Circular RNAs as disease modifiers of complex neurologic disorders

Abstract

Circular RNAs are a large class of non-coding RNA molecules, conserved across species and produced by back-splicing. While their molecular functions are still elusive, the ones primarily retained in the nucleus are usually associated to regulation of transcription and mRNA processing patterns. Instead, the majority, are transported to the cytoplasm where they elicit micro-RNA (miRNA) or RNA binding protein (RBP)-sponging functions, or could be translated. CircRNAs are abundantly expressed in brain tissue, where they do not only act as regulators of brain development and physiology, but can also contribute to complex neurological conditions. In fact, deregulated circRNA expression levels were described in neurodevelopmental and neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease and Huntington's disease. Because of their described roles in pathology, these molecules may not only represent possible disease bio-markers, but they could even function as disease modifiers. As such, they could be targeted or protected in search of novel routes of therapeutic intervention. In this review, we highlight recent developments in the field, first discussing circRNAs involved in physiologic brain development and function, then reviewing studies that implicate circRNAs in neurodevelopmental and neurodegenerative disorders, with major attention to experimental studies exploring circRNA function and their role in neuropathologic processes. Such experimental strategies are mainly based on depletion or over-expression approaches and provide important insights into the modulatory potential of these molecules. They are relevant for clinical translation of basic research findings to drug development, possibly generating a positive impact for patients' quality of life.

Keywords: alternative splicing; circRNA; neurodegeneration; neurodevelopment; non-coding RNA.

FIGURE 1

CircRNAs as modifiers of complex brain disorders. (A) Schematic of linear splicing (top) vs back-splicing (bottom). Back-splicing leads to the formation of a covalent bond between the 3′-splice donor and a 5′-splice acceptor site of unprocessed pre-mRNAs, generating the unique back-splicing sequence and rendering the resulting circRNAs extremely resistant against degradation through exoribonucleases. (B) CircRNAs are evolutionarily conserved and especially abundant in the nervous system of a large number of different species and especially mammals. Globally, circRNA expression increases during brain development, maturation and aging (bottom). In the last decade, numerous studies have linked deregulation of a variety of circRNAs with both, neurodevelopmental disorders (NDDs) such as Autism Spectrum Disorders and Schizophrenia (left); neurodegenerative (NDG) processes such as Alzheimer’s, Parkinson’s and Huntington’s Disease, as well as Frontotemporal dementia and Amyotropic lateral sclerosis (right); and other complex brain disorders such as Epilepsy, Depression and brain tumor development (top). Relevant circRNAs (in red) have been shown to regulate the cell cycle of neural stem cells (radial glia -RG, intermediate progenitors–IP), the migration of newborn neurons (MN), the differentiation into inhibitory (IN) and excitatory (EN) post-mitotic neurons, astrocytes (A), microglia (MG), and oligodendrocytes (O), synaptogenesis, and mechanisms involved in mature brain function, homeostasis and survival- synaptic transmission and plasticity, neuronal and dendritic morphology, as well as brain immune activation.

FIGURE 2

Moving forward: steps towards integrating results from circRNA basic research with the clinics of complex neurologic disorders. 1. Numerous RNA sequencing studies have found deregulated circRNA expression levels in samples derived from human patients. However, much fewer studies were able to experimentally validate a functional role and its molecular mechanism for the identified circRNAs in the pathophysiologic processes of the respective disorder. Future studies need to address this current gap in knowledge to avoid fallacy of causation. 2. This requires high quality follow up-experiments to identify circRNA regulatory functions. Experimental approaches can be based on circRNA depletion as well as over-expression. Both strategies face methodological difficulties unique to the circRNA field. CircRNA depletion approaches need to be highly specific to exclusively target the circRNA without simultaneously changing the linear RNA counterparts, or other unintended off-targets. Targeting circRNAs is especially difficult as sequence specificity is restricted to the back-splice junction, while genomic removal of sites required for back-splicing may unintentionally also alter linear splicing dynamics. Over-expression approaches, instead, can lead to difficult interpretation, since linear cognate RNAs can be produced (from plasmids), or co-transfected with in vitro transcribed or synthetized circRNAs; unintended immune activation may be induced, while over-expressed circRNA levels may reach extremely high levels leading to cellular effects that could be irrelevant in the physiologic context. 3. If depletion or over-expression of a circRNA of interest produces an effect, the underlying molecular mechanism should be investigated. A number of different molecular mechanisms have been proposed for circRNAs, including sequestration of RNA-binding proteins or activity as protein scaffolds, sponging of miRNAs as well as the production of circRNA related peptides through translation. When molecular mechanisms of function are investigated, it is of utmost importance to first determine endogenous expression levels of the involved components in the disorder of interest, to be able to draw physiologically relevant conclusions. 4. Finally, findings obtained from studying in vivo and in vitro model systems need to be translated back to the clinics in order to be useful for patients suffering from complex neurologic disorders. To that end, evaluation of a true contribution of the circRNA molecular function to the pathophysiologic processes, either on initiation or progression of the disease or modulation thereof, and/or its potential as biomarker as well as novel drug target need to be carefully assessed and tested.