Prameet Kaur

Circulating MicroRNAs as Biomarkers of Acute Stroke

MicroRNAs have been identified as key regulators of gene expression and thus their potential in disease diagnostics, prognosis and therapy is being actively pursued. Deregulation of microRNAs in cerebral pathogenesis has been reported to a limited extent in both animal models and human. Due to the complexity of the pathology, identifying stroke specific microRNAs has been a challenge. This study shows that microRNA profiles reflect not only the temporal progression of stroke but also the specific etiologies. A panel of 32 microRNAs, which could differentiate stroke etiologies during acute phase was identified and verified using a customized TaqMan Low Density Array (TLDA). Furthermore we also found 5 microRNAs, miR-125b-2*, -27a*, -422a, -488 and -627 to be consistently altered in acute stroke irrespective of age or severity or confounding metabolic complications. Differential expression of these 5 microRNAs was also observed in rat stroke models. Hence, their specificity to the stroke pathology emphasizes the possibility of developing these microRNAs into accurate and useful tools for diagnosis of stroke.

microRNAs Involved in Regulating Spontaneous Recovery in Embolic Stroke Model

To date, miRNA expression studies on cerebral ischemia in both human and animal models have focused mainly on acute phase of ischemic stroke. In this study, we present the roles played by microRNAs in the spontaneous recovery phases in cerebral ischemia using rodent stroke models. Brain tissues were harvested at different reperfusion time points ranging from 0–168 hrs after middle cerebral artery occlusion using homologous emboli. MiRNA and mRNA expression profiles were investigated by microarray followed by multiple statistical analysis. Candidate transcripts were also validated by quantitative RT-PCR. Three specific groups of miRNAs were observed among a total of 346 differentially expressed miRNAs. miRNAs, miR-21, -142-3p, -142-5p, and -146a displayed significant upregulation during stroke recovery (48 hrs to 168 hrs) compared with those during acute phases (0 hrs to 24 hrs). On the other hand, an opposite trend was observed in the expression of miR-196a/b/c, -224 and -324-3p. Interestingly, miR-206, -290, -291a-5p and -30c-1*, positively correlated with the infarct sizes, with an initial increase up to 24hrs followed by a gradual decrease from 48 hrs to 168 hrs (R = 0.95). Taken together with the expression levels of corresponding mRNA targets, we have also found that Hedgehog, Notch, Wnt and TGF-β signaling pathways could play significant roles in stroke recovery and especially in neuronal repair.

MicroRNAs in Neurotoxicity

MicroRNAs are gaining importance as regulators of gene expression with the capability to fine-tune and modulate cellular events. The complex network with their selective targets (mRNAs/genes) pave way for regulation of many physiological processes. Dysregulation of normal neuronal activities could result in accumulation of substances that are detrimental to neuronal functions and subsequently result in neurotoxicity. Neurotoxicity-mediated pathophysiological conditions could then manifest as diseases or disabilities like Parkinsons and Alzheimers which have debilitating implications. Such toxicity can be a result of individuals predisposed due to genetic inheritance or from other sources such as brain tumours. Neurotoxicity can also be brought about by external agents like drugs and alcohol as well as brain injury with miRNAs playing a pivotal role in diseases. It is therefore vital to understand the expression of these microRNAs and their impact on neuronal activities. In this paper, we discuss some of the neuronal pathophysiological conditions that could be caused by dysregulated microRNAs.

MiR-335 Regulates Hif-1α to Reduce Cell Death in Both Mouse Cell Line and Rat Ischemic Models.

Hypoxia inducible factor-1α facilitates cellular adaptation to hypoxic conditions. Hence its tight regulation is crucial in hypoxia related diseases such as cerebral ischemia. Changes in hypoxia inducible factor-1α expression upon cerebral ischemia influence the expression of its downstream genes which eventually determines the extent of cellular damage. MicroRNAs are endogenous regulators of gene expression that have rapidly emerged as promising therapeutic targets in several diseases. In this study, we have identified miR-335 as a direct regulator of hypoxia inducible factor-1α and as a potential therapeutic target in cerebral ischemia. MiR-335 and hypoxia inducible factor-1α mRNA showed an inverse expression profile, both in vivo and in vitro ischemic conditions. Given the biphasic nature of hypoxia inducible factor-1α expression during cerebral ischemia, miR-335 mimic was found to reduce infarct volume in the early time (immediately after middle cerebral artery occlusion) of embolic stroke animal models while the miR-335 inhibitor appears to be beneficial at the late time of stroke (24 hrs after middle cerebral artery occlusion). Modulation of hypoxia inducible factor-1α expression by miR-335 also influenced the expression of crucial genes implicated in neurovascular permeability, cell death and maintenance of the blood brain barrier. These concerted effects, resulting in a reduction in infarct volume bring about a beneficial outcome in ischemic stroke.

Non-Coding RNAs as Potential Neuroprotectants against Ischemic Brain Injury.

