), located at the hydrophobic-hydrophilic interface of the membrane and 1,6 diphenyl-1,3,5-hexatriene, incorporated in the hydrophobic lipid region. A significant increase in membrane fluidity was found in myelin membrane core in SD relative to S (DPH anisotropy: S [0.231 ± 0.008]; SD [0.222 ± 0.005], p <0.00019), while no differences have been detected at the polar headgroups level. Thus, increased fluidity of the inner myelin membrane region could contribute to morphological modifications of myelin induced by sleep loss. Additionally, using the same micrographs used for the ER network, a visual inspection of hundreds of manually segmented PAPs around the excitatory synapses of the somatosensory and cingulate cortex was performed. Several distinct organelles within PAPs were detected, including empty and filled endosomes, phagosomes, mitochondria, and endoplasmic reticulum cisternae, distributed within three categories of PAP (branches, twigs, and leaflets). Most PAPs belonged to the leaflet category (~ 60%), with twigs representing a minority (~ 37%). The branches were rarely in contact with the synapses (<3%). Branches had a higher density of mitochondria and cisterns than ER at twigs and leaflets. Also, branches and twigs showed organelles more frequent than leaflets. Endosomes and phagosomes, which accounted for over 60% of all detected organelles, were often associated with the same PAP. Likewise, mitochondria and ER cisterns, which account for approximately 40% of all organelles, were associated with them. No differences were noted between the distribution of organelles in the somatosensory cortex and the anterior cingulate cortex. Finally, the distribution of organelles in PAPs did not depend on the presence of a spinal apparatus or presynaptic mitochondrion in the synapse enveloping PAPs, with some exceptions regarding the presence of phagosomes and ER cisterns, slightly more represented around synapses. lacking respectively a spinal apparatus and a presynaptic mitochondrion. Therefore, PAPs contain several subcellular organs that could underlie the different astrocytic functions performed centrally

Additional file 5 of Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation

Additional file 5. Quantification of western blot data for S (n = 5) and SD (n = 5) mice

Additional file 3 of Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation

Additional file 3. Functional enrichment of Mito genes not affected by SD (top 50 biological processes with strength of enrichment >1)

Additional file 2 of Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation

Additional file 2. Functional enrichment of ER genes not affected by SD (top 50 biological processes with strength of enrichment >1)