Research

Recent research projects

microRNAs and circRNAs in the blood as biomarkers for Parkinson’s disease
There is a pressing need to identify and validate, minimally invasive, molecular biomarkers that will complement current practices and increase the diagnostic accuracy in Parkinson's disease (PD). miRNAs and circRNAs are stable noncoding RNAs that are increasingly shown to regulate all aspects of neuronal development and function. Τhe objectives here were to identify differentially expressed brain-derived miRNAs in the plasma and circRNAs in PBMCs of patients with PD and recognize the molecular pathways affected by these deregulated ncRNAs. In independent discovery and validation studies we identified five miRNAs while in a discovery study identified six circRNAs that are differentially expressed in PD (Figure 1).

 

Figure 1. Swarm plots of the (A) miRNAs and (B) circRNAs whose expression is significantly altered in the plasma and PBMCs of idiopathic PD patients, respectively. Mean levels +/- SEM are included below each graph.
 

Pre-mRNA splicing of transcripts
Alternative pre-mRNA splicing (AS) allows the functional proteome to qualitatively expand; new proteins are generated from the same pre-mRNA with different binding partners and functions. We have sought to determine the splicing of amyloid precursor protein (APP), a protein that plays pivotal role in the pathogenesis of Alzheimer's disease (AD). APP is spliced in three main isoforms, the APP695 which is neuronally-enriched and the more ubiquitous APP751 and APP770. APP695 is less amyloidogenic by generating lower levels of Aβ42 catabolite but its levels are decreased in AD brains. Thus, we looked for RBPs that are involved in APP splicing and discovered that the neuronal-only members of the Hu RBP family, HuB, HuC and HuD, induce APP695 generation via binding to specific intronic sites on the APP pre-mRNA (Figure 2).




Figure 2. nHu proteins promote the exclusion of exons 7 and 8 from APP mRNA (A) Schematic representation of APP AS in neurons. (B) Expression of nHu proteins correlates with the APP695-specific mRNA processing in different cell types. (C-D) Splicing products determined by RT-PCR and immunoblotting following transfection of nHu plasmids in SK-N-SH cells. (E-F) AUF1 facilitates nHu AS.


Cis-acting mRNA elements in the regulation of translation
The 5' untranslated region (UTR) of several mRNAs, particularly those with long and complex 5'UTRs are critical determinant of translation initiation. We have sought to determine the role in translation of alpha-synuclein (SNCA) 5'UTR, a protein whose high levels of expression are linked to the pathological process in Parkinson disease. We found that the long and GC-rich SNCA 5'UTR promotes translation despite the presence of multiple inhibitory motifs. This effect was not associated with nucleocytoplasmic shuttling. Additionally, we revealed an internal ribosome entry site (IRES) element that enhanced SNCA synthesis when cap-dependent translation was attenuated or when stress signals were applied. These data support the idea that the 5'UTR is an important positive regulator of SNCA synthesis under both physiological and pathological conditions explaining in part the abundance of SNCA in healthy neurons and its accumulation in degenerative cells (Figure 3).

Figure 3. SNCA 5'UTR promotes translation and harbors an IRES element (A-B) Inclusion of 5'UTR enhances CDS-3' translation without altering mRNA levels. (C) The 5'UTR mediates cap-independent translation. (D) The 5'UTR harbors an IRES element. The 5'UTR was inserted in the intercistronic region of pRF plasmid. (E) The 5'UTR mediates translation under stress.


microRNA-mediated target mRNA translation
Alternative polyadenylation allows the generation of transcripts with different 3'UTR sizes that can be quantitatively regulated by RBPs and/or miRs. Neuronally-expressed transcripts display the longest possible 3'UTR isoforms indicating that mRNA translation is tightly regulated in this cell population. SNCA mRNA is a typical such transcript with a 2,5kb long 3'UTR. Using multiple assays we demonstrated that SNCA translation is negatively modulated by the binding of the neuronally-enriched miR-7 and miR-153 on its 3'UTR (Figure 4).





Figure 4. miR-7 and miR-153 target SNCA 3'UTR to directly reduce SNCA levels. (A-B) HEK293 cells were co-transfected with different reporter and miR plasmids.

Therapeutic potential of microRNAs
Successful medical interventions based on miRs have been shown in cornerstone studies to lower plasma cholesterol levels in rodents and primates. Currently, several miRs are studied in preclinical and clinical settings. Previously, we have identified that miR-7 and miR-153 target SNCA to reduce its protein levels. To test if these miRs could, in principle, be used for therapeutic purposes we evaluated their ability to protect neurons from the MPP+ parkinsonian toxin. We found that miR-7 and miR-153 protected neurons against MPP+-induced toxicity by preserving the activation of the downstream master integrating signaling pathway of mTOR. Blocking the miR-7/153 -mediated mTORC1 activation by the inhibitor rapamycin abolished the neuroprotective response (Figure 5).



Figure 5. miR-7 and miR-153 protect neurons against MPP+- induced death via mTORC1. Cortical neurons were transduced using miR adenoviral vectors. 24h later, they were exposed for another 24h to 5-50μΜ MPP+. (A) Neuronal viability was monitored using MTT. (B-C) Protein lysates were analyzed by immunoblotting. (D) Transduced neurons were exposed to MPP+ in the absence or presence of rapamycin.