Role of autophagy and transcriptome regulation in acute brain injury

Abstract

Autophagy is an evolutionarily conserved intracellular system that routes distinct cytoplasmic cargo to lysosomes for degradation and recycling. Accumulating evidence highlight the mechanisms of autophagy, such as clearance of proteins, carbohydrates, lipids and damaged organelles. The critical role of autophagy in selective degradation of the transcriptome is still emerging and could shape the total proteome of the cell, and thus can regulate the homeostasis under stressful conditions. Unregulated autophagy that potentiates secondary brain damage is a key pathological features of acute CNS injuries such as stroke and traumatic brain injury. This review discussed the mutual modulation of autophagy and RNA and its significance in mediating the functional consequences of acute CNS injuries.

Introduction

Acute brain injuries such as stroke (ischemic or hemorrhagic) and traumatic brain injury (TBI) are leading causes of mortality and morbidity worldwide. It is estimated that ~3 million Americans are affected annually by stroke and TBI, with the incidence of a stroke-related death every 4 minutes and an overall economic cost of >110 billion dollars per year (Kandell et al., 2020; Virani et al., 2020). In 2017, TBI alone contributed to >60,000 deaths with a substantial increase in disability-adjusted life years in the United States (Daugherty et al., 2019). Moreover, the available treatment options for the management of these conditions are limited and are merely palliative, rather than therapeutic. One of the reasons for the lack of therapeutic modalities for acute central nervous system (CNS) injuries might be due to complex molecular and cellular events that include, but not limited to, oxidative stress, neuroinflammation, excitotoxicity, impaired cerebrovascular integrity, apoptotic/necrotic signaling, altered transcriptome and uncontrolled autophagy; all these direct the secondary brain damage following either a physiological injury in case of stroke or a mechanical injury in case of TBI (George and Steinberg, 2015; Kim et al., 2018; Mehta et al., 2007; Samal et al., 2015; VanGilder et al., 2012). There is a need to find newer mechanisms to design effective new therapies. In this review, we focused on the possibility of autophagy and transcriptome regulation in modifying biochemical and functional outcomes following stroke and TBI.

Autophagy is an evolutionarily conserved intracellular response that is crucial for maintaining cellular homeostasis and will be triggered upon nutrient deprivation or altered cellular homeostasis, where cytoplasmic cargo is routed to lysosomes for degradation or recycling (Nakatogawa et al., 2009). Autophagy is categorized into 3 forms viz., macroautophagy, microautophagy and chaperone-mediated autophagy (Glick et al., 2010). Macroautophagy is a multistep process mediated by autophagy-related proteins (Atg’s) and vacuoles that deliver cargo to lysosomes. In microautophagy, cytoplasmic constituents are sequestered into intralysosomal vesicles and in chaperone-mediated autophagy, chaperones bind and translocate soluble cytosolic proteins to lysosomes (Mizushima, 2007). Dysregulated autophagy is linked to diverse pathological conditions such as infections, malignancy, metabolic diseases, age-related disorders and neurodegeneration (Levine and Kroemer, 2008). In the context of acute brain injuries such as stroke, cerebral hemorrhage and TBI, autophagy acts as a double-edged sword in regulating neuronal homeostasis (Galluzzi et al., 2016). On the other hand, altered transcriptome (the entire RNA repertoire) also regulates the behavioral, biochemical and functional outcomes after acute brain injuries (Cho et al., 2020). Recent evidence suggests that autophagy (RNautophagy) influences many classes of RNAs, and thus impacts RNA landscape of a cell under stressful conditions (Frankel et al., 2017). Furthermore, noncoding RNAs (ncRNAs) regulate autophagy in the pathogenesis of stroke and TBI (Luo et al., 2020; Sun et al., 2018). Hence, understanding the molecular mechanisms by which this bidirectional interaction of autophagy and transcriptome alters secondary brain damage following stroke and TBI is essential to design therapeutic candidates to minimize severity of functional consequences.

RNautophagy shapes the transcriptome abundance

Autophagy maintains energy balance during development and in response to nutrient deprivation that ensure cellular homeostasis. Of the different biological macromolecules degraded or recycled by autophagy, much attention has been focused to proteins than nucleic acids (Fujiwara et al., 2017). However, amino acid deprivation can induce RNA degradation by autophagy, and autophagic bodies concentrated with ribosomes along with rough endoplasmic reticulum, mitochondria, granules of lipids and glycogen suggest the role of autophagy in RNA degradation (Mortimore et al., 1989; Takeshige et al., 1992). At present, the precise mechanisms by which autophagy regulates RNA homeostasis is poorly characterized.