Over the past decade, scientific discoveries have highlighted new roles for a unique class of non-coding RNAs. Transcribed from the genome, these non-coding RNAs have been implicated in determining the biological complexity seen in mammals by acting as transcriptional and translational regulators. Non-coding RNAs, which can be sub-classified into long non-coding RNAs, microRNAs, PIWI-interacting RNAs and several others, are widely expressed in the nervous system with roles in neurogenesis, development and maintenance of the neuronal phenotype. Perturbations of these non-coding transcripts have been observed in ischemic preconditioning as well as ischemic brain injury with characterization of the mechanisms by which they confer toxicity. Their dysregulation may also confer pathogenic conditions in neurovascular diseases. A better understanding of their expression patterns and functions has uncovered the potential use of these riboregulators as neuroprotectants to antagonize the detrimental molecular events taking place upon ischemic-reperfusion injury. In this review, we discuss the various roles of non-coding RNAs in brain development and their mechanisms of gene regulation in relation to ischemic brain injury. We will also address the future directions and open questions for identifying promising non-coding RNAs that could eventually serve as potential neuroprotectants against ischemic brain injury.

Expression profiling of RNA transcripts during neuronal maturation and ischemic injury.

Neuronal development is a pro-survival process that involves neurite growth, synaptogenesis, synaptic and neuronal pruning. During development, these processes can be controlled by temporal gene expression that is orchestrated by both long non-coding RNAs and microRNAs. To examine the interplay between these different components of the transcriptome during neuronal differentiation, we carried out mRNA, long non-coding RNA and microRNA expression profiling on maturing primary neurons. Subsequent gene ontology analysis revealed regulation of axonogenesis and dendritogenesis processes by these differentially expressed mRNAs and non-coding RNAs. Temporally regulated mRNAs and their associated long non-coding RNAs were significantly over-represented in proliferation and differentiation associated signalling, cell adhesion and neurotrophin signalling pathways. Verification of expression of the Axin2, Prkcb, Cntn1, Ncam1, Negr1, Nrxn1 and Sh2b3 mRNAs and their respective long non-coding RNAs in an in vitro model of ischemic-reperfusion injury showed an inverse expression profile to the maturation process, thus suggesting their role(s) in maintaining neuronal structure and function. Furthermore, we propose that expression of the cell adhesion molecules, Ncam1 and Negr1 might be tightly regulated by both long non-coding RNAs and microRNAs.

Pro-domain in precursor nerve growth factor mediates cell death.

Nerve growth factor (NGF) is synthesized as a precursor, proNGF that undergoes post-translational processing to generate the biologically active mature NGF. While the neurotrophic function of NGF is well established, the activity of the proNGF precursor is still unclear. In this study, we have cloned the pro-domain of the precursor NGF molecule and have elucidated its function. We have used both mature and the furin resistant pro((R/G))NGF as controls in our experiments. Both pro((R/G))NGF and mature NGF (NGF) exhibited neurotrophic activity on PC12 cells while the pro-domain itself promoted cell death. The pro-domain, has been found to mediate apoptosis possibly by promoting the formation of a signaling complex comprising of endogenous p75(NTR) receptor, Bim/Bcl2 group of proteins and JNK and MEK1/2 signaling pathways.

Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis

Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of Drosophila embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila β–catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of β–catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

An embryonic system to assess direct and indirect Wnt transcriptional targets

During animal development, complex signals determine and organize a vast number of tissues using a very small number of signal transduction pathways. These developmental signaling pathways determine cell fates through a coordinated transcriptional response that remains poorly understood. The Wnt pathway is involved in a variety of these cellular functions, and its signals are transmitted in part through a β-catenin/TCF transcriptional complex. Here we report an in vivo Drosophila assay that can be used to distinguish between activation, de-repression and repression of transcriptional responses, separating upstream and downstream pathway activation and canonical/non-canonical Wnt signals in embryos. We find specific sets of genes downstream of both β-catenin and TCF with an additional group of genes regulated by Wnt, while the non-canonical Wnt4 regulates a separate cohort of genes. We correlate transcriptional changes with phenotypic outcomes of cell differentiation and embryo size, showing our model can be used to characterize developmental signaling compartmentalization in vivo.

Importance of miRNA stability and alternative primary miRNA isoforms in gene regulation during Drosophila development

Mature microRNAs (miRNAs) are processed from primary transcripts (pri-miRNAs), and their expression is controlled at transcriptional and post-transcriptional levels. However, how regulation at multiple levels achieves precise control remains elusive. Using published and new datasets, we profile a time course of mature and pri-miRNAs in Drosophila embryos and reveal the dynamics of miRNA production and degradation as well as dynamic changes in pri-miRNA isoform selection. We found that 5’ nucleotides influence stability of mature miRNAs. Furthermore, distinct half-lives of miRNAs from the mir-309 cluster shape their temporal expression patterns, and the importance of rapid degradation of the miRNAs in gene regulation is detected as distinct evolutionary signatures at the target sites in the transcriptome. Finally, we show that rapid degradation of miR-3/–309 may be important for regulation of the planar cell polarity pathway component Vang. Altogether, the results suggest that complex mechanisms regulate miRNA expression to support normal development.