Extracellular clusterin limits the uptake of α-synuclein fibrils by murine and human astrocytes.

The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α-synuclein. Clearance of extracellular α-synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α-synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α-synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α-synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human-induced pluripotent stem cell (iPSC)-derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α-synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α-synuclein preformed fibrils (pffs) through dynamin-dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α-synuclein pffs exposure. Specifically, we found that clusterin interacts with α-synuclein pffs in the extracellular compartment and the clusterin/α-synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock-out primary astrocytes and clusterin knock-down hiPSC-derived astrocytes we observed that clusterin limits the uptake of α-synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno-associated virus- and the α-synuclein pffs- injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α-synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology.

Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables.

BACKGROUND: A-to-I RNA editing is a co-/post-transcriptional modification catalyzed by ADAR enzymes, that deaminates Adenosines (A) into Inosines (I). Most of known editing events are located within inverted ALU repeats, but they also occur in coding sequences and may alter the function of encoded proteins. RNA editing contributes to generate transcriptomic diversity and it is found altered in cancer, autoimmune and neurological disorders. Emerging evidences indicate that editing process could be influenced by genetic variations, biological and environmental variables. RESULTS: We analyzed RNA editing levels in human blood using RNA-seq data from 459 healthy individuals and identified 2079 sites consistently edited in this tissue. As expected, analysis of gene expression revealed that ADAR is the major contributor to editing on these sites, explaining ~ 13% of observed variability. After removing ADAR effect, we found significant associations for 1122 genes, mainly involved in RNA processing. These genes were significantly enriched in genes encoding proteins interacting with ADARs, including 276 potential ADARs interactors and 9 ADARs direct partners. In addition, our analysis revealed several factors potentially influencing RNA editing in blood, including cell composition, age, Body Mass Index, smoke and alcohol consumption. Finally, we identified genetic loci associated with editing levels, including known ADAR eQTLs and a small region on chromosome 7, containing LOC730338, a lincRNA gene that appears to modulate ADARs mRNA expression. CONCLUSIONS: Our data provides a detailed picture of the most relevant RNA editing events and their variability in human blood, giving interesting insights on potential mechanisms behind this post-transcriptional modification and its regulation in this tissue.

Differential Enzymatic Activity of Rat ADAR2 Splicing Variants Is Due to Altered Capability to Interact with RNA in the Deaminase Domain.

In mammals, adenosine (A) to inosine (I) RNA editing is performed by adenosine deaminases acting on RNA (ADAR), ADAR1 and ADAR2 enzymes, encoded by mRNAs that might undergo splicing process. In rat, two splicing events produce several isoforms of ADAR2, called ADAR2a, ADAR2b, ADAR2e, and ADAR2f, but only ADAR2a and ADAR2b are translated into an active protein. In particular, they differ for ten amino acids located in the catalytic domain of ADAR2b. Here, we focused on these two isoforms, analyzing the splicing pattern and their different function during rat neuronal maturation. We found an increase of editing levels in cortical neurons overexpressing ADAR2a compared to those overexpressing ADAR2b. These results indicate ADAR2a isoform as the most active one, as reported for the homologous human short variant. Furthermore, we showed that the differential editing activity is not due to a different dimerization of the two isoforms; it seems to be linked to the ten amino acids loop of ADAR2b that might interfere with RNA binding, occupying the space volume in which the RNA should be present in case of binding. These data might shed light on the complexity of ADAR2 regulations.

Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse.

The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. To evaluate the ADAR2-FMRP interaction in mammals we analyzed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP leads to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes.

The Good and the Bad of Glutamate Receptor RNA Editing.

Glutamate receptors play a key role in excitatory synaptic transmission and plasticity in the central nervous system (CNS). Their channel properties are largely dictated by the subunit composition of tetrameric receptors. Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate channels are assembled from GluA1-4 AMPA or GluK1-5 kainate receptor subunits. However, their functional properties are highly modulated by a post-transcriptional mechanism called RNA editing. This process involves the enzymatic deamination of specific adenosines (A) into inosines (I) in pre-messenger RNA. This post-transcriptional modification leads to critical amino acid substitutions in the receptor subunits, which induce profound alterations of the channel properties. Three of the four AMPA and two of the five kainate receptor subunits are subjected to RNA editing. This study reviews the advances in understanding the importance of glutamate receptor RNA editing in finely tuning glutamatergic neurotransmission under physiological conditions and discusses the way in which the dis-regulation of RNA editing may be involved in neurological pathology.

Chronic glutamate treatment selectively modulates AMPA RNA editing and ADAR expression and activity in primary cortical neurons.

Adenosine-to-inosine RNA editing is a post-transcriptional process, catalyzed by ADAR enzymes, with an important role in diversifying the number of proteins derived from a single gene. In neurons, editing of ionotropic AMPA glutamate receptors has been shown to be altered under several experimental conditions, including severe pathologies, thus highlighting the potential significance of its modulation. In this study, we treated rat primary cortical cell cultures with a sub-lethal dose of glutamate (10 µM), focusing on RNA editing and ADAR activity. We found that chronic glutamate treatment down-regulates RNA editing levels at the R/G site of GluA2-4 subunits of AMPA receptors and at the K/E site of CYFIP2. These changes are site-specific since they were not observed either for the GluA2 Q/R site or for other non-glutamatergic sites. Glutamate treatment also down-regulates the protein expression levels of both ADAR1 and ADAR2 enzymes, through a pathway that is Ca(2+)- and calpain-dependent. Given that AMPA receptors containing unedited subunits show a slower recovery rate from desensitization compared to those containing edited forms, the reduced editing at the R/G site may, at least in part, compensate for glutamate over-stimulation, perhaps through the reduced activation of postsynaptic receptors. In summary, our data provide direct evidence of the involvement of ADAR1 and ADAR2 activity as a possible compensatory mechanism for neuronal protection following glutamate over-stimulation.