Autophagy-dependent RNA catabolism was demonstrated in yeast deprived of nitrogen (Huang et al., 2015). Nitrogen starvation in wild-type yeast resulted in depletion of amino acids that increased intracellular levels of nucleosides up to 1h and subsequent decline after 4h, however, such a transient elevation in nucleosides was not seen in the atg2 defective cells (Huang et al., 2015). Autophagy induction due to nitrogen starvation leads to sequestration of rRNA to the vacuoles via autophagosomes followed by RNA cleavage by T2-like ribonuclease, Rny1 that generates 3´ nucleotides (Huang et al., 2015). Subsequently, alkaline phosphatase, Pho8 converts nucleotides to nucleosides that were further cleaved to purine and pyrimidine bases by purine nucleoside phosphorylase 1 and uridine-ribohydrolase 1, and ultimately converted to de-aminated forms, xanthine and uracil that are excreted into the extracellular medium (Huang et al., 2015). These dynamic changes in bulk RNA degradation by autophagy in higher organisms needs to be explored. Moreover, the selective autophagy of other type of RNA species, including tRNA, mRNA, and various classes of non-coding RNAs is yet to be identified.

Defects in RNA turnover due to functional mutations in ribonucleases like Ribonuclease T2 are linked to the development of neurological lesions, leukoencephalopathy and severe lysosomal storage disorders (Haud et al., 2011; Henneke et al., 2009). Some of the defective RNAs are trafficked to lysosomes for degradation by RNautophagy, which is an ATP-dependent process that uses a lysosomal membrane protein, lysosome-associated membrane protein 2C, which is highly conserved and was shown to bind to all types of RNAs from mouse brain (Fujiwara et al., 2013). Similarly, another lysosomal transmembrane protein, systemic RNA interference deficient-1 transmembrane family, member 2 (SIDT2) was found to be essential for delivering the RNA directly into lysosomes (Aizawa et al., 2016). LAMP2 is a type I transmembrane protein with short cytosolic C-terminus and heavily glycosylated luminal N-terminus domain, which maintains integrity of lysosomal membrane and regulates autophagy (Saftig and Klumperman, 2009). Enhanced expression of SIDT2 was shown to upregulate RNautophagy even in LAMP2 knockout cells suggesting that RNA translocation into lysosomes by SIDT2 is independent of LAMP2 (Aizawa et al., 2016). Importantly, SIDT2 knockdown attenuated ~50% of total RNA degradation, suggesting the critical role of SIDT2 in RNautophagy (Aizawa et al., 2016).

Autophagy-driven degradation of mRNAs was also demonstrated in yeast challenged with rapamycin (Makino et al., 2021). In yeast, mRNAs encoding amino acid biosynthesis and ribosomal proteins were preferentially transferred to the vacuole for degradation by autophagy (Makino et al., 2021). Furthermore, continuous ribosomal interaction is associated with selective mRNA trafficking to vacuoles, and autophagic degradation of proteasome and ribosome complexes is dependent on Atg24 sorting nexin complex (Makino et al., 2021). While selective degradation of ribosomes (ribophagy) was found to occur after 24h of nitrogen starvation, delivery of mRNAs has started as early as 3h after target of rapamycin complex 1 (TORC1) inhibition (Makino et al., 2021; (Huang et al., 2015). TORC1 inactivation increases autophagy by activation of unc-51-like autophagy activating kinase 1, suggesting that mRNAs are subjected to autophagic degradation prior to ribosomes. Additionally, RNAs are delivered into vacuoles even in the absence of ubiquitin-specific protease 3/brefeldin A sensitivity 5 deubiquitination complex system, which is essential for ribophagy (Kraft et al., 2008). Hence, in yeast, RNA degradative capacity of autophagy is substantially selective and independent of ribophagy. These studies indicate the critical role of autophagy in RNA degradation and suggest that regulatory control of RNautophagy and ribophagy might offer promising directions in diseases modifying strategies. Canonical and non-canonical roles of autophagy are illustrated in Fig. 1 and Fig. 2, respectively.

Fig. 1. Canonical functions of autophagy.

