Analysis of the Circular Transcriptome in the Synaptosomes of Aged Mice.

Recently, circular RNAs (circRNAs) have been revealed to be an important non-coding element of the transcriptome. The brain contains the most abundant and widespread expression of circRNA. There are also indications that the circular transcriptome undergoes dynamic changes as a result of brain ageing. Diminished cognitive function with increased age reflects the dysregulation of synaptic function and ineffective neurotransmission through alterations of the synaptic proteome. Here, we present changes in the circular transcriptome in ageing synapses using a mouse model. Specifically, we observed an accumulation of uniquely expressed circular transcripts in the synaptosomes of aged mice compared to young mice. Individual circRNA expression patterns were characterized by an increased abundance in the synaptosomes of young or aged mice, whereas the opposite expression was observed for the parental gene linear transcripts. These changes in expression were validated by RT-qPCR. We provide the first comprehensive survey of the circular transcriptome in mammalian synapses, thereby paving the way for future studies. Additionally, we present 16 genes that express solely circRNAs, without linear RNAs co-expression, exclusively in young and aged synaptosomes, suggesting a synaptic gene network that functions along canonical splicing activity.

RNA sequencing reveals pronounced changes in the noncoding transcriptome of aging synaptosomes.

Normal aging is associated with impairments in cognitive functions. These alterations are caused by diminutive changes in the biology of synapses, and ineffective neurotransmission, rather than loss of neurons. Hitherto, only a few studies, exploring molecular mechanisms of healthy brain aging in higher vertebrates, utilized synaptosomal fractions to survey local changes in aging-related transcriptome dynamics. Here we present, for the first time, a comparative analysis of the synaptosomes transcriptome in the aging mouse brain using RNA sequencing. Our results show changes in the expression of genes contributing to biological pathways related to neurite guidance, synaptosomal physiology, and RNA splicing. More intriguingly, we also discovered alterations in the expression of thousands of novel, unannotated lincRNAs during aging. Further, detailed characterization of the cleavage and polyadenylation factor I subunit 1 (Clp1) mRNA and protein expression indicates its increased expression in neuronal processes of hippocampal stratum radiatum in aging mice. Together, our study uncovers a new layer of transcriptional regulation which is targeted by aging within the local environment of interconnecting neuronal cells.

Vascular endothelial growth factor (VEGF) affects processing of amyloid precursor protein and beta-amyloidogenesis in brain slice cultures derived from transgenic Tg2576 mouse brain.

The up-regulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of Alzheimer patients in close relationship to beta-amyloid (Abeta) plaques, suggests a link of VEGF action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may affect APP processing, brain slices derived from 17-month-old transgenic Tg2576 mice were exposed with 1ng/ml VEGF for 6, 24, and 72h, followed by assessing cytosolic and membrane-bound APP expression, level of both soluble and fibrillar Abeta-peptides, as well as activities of alpha- and beta-secretases in brain slice tissue preparations. Treatment of brain slices with VEGF did not significantly affect the expression level of APP, regardless of the exposure time studied. In contrast, VEGF exposure of brain slices for 6h reduced the formation of soluble, SDS extractable Abeta(1-40) and Abeta(1-42) as compared to brain slice cultures incubated in the absence of any drug, while the fibrillar Abeta peptides did not change significantly. This effect was less pronounced 24h after VEGF exposure, but was no longer detectable when brain slices were exposed by VEGF for 72h, which indicates an adaptive response to chronic VEGF exposure. The VEGF-mediated reduction in Abeta formation was accompanied by a transient decrease in beta-secretase activity peaking 6h after VEGF exposure. To reveal whether the VEGF-induced changes in soluble Abeta-level may be due to actions of VEGF on Abeta fibrillogenesis, the fibrillar status of Abeta was examined using the thioflavin-T binding assay. Incubation of Abeta preparations obtained from Tg2576 mouse brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing incubation time up to 72h. The data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of VEGF in the pathogenesis of Alzheimer's disease.

New approach to calculating and predicting the ionic strength generated during carrier ampholyte isoelectric focusing.

A serious drawback of the carrier ampholyte isoelectric focusing is the undetermined ionic strength at which the proteins separate. We tried to study its effect in order to bridge the gap between theory and practice giving a quantitative and qualitative description. Using certain electrode systems, we managed to calculate the discrete values of the ionic strength in a random position between the electrodes. As a rule, higher values of up to 10(-3)mol/l were obtained near the electrodes, which gradually decreased towards the middle of the carrier reaching 10(-7)mol/l. A relationship between the measured isoelectric points (pI) of some proteins and the ionic strength was found. In the present paper, we report our findings on the dynamics of the process referring to the origin of ionic strength, its formation and alteration during the isoelectric focusing process.