Small ncRNA binding protein, PIWI: A potential molecular bridge between blood brain barrier and neuropathological conditions

Journal Pre-proofs Small ncRNA binding protein, PIWI: A potential molecular bridge between Blood Brain Barrier and Neuropathological conditions Rupa Roy, Sambhavi Pattnaik, Suganya Sivagurunathan, Subbulakshmi Chidambaram PII: DOI: Reference:

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Please cite this article as: R. Roy, S. Pattnaik, S. Sivagurunathan, S. Chidambaram, Small ncRNA binding protein, PIWI: A potential molecular bridge between Blood Brain Barrier and Neuropathological conditions, Medical Hypotheses (2020), doi: https://doi.org/10.1016/j.mehy.2020.109609

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Small ncRNA binding protein, PIWI: A potential molecular bridge between Blood Brain Barrier and Neuropathological conditions Rupa Roy1, Sambhavi Pattnaik1, Suganya Sivagurunathan2 and Subbulakshmi Chidambaram1* 1Department

of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, India.

2R.S.

Mehta Jain Department of Biochemistry and Cell Biology, KBIRVO, Vision Research Foundation, Chennai, India

*Correspondence: [email protected]

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ABSTRACT The blood brain barrier (BBB) is a neuroprotective layer that maintains the homeostasis of central nervous system and provides an appropriate environment for neurons to execute their functions. The fundamental role of the dynamic semi-permeable BBB is selective and stringent transport of molecules from circulating blood and surrounding extracellular matrix across brain. Disruption of BBB has critical implications that can lead to various neuropathological disorders (NPDs) namely multiple sclerosis, Alzheimer’s disease, epilepsy, traumatic brain injuries and neuropsychiatric disorders, etc. Therapeutic management of NPDs is still a daunting challenge in the field of neuromedicine and there is a great need for identifying novel drug targets and biomarkers. Recently, noncoding RNAs (ncRNA) have emerged as promising prognostic markers in NPDs. Piwi interacting RNAs (piRNA), a family of short noncoding RNAs which in association with PIWI-like proteins have shown to regulate neuronal function and memory formation. In addition, piRNAs are differentially expressed in Alzheimer’s brain tissues and studies also revealed the association of denovo mutations in PIWI genes with autism. Moreover, the role of PIWI-like proteins in neuronal long-term potentiation and neurite outgrowth is now evident, confirming their importance in normal physiology of the brain. Notably, we have reported the significance of PIWI-like proteins in the maintenance of Blood Retinal Barrier (BRB) and its potential role in diseases like diabetic retinopathy through modulation of tight junction proteins. Further studies in hydra and cancer cells confirmed the important function of PIWI-like proteins in the organization of tight junctions. Interestingly, we also observed that loss of PIWI-like proteins affected the activity of Ephrin receptors which have an established functional link to tight junction assembly. Collectively, the evidences profoundly support the novel concept that piRNAs/PIWI-like proteins may have a potential role on the governance of 2

BBB by altering the tight junctions through Ephrin effectors, commotion of which could be the preceding event to various NPDs. Here, we propose PIWI-like proteins and associated piRNAs as potential restorative drug targets for combating neuropathological conditions. INTRODUCTION BBB is an integral part of neurovascular unit (NVU) basically composed of endothelial cells, pericytes, astrocytes and a non-cellular component, the basement membrane [1]. These essential components of BBB are involved in the biology of vascular and brain pathologies [2]. Endothelial Cells (ECs) are connected by tight junctions (TJs) which include integral membrane proteins like occludin, claudins and junctional adhesion molecules (JAMs) which make the intercellular contacts and interact with cytoplasmic scaffolding proteins such as zonula occludens (ZO), actin cytoskeleton and other associated proteins, such as protein kinases, small GTPases and heterotrimeric G-proteins [3][4][5][6]. ZO-1 binds with most of the transmembrane proteins [7][8] and connects actin cytoskeleton for stabilizing the TJs [9]. Dissociation of ZO-1 from the junctional complex often results in increased barrier permeability [10]. TJs are enriched in the endothelium of the brain microvasculature; it seals the paracellular space, limits permeability and maintains polarity [5]. Integrity of BBB is altered in many neurological conditions and has severe impact on the cognitive and behavioral phenotypes [11][3]. Blood Brain Barrier and Neuropathological Disorders There are evidences of BBB leakiness in NPDs with elevation of inflammatory molecules indicating it as a hallmark [12][13][14][15]. Recent studies have suggested the implication of neurovascular unit (NVU) dysfunction in the etiology of mood disorders, autism spectrum disorder (ASD), and schizophrenia [11][15][16][17]. Clinical and pre-clinical research has 3

shown an association of claudin-5, a TJ protein to above mentioned disorders [18][19][20]. Suppression of claudin-5 has been attributed with psychosis like symptoms including memory impairment and increase in anxiety [21]. Furthermore, it has been recently reported that stress induces changes in BBB permeability and contributes to depression pathology [22]. These findings have suggested a link between BBB and neurovascular alteration, peripheral immune activation and depression. Another vital study reported that the loss of claudin-5 altered BBB and this in turn allowed the passage of IL-6 into brain parenchyma, inducing depression like phenotypes [21]. Similarly, the connection between BBB and NVU dysfunction with depression has been reported in various animal models [11][23][24][25] reinstating that disruption of BBB has serious implications on neurons. Hadar Shalev and his team have shown that loss of BBB integrity causes leakage of serum derived components into the brain tissue, abnormal blood-brain communication and neuronal dysfunction. This eventually leads to disturbed thinking process, mood, and behavior [26]. Although, there is a potential correlation between BBB and NPDs, there are only very few studies that validated the concept. Therefore, deciphering the unraveled molecular mechanisms governing BBB homeostasis is important for developing better therapeutic approach to curb NPDs. PIWI like proteins and Piwi-interacting piRNA in CNS PIWI (P-element Induced Wimpy Testis), a subset of argonaute family proteins play a key role in the biogenesis and functions of piRNAs, which belong to small non coding RNAs [27][28]. In human, four orthologues of PIWI-like proteins namely Hili, Hiwi1, Hiwi2 and Hiwi3 are present [29]. PIWI-like protein orthologues are widely expressed in many organs, however, testis and 4

brain have highest expression [30][29][31][32][33][34]. Earlier studies demonstrated the significance of PIWI-like proteins and piRNAs extensively in germ line cells [35] but recent reports have indicated their expression in somatic cells, especially neurons, and their role in gene expression, cell cycle progression, proliferation, differentiation and cell survival signaling etc. [34][36][37][38][39][40][41][42]. They regulate diverse functions like memory and synaptic plasticity, regenerative processes, fat metabolism homeostasis and insulin secretion [30][43][44][45][46]. Rajasethupathy et al., deciphered the importance of Piwi/piRNAs in Aplysia brain, in the regulation of long term changes in neurons for the persistence of memory through epigenetic modifications [45]. Expression of Miwi (Piwi homologue in mouse) has been observed in several brain regions including hippocampus [47]. Knockdown studies revealed that Piwi/piRNA can also act as modulators of dendritic spine development [41]. Astrotactin, one of the targets of Piwi/piRNA complex has been shown to be responsible in neuronal migration. In addition, Piwi/piRNA regulates spinogenesis through Cdk5rap1, microtubule affinity-regulating kinase 1/2 (Mark1/2) and AKAP79/150 [48][49][50]. Recently, a group of researchers performed genome-wide association studies (GWAS) and genome-wide by environment interaction studies (GWEIS) of depressive symptoms and stressful life events (SLE) in two UK population-based cohorts where it was found that PIWIL4 harnessed mutation. They have also speculated that as PIWIL4 is expressed in brain and involved in the modification of chromatin, it may exaggerate the effects of stress induced depression [51]. Recently, we demonstrated the critical role of PIWI-like proteins in the regulation of BRB via TJs in human retinal pigment epithelium [40]. Further, we showed the increased level of PIWIL4 in the vitreous from patients with proliferative diabetic retinopathy (PDR) [52]. In addition, we also reported that PIWIL4 is highly elevated in retinoblastoma cells and also controls cell cycle 5

[53]. Our recent work showed that Piwi/piRNA could have a role in retinal degeneration [54]. Interestingly, our preliminary studies found a functional link between Piwi-like proteins with receptor tyrosine kinase (RTK) family receptors, Ephrin (Unpublished data). EphR/ephrin signaling system The Erythropoietin-Producing-Hepatocellular Carcinoma Receptors (EphR) are the largest subfamily of RTK that interact with their corresponding Eph receptor-interacting signal ligands (ephrin) which are cell surface proteins. EphR is categorized into two classes: EphA and EphB. Human genome includes nine EphA receptors that promiscuously binds to five ephrin-A ligands and five EphB receptors binding to three ephrin-B ligands [55]. EphA receptors can also cross talk with few ephrin-B ligands and are attached to the cell surface through a glycosylphosphatidylinositol (GPI) linkage whereas EphB receptors are transmembrane proteins with a short cytoplasmic tail [56]. A characteristic feature of EphR/ephrin complexes is their ability to produce bidirectional signaling. It involves forward signaling generated in the Eph (receptor) expressing cell upon activation of kinase domain and reverse signaling is generated in the ephrin (ligands) expressing cells by phosphorylation by Src family kinase [57][58][59]. Diverse expression of EphR/ephrin complexes has been observed in variety of tissues of a developing embryo with roles in cardiovascular and skeletal development, axon guidance and tissue patterning [60]. They are also involved in an array of physiological functions in adult namely, learning and memory, cytoskeleton dynamics, neuronal plasticity, spine morphogenesis and dendrite growth [56][59][61][62][63][64][65][66]. EphR/ephrin complexes control these biological functions and modulate cell behavior by cross regulating various signaling pathways. Dysregulation of EphR/ephrin signaling has been implicated in various neurological diseases like Alzheimer’s

disease,

amyotrophic

lateral

sclerosis

6

(ALS)

and

parkinson’s

disease

[59][67][68][69]. It has been also reported that stimulated EphB2 attenuates phosphorylation of tau proteins via PI3K/AKT pathway in Alzheimer’s disease [70]. Malick et al., documented various roles of EphR/ephrin system in BBB and their implication in neuropsychiatric disorders. However, molecular mechanism by which these proteins regulate BBB is still not clear [3]. Therefore, it is important to investigate the mechanisms and signaling pathways that link BBB and NPDs. HYPOTHESIS BBB breakdown has already been shown to have a link with many NPDs though the exact mechanism is not elucidated so far. Our recent work demonstrated that PIWIL4 (HIWI2) regulates TJ assembly through modulation of AKT/GSK3 pathway in retinal cells [40]. In addition, we showed that PIWIL4 was highly elevated in the vitreous samples from patients with PDR where BRB breakdown is the initial event [51]. Another important observation was that PIWIL4 knockdown showed altered expression of proteins involved in phagocytosis and vesicular transport that are important for proper tight junction assembly [54]. Moreover, we observed that loss of PIWIL4 strongly altered the activity of specific EphR which are known modulators of TJ organization. Taken together, we hypothesize that PIWI-like proteins might be regulating the activation of EphR/ephrin signaling to effect alterations in TJ to maintain the functional integrity of BBB. During pathological conditions, for instance in retinopathy, PIWIL4 levels are elevated which could compromise BRB/BBB homeostasis resulting into increased infiltration of inflammatory molecules which may finally lead to an array of neurovascular diseases. Further, molecular level link between TJ and PIWI-like proteins could be explained based on earlier studies demonstrating PIWI/piRNAs control on gene regulation through epigenetic, transcriptional and post transcriptional modifications [35][45][46]. We

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speculate that PIWI/piRNA could be the master regulator of TJ assembly and BBB homeostasis acting through the downstream effectors like EphR/ephrin. As therapeutic approaches against NVDs are still in infancy, this new concept could be useful as future target in treating NVDs or diseases associated with BBB.

RATIONALE BEHIND THE HYPOTHESIS The role of EphR/ephrin in regulating the growth and differentiation of embryonic cells and formation of efficient BBB has been well established. Expression levels of EphA2 regulates the switch between angiogenic and barrigenic effects of EphA2 in ECs of brain [71]. During embryonic development, astrocytes release VEGF that has vital role in tight junction formation. In the later stages of development, inhibition of EphA2 activity in microvessel ECs of human brain is required to promote tight junction strengthening [3], glial cell activation and its interaction with ECs lead to BBB disintegration by impairment of TJs [72]. It has been reported that ephrinA1 ligand induced EphA2 phosphorylation altered the distribution of occludin and ZO-1 in human brain micro vascular endothelial cells (HMBEC). These are important tight junction associated molecules which showed discontinuous cellular distribution on EphA2 activation, confirming EphA2/ephrinA1 involvement in the regulation of endothelial TJ in HMBEC and thus paracellular permeability [71]. EphA2 has also shown to regulate claudin, a fundamental component of the endothelial TJs in BBB. EphA2 phosphorylates the cytoplasmic tail of claudin-4 at tyrosine residues, attenuates the association of claudin-4 and ZO1 and decreases the integration of claudin-4 at cell-cell contact which affects cell permeability [73].

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Similarly, it has been reported that the cells expressing altered levels of EphA4 lack ZO proteins, thus TJ formation was disrupted by failed polymerization of claudin. Studies have shown that over expression of EphR/ephrin disturbs brain homeostasis by influencing barrier integrity. Putative interactions between EphA2 expressing ECs with ephrin-A1 expressing perivasular astrocytes or pericytes confirmed their role in modulating the TJ formation [72]. In addition, knockdown of EphB2 increases the vulnerability to stress and depressive condition. These are further associated with alterations in phosphorylation of cofilin and synaptic proteins like PSD95, synapsin I, GluR1 and GluR2 in medial prefrontal cortex [74]. Reduced expression of claudin-5 has been observed in chronic social stress mouse model with reduced BBB integrity and infiltration of IL-6 cytokines. This led to subsequent presentation of depression-like behaviors [21]. These studies suggest a link between EphB2 and stress with regard to BBB sealing properties and effects on behavioral phenotypes which is yet to be elucidated in detail. Thus, over activation of EphR/ephrin signaling and increased stress is correlated with barrier permeability and impaired TJs which induces the onset of neurovascular disorders. However, the prime mediators modulating the signaling pathways are still unknown [3]. The triad connection between PIWI-like proteins, EphR/ephrin and BBB: Importance of PIWI-like proteins in modulating tight junction through AKT pathway has already been reported by our group. HIWI2 knockdown altered the expression of TJs, claudin1 and TJP1 by escalating the phosphorylation status of AKT and GSK3β [40]. We have also shown an association of HIWI2 with PDR where breakdown of BRB plays a critical role; however, there are no studies on the potential role of PIWI-like proteins in BBB and the effect on NPDs. Evidences point out the significance of EphA4 and its downstream protein ephexin in the

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implication of depression [75]. Furthermore, denovo mutation of PIWIL2 and PIWIL4 has been reported in ASD [76]. Also, it has been found that deletion of MILI (Piwi-like proteins in mouse) contributed to hyperactive and reduced anxiety like behavior in mouse [31]. Moreover, disruption of PIWI-like proteins increased the contextual fear memory showing their importance in the hippocampus region [47]. A recent study has depicted that non-coding RNAs do have a significant role in glioma conditioned BBB [77]. Mechanism connecting PIWI-like proteins and EphR/ephrin or their combined effect in maintaining BBB is yet to be elucidated. Also, the role of PIWI-like proteins in NPDs via BBB is still not deciphered. Based on available reports and our preliminary experimental data, we speculate that PIWI-like proteins could modulate the TJs and sealing properties of BBB via Eph/ephrin signaling. NPDs, including stress and depression are the increasing problems in current scenario. In future, these diseases can have a huge impact in mass population, thus deeper insight and new convincing approaches are needed to curb or reduce the health issues. Therefore, it would be interesting to unearth the underlining mechanisms of PIWI-like proteins and EphR/ephrin in the regulation of TJs and thus BBB. VALIDATION OF HYPOTHESIS To justify our hypothesis, genetic manipulations of EphR and PIWI in animal models could be generated to recapitulate the effects of NPDs to study the molecular and behavioral phenotypes. Further rescue experiments and BBB permeability assays in the animals will substantiate the perturbed triad molecular link to NPDs. In addition, a detailed analysis of genomic and proteomics data from patients with NPDs can shed more light and strengthen our concept. In

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vitro experiments using BBB models like spheroid or organoid cultures can provide further insights into molecular changes and identify the novel effector molecules. CONCLUSION BBB is one of the critical and vulnerable barriers of the brain which should be stabilized and synchronized to maintain the physiological milieu. Previous studies have shown that PIWI-like proteins are involved in stabilizing BRB and Brain Tumor Barrier (BTB) through alteration of TJ proteins. However, the underlining mechanism is largely unexplored. Here, we propose a novel link between the small noncoding RNA binding PIWI-like proteins and the bidirectional signaling receptors, EphR in the regulation of TJ and BBB integrity which is compromised in various neuropathological conditions (Fig 1). Furthermore, it would be riveting to check whether PIWI-like proteins and EphR/ephrin can be used as a drug target for various brain and mood related disorders. Acknowledgement: We acknowledge the financial assistance provided by Department of Biotechnology under the project DBT-Neurobiology Taskforce - “BT/PR5055/MED/30/761/2012” and Indian Council of Medical Research (ICMR) project NO. 5/4/6/1/OPH/2015-NCD-II. The funding body is not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. RR thanks the Department of Science and Technology (DST) for the fellowship under the project NO.SR/WOS-A/LS-388/2018. CS thanks the support from University Grants Commission, New Delhi for the award of Assistant Professorship under its Faculty Recharge Program (UGC-FRP).

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Figure Legend: Fig.1: Proposed role of PIWI-like proteins in NPDs: We hypothesize that the integrity of Blood Brain Barrier (BBB) might be regulated by PIWI-like proteins through the modulation of tight junction molecules via EphR/ephrin, the bidirectional receptor system. Besides, the effect of PIWI-like proteins on BBB may have a significant impact on neuropathological disorders.

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Conflict of Interest

All the authors have declared no conflict of Interest.

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