Autophagic degradation of dysfunctional proteins and damaged organelles such as ER (ER phagy), mitochondria (mitophagy) and ribosomes (ribophagy) is considered as canonical functions of autophagy. 1) In macroautophagy, dysfunctional organelles and proteins are sequestered by the phagophore near the ER and subsequently undergo maturation into autophagosome. Furthermore, autophagosome fuses with lysosome to form autolysome where the sequestered cargo subjected to degradation by lysosomal hydrolases. 2) In chaperone-mediated autophagy, cytosolic proteins will be trafficked into lysosomes in HSC70 (one of the active chaperones)-dependent manner. 3) In microautophagy, cargo will be sequestration into lysosome or vacuole without the involvement of autophagosome. HSC70, heat shock cognate 71 kDa protein; LAMP2A, lysosome-associated membrane protein 2A.

Fig. 2. Non-canonical functions of autophagy.

Regulation of RNA abundance either by selective degradation (RNautophagy) or non-selective bulk degradation of RNA granules (stress granules) influence RNA levels and functions. Additionally, tethering of RNAs to lysosomes for long distance trafficking inside the cells ensure spatiotemporal protein expression which is vital for homeostasis of polarized cells like neurons. On the other hand, secretory autophagy where LC3-dependent loading and secretion of RNA binding proteins, ncRNAs in extracellular vesicles influence RNA functions and also mediate extracellular communication. AGO1, argonaute RISC component 1; p62, sequestome 1; ub, ubiquitin; ANXA11, annexin A11; LAMP2C, lysosome-associated membrane protein 2C; LC3-II, microtubule-associated protein 1A/1B-light chain 3-II; HNRNPK, heterogeneous nuclear ribonucleoprotein K; SAFB, scaffold-attachment factor B.

Implications of mutual regulation of RNA and autophagy in acute brain injuries

The impact of autophagy on cellular RNA homeostasis and gene expression is poorly characterized. Non-canonical roles of autophagy, including selective secretion and transport of RNAs, RNA binding proteins (RBPs) and ribonucleoprotein (RNP) complexes, modulate the spatiotemporal expression of genes. Importantly, altered kinetics and dynamics of mRNA and RBPs directly influence the intracellular proteome distribution, which is vital for the homeostasis of polarized cells such as neurons (Wong et al., 2017). Trafficking of RNA granules by cellular organelles such as lysosomes mediated through the RNA granule-associated phosphoinositide-binding protein Annexin A11 and mutations of this gene are linked to the pathogenesis of amyotrophic lateral sclerosis (Liao et al., 2019). Hence, the interplay between autophagy and RNA has a crucial role in CNS health and disease. Many ncRNAs were shown to differentially modulate autophagy in stroke and TBI (Table 1).

Table 1.

Regulation of autophagy by ncRNAs in stroke and TBI

Model	ncRNA involved	Mechanism	Functional outcome
pMCAO in SD rats; OGD/R in primary microglial cells (Jiang et al., 2018b)	miR-30d-5p⬇	miR-30d-5p inhibits autophagy by targeting Beclin-1, ATG5, LC3-II and p62	miR-30d-5p mimic enriched ADSC-derived exosomes ameliorated infarction; decreased microglial polarization to M1 phenotype
Distal tMCAO in SD rats (Chen et al., 2019a)	miR-497⬆	Repression of miR-497 target LC3 suppress autophagy	antagomiR-497 decreased infarct volume and NSS, and improved motor function
tMCAO in SD rats; OGD/R in N2a cells (Li et al., 2020a)	miR-202–5p⬇	miR-202–5p represses eIF4E induced autophagy	miR-202–5p mimic repressed eIF4E leading to reduced LC3-II/LC3-I ratio and decreased infarct volume in rats; promoted N2a cell survival
tMCAO in C57BL/6 mice; OGD/R in PC12 cells (Miao et al., 2020)	miR-124⬆	miR-124 inhibits PI3K/Akt/mTOR signalling to activate autophagy	AntagomiR-124 decreased infarct volume in mice; attenuated PC12 cell apoptosis
ICH in C57BL/6 mice; hypoxic PC in MSCs (Liu et al., 2021a)	miR-326⬇	miR-326 induces autophagy by repressing PTBP-1/PI3K signaling	miR-326 mimic attenuated the senescence of OM-MSCs.
ICH in SD rats (Huan et al., 2020)	miR-146a⬇	miR-146a inhibits autophagy by decreasing the levels of LC3-II and Beclin-1	AgomiR-146a improved nerve function, decrease brain water content and attenuated hippocampal neuronal apoptosis
ICH in wistar rats; hemin stimulation in BV2 microglia and HT22 neuronal cells (Sun et al., 2018)	miR-23b⬇	miR-23b inhibits autophagy by targeting ATG12 and activating Akt/mTOR pathway	LV-miR-23b improved motor function and decreased brain edema, hematoma area and neuronal death in mice; supressed inflammatory response in BV2 cells and apoptosis of HT22 cells
tMCAO in C57BL/6 mice; OGD/R in N2a cells (Yu et al., 2019a)	lncRNA KCNQ1OT1⬆	KCNQ1OT1 elevates autophagy by releasing ATG7 transcript from repression by miR-200a	sh-KCNQ1OT1 decreased infarct volume in mice; curtailed N2a cell apoptosis
tMCAO in C57BL/6 mice; OGD/R in SH-SY5Y cells (Yao et al., 2019)	lncRNA SNHG12⬆	SNHG12 activates autophagy by increased conversion of LC3-I to LC3-II and enhanced Beclin-1 levels	pcDNA-SNHG12 attenuated apoptosis in SH-SY5Y cells
tMCAO in C57BL/6 mice; OGD/R in N2a cells (Cao et al., 2020)	lncRNA SNHG3⬆ miR-485⬇	SNHG3 mitigates autophagy by acting as ceRNA for miR-485 which targets ATG7	miR-485 mimic decreased N2a cell apoptosis
tMCAO in C57BL/6 mice; OGD/R in N2a cells (Xu et al., 2021)	lncRNA C2dat2⬆	C2dat2 augments autophagy via miR-30d-5p/DDIT4/mTOR axis	siRNAs/snoRNAs against C2dat2 mitigated N2a cell apoptosis
tMCAO in SD rats; OGD/R injury in PC12 cells (Guo et al., 2021)	lncRNA MIAT⬆	MIAT found to stabilize REDD1 and promotes autophagy through activation of mTOR	sh-MIAT or si-MIAT reduced neurological deficit scores and infarct volume in rats; decreased apoptosis of PC12 cells
tMCAO in SD rats; OGD/R injury SH-SY5Y cells (Liu et al., 2021b)	lncRNA AC136007.2⬇	AC136007.2 counteracts autophagy via inhibition of AMPK/mTOR signaling	Lenti-lncRNA AC136007.2 decreased cerebral infarction and edema in rats; promoted SH-SY5Y cell survival
tMCAO in C57BL/6 mice; OGD/R in primary neurons and astrocytes (Chen et al., 2020)	circSHOC2⬆	circSHOC2 augments autophagy by sponging miR-7670–3p and regulating Beclin-1, LC3-II and SQSTM1 levels	circSHOC2 in IPAS-EXOs administration decreased infarct volume in mice; attenuated astrocyte and neuronal apoptosis.
tMCAO in C57BL/6 mice; OGD/R in primary neurons and astrocytes (Xu et al., 2020a)	circAkap7⬇	circAkap7 acts as a miR-155–5p sponge to induce ATG12-mediated autophagy	Exosomes derived from circAkap7-modified ADSCs decreased infarct volume and improved motor function in mice; mitigated apoptosis of astrocytes and neurons
tMCAO in C57BL/6 mice; OGD/R in SH-SY5Y cells (Shang et al., 2020)	circRNA_0001449 ⬆	circRNA_0001449 increases autophagy via miR-124–3p/miR-32–5p/ORP-5/Akt pathway	Sh-Circ_0001449 decreased NSS and improved motor function in mice; attenuated SH-SY5Y cell death
CCI injury in SD rats (Li et al., 2020b)	circRNA_010705 (circLrp1b)⬆	circLrp1b activates autophagy by acting as a miR-27a-3p sponge to regulate Dram2-mediated autophagy	circLrp1b inhibition by sh-circLrp1b decreased NSS and improved cognitive function

In all ICH studies listed, collagenase was used to induce ICH.

MCAO, middle cerebral artery occlusion; pMCAO, permanent MCAO; tMCAO, transient MCAO; OGD/R, oxygen-glucose deprivation/reperfusion; SD, sprague dawley; ATG5, autophagy related 5; LC3-II, Microtubule-associated protein 1A/1B-light chain 3-II; ADSC, adipose-derived stem cells; NSS, neurological severity score; eIF4E, eukaryotic translation initiation factor 4E; N2a cells, neuroblastoma 2 a cells; PC12 cells, adrenal phaeochromocytoma cell line; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; mTOR, mechanistic target of rapamycin; ICH, intracerebral hemorrhage; PC, preconditioning; PTBP-1, polypyrimidine tract-binding protein 1; OM-MSCs, olfactory mucosa-mesenchymal stem cells; ATG12, autophagy related 12; ceRNA; competing endogenous RNA; ATG7, autophagy related 7; DDIT4, DNA damage-inducible transcript 4; siRNAs, small interfering RNA; snoRNAs, small nucleolar RNAs; REDD1, regulated in development and DNA damage 1; AMPK, adenosine 5′ monophosphate-activated protein kinase; SQSTM1, sequestosome-1; IPAS-EXOs, ischemic-preconditioned astrocyte-derived exosomes; ORP-5, OSBP-related protein-5; Dram2, DNA damage regulated autophagy modulator 2; CCI, controlled cortical impact;

Ischemic stroke

Acute ischemic stroke is the leading cause of death and disability in adult population worldwide. Many mechanisms that include oxidative stress, excitotoxicity, unregulated peri-infarct depolarization, neuroinflammation, and necrosis/apoptosis aggravate cell death after stroke (Khoshnam et al., 2017). In addition, autophagy has been shown to promote cell death following ischemic injury (Wang et al., 2018b). Non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are known to modulate the ischemic pathophysiology (Mehta et al., 2021; Wang et al., 2018c). Their role in autophagy is still evolving. The miRNA, miR-224 was shown to preferentially recruited to autophagosome and a low autophagic activity correlated with increased miR-224 expression that suppressed its target gene Smad4 leading hepatocellular carcinoma growth (Lan et al., 2014). Furthermore, treatment with amiodarone suppressed tumorigenesis in rats via enhanced autophagic degradation of miR-224 (Lan et al., 2014).

Human brain cortical neuronal HCN-2 cells showed elevated levels of miR-224 following oxygen-glucose deprivation (OGD) and inhibition of miR-224 resulted in decreased neuronal apoptosis following ischemic injury (Fu et al., 2016). Similarly, primary cortical neurons showed increased expression of miR-224-5p after OGD. Whereas, treatment with anti-miR-224-5p significantly promoted neuronal viability and attenuated apoptosis following OGD (Liu et al., 2020). Upregulation of miR-224 was also observed in the peri-infarct cortex of rats following transient focal ischemia (Liu et al., 2020) and in the hippocampus of rats following global ischemia (Deng et al., 2013). Although, there is no evidence of the impact of autophagy on miR-224 degradation in these in vivo models of cerebral ischemia, negative regulation of miR-224 by autophagy might be a possible mechanism in reducing deleterious stroke outcomes that need to be further investigated.

Target gene silencing by miRNAs is mediated through argonaute (AGO) subfamily of proteins, where AGO loads miRNAs to form effector RNP complex known as the RNA-induced silencing complex (RISC) (Kawamata et al., 2009). Inside RISC, miRNAs are directed to their complementary target mRNAs by AGO leading to translational repression and/or decay of mRNAs (Iwakawa and Tomari, 2015). Intracerebral injection of lentiviral-miR-126 showed decreased infarct volume and edema in mice at 3 days after permanent middle cerebral artery occlusion (MCAO). miR-126 upregulation has been shown to maintain blood brain barrier (BBB) integrity by repressing the endothelial adhesion molecules vascular cell adhesion molecule-1 and E-selectin (Pan et al., 2020). Notably, a recent report showed that autophagy induction resulted in differential trafficking of miR-126. While autophagy promoted the degradation of miR-126-3p, miR-126-5p gets translocated into the nucleus as a ternary complex with RNA-binding protein Mex3a and AGO2. Interestingly, within the nucleus, miR-126-5p, once dissociated from AGO2, interferes with caspase 3 activity by preventing its active site formation. In contrast, deletion of Mex3a or suppression of autophagy exacerbated endothelial apoptosis and atherosclerosis progression, the hallmark pathogenetic factor of ischemic stroke (Santovito et al., 2020). It has been shown that AGO2-associated miRNAs are differentially altered in adult neural progenitor cells (NPCs) after MCAO in rats (Liu et al., 2017). Selective autophagic degradation of DICER (miRNA processing enzyme) and AGO2 was associated with regulation of miRNA synthesis, biological activity and degradation in HeLa cells (Gibbings et al., 2012). This way, autophagy serves as a checkpoint for the synthesis and continuous loading of miRNAs into AGO2 to exert their physiological functions. Role of autophagy in selective degradation of DICER and AGO2 in the context of ischemia needs to be elucidated.

Several ncRNAs were shown to modulate autophagy in ischemic stroke. miRNA-30a levels were markedly increased after 6h of permanent MCAO, but significantly decreased in the peri-infarct cortex of mice after 24h of reperfusion following transient MCAO (Wang et al., 2014). Decreased miRNA-30a was accompanied with the increased conversion of LC3-II from LC3-I and elevated levels of Beclin-1 in N2A cells and primary cortical neurons after OGD (Wang et al., 2014). Whereas, anti-miRNA-30a attenuated neuronal death in N2A cells after OGD, and improved behavioral outcomes in mice following transient focal ischemia. These results suggest the potential of miRNA-30a downregulation in the alleviation of ischemic injury via autophagy induction (Wang et al., 2014).

Transient focal ischemia in rats significantly decreased the levels of miR-207 in the peri-infarct cortex and treatment with miR-207 mimic improved neurological functional recovery and reduced infarct volume (Tao et al., 2015). miR-207 was shown to interfere with later stages of autophagy pathways, such as the formation of the auto-lysosomal system via regulation of LAMP2 (Tao et al., 2015). It was also shown that overexpression of miR-9a-5p inhibited autophagy, and thus protected brain following transient MCAO in rats (Wang et al., 2018a). Mechanistically, miR-9a-5p was shown to suppress ATG5 and thus autophagy in SY-5Y cells challenged with OGD (Wang et al., 2018a).

Long non-coding RNA (lncRNA) H19 levels were shown to be upregulated in SH-SY5Y cells subjected to OGD/reperfusion (Wang et al., 2017c). H19 activation augmented autophagy via dual-specificity phosphatase 5- extracellular signal-regulated protein kinase 1/2 signaling to promote apoptosis in SH-SY5Y cells after OGD (Wang et al., 2017c). High glucose levels in diabetic rats decreased H19 expression that correlated with increased cardiomyocyte autophagy and overexpression of H19 epigenetically repressed GTP-binding RAS-like 3 (DIRAS3) to attenuate autophagy (Zhuo et al., 2017). This suggests that H19 controls autophagy in opposing ways in different conditions. A recent study showed that rs217727 polymorphism of the H19 was associated with increased susceptibility to ischemic stroke in the Iranian population (Rezaei et al., 2021). H19 levels were also shown to be induced following transient MCAO in rats (Wang et al., 2017c) but its significance in post-stroke autophagy is not yet evaluated. Expression of the lncRNA MALAT1 was reported to be high in the cerebral cortex after transient MCAO in mice along with increased conversion of LC3-I to LC3-II, enhanced Beclin-1 and decreased levels of miR-30a which targets Beclin-1 (Guo et al., 2017). However, MALAT1 knockdown together with administration of autophagy inhibitor 3-methyl adenine (3-MA) reversed neuronal death following transient MCAO, underscoring the potential MALAT1/miR-30a/Beclin-1 regulatory network in ischemic stroke pathogenesis (Guo et al., 2017). MALAT1 was also shown to enhance autophagy by sponging miR-26b to increase the activity of its target Unc-51 like autophagy activating kinase 2, an upstream signaling molecule of autophagy in cultured brain microvascular endothelial cells following OGD (Li et al., 2017). The lncRNA MEG3 sponges miR-378, which represses growth factor receptor bound protein 2 (GRB2) mRNA, which in turn suppresses the activation of protein kinase B (Akt)/ mechanistic target of rapamycin (mTOR) to fuel autophagy (Luo et al., 2020). MEG3 interaction with miR-378 resulted in activation of autophagy that ultimately promoted neuronal death after MCAO in mice and OGD in primary cortical neurons (Luo et al., 2020). In mice subjected to transient MCAO, either miR-378 overexpression or GRB2 knockdown decreased autophagy leading to better functional recovery (Luo et al., 2020). This further affirms the dual role of autophagy in maintaining cerebral homeostasis after ischemic injury.

circRNAs are highly stable ncRNAs that are abundant in CNS (Mehta et al., 2020). A recent study reported that levels of circHECTD1 are high in plasma samples of acute ischemic stroke patients as well as in the cerebral cortex of mice subjected to transient MCAO (Han et al., 2018). They further showed that circHECTD1 knockdown following OGD in mouse primary astrocytes resulted in decreased autophagic flux that counteracted astrocyte activation (Han et al., 2018). Autophagy induction in astrocytes by circHECTD1 was shown to be mediated through miR142/TIPARP axis, where circHECTD1 sponges miR142 that controls TIPARP, which induces autophagy (Han et al., 2018). In human astrocytes subjected to OGD, circ_0025984 upregulation has been shown to sponge miR-143-3p, thereby elevating the levels of its target ten-eleven translocation 1 that resulted in attenuation of astrocytic apoptosis and suppression of autophagy by increased ORP150 expression via its promoter demethylation that suppressed GRP78 induced ATG7 mediated autophagy (Zhou et al., 2021). However, autophagy inhibition by circ_0025984 in cultured astrocytes was not observed in cells in which ATG7 was silenced (no effect on LC3-I) or overexpressed (no effect on LC3-II), suggesting that the circ_0025984 regulates autophagy under ischemic conditions in an ATG7 dependent manner (Zhou et al., 2021).

We previously showed that circRNA expression profiles alter significantly in the peri-ischemic cortex following transient MCAO in adult mice (Mehta et al., 2017). However, the role of circRNAs in modulating post-ischemic autophagy is not yet evaluated in detail.

The circSCMH1 levels were reported to be very high in the plasma of acute ischemic stroke patients and in the peri-infarct cortex of mice subjected to photothrombotic ischemia (Yang et al., 2020b). Increased expression of circSCMH1 by intravenous administration of circSCMH1 overexpressed vector significantly improved functional recovery in mice and rhesus monkeys after photothrombotic stroke. Mechanistically, circSCMH1 attenuated glial activation in peri-infarct cortex of mice after stroke by transcriptional regulation of methyl CpG binding protein 2 (MeCP2) target genes (Yang et al., 2020b). Transplantation of bone-marrow-derived endothelial progenitor cells (EPCs) are known to promote vascular homeostasis in stroke (Esquiva et al., 2018; Sobrino et al., 2007). MeCP2 overexpression in EPCs disrupted autophagy via suppression of forkhead box protein O3a (FoxO3a), thereby altered the migratory and adherent properties and the viability of transplanted EPCs (Zha et al., 2019). Although the interaction of circSCMH1/MeCP2/FoxO3a/autophagy has not been studied in stroke, it may serve as a potential signaling axis to modulate autophagy in ischemic stroke.

Hemorrhagic stroke

Hemorrhagic stroke accounts for ~13% of all stroke cases (Li et al., 2018a). Both intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH) contribute to hemorrhagic stroke (Fang et al., 2020). Hematoma formation after ICH is a major proponent of secondary brain damage which leads to degradation of hemoglobin and iron release (Wu et al., 2003). Iron-induced toxicity following ICH is mediated through autophagy induction, and suppression of ferrous citrate-induced autophagy after ICH in rats attenuated the severity of secondary brain injury (Chen et al., 2012). Recently, transcriptomic analysis of ICH patients showed the role of autophagic dysregulation as the proponent of ICH pathology (Durocher et al., 2021). Similarly, increased conversion of LC3-I to LC3-II and elevated beclin-1 levels indicate enhanced autophagy observed in the ipsilateral fronto-basal cortex of rats after SAH (Lee et al., 2009). Several studies evaluated the role of ncRNAs in modulating autophagy after hemorrhagic stroke. ICH in mice after autologous blood injection markedly enhanced miR-144 expression that resulted in suppression of mTOR activity, which is an upstream inhibitor of autophagy leading to increased autophagy of microglia and compromise neurological functions, which were reversed by treatment with anti-miR-144 (Yu et al., 2017). Although ICH patients showed low levels of miR-21 in blood and brain tissues (Wang et al., 2016), its role in ICH progression is not yet evaluated. Activation of PI3K/AKT/mTOR signaling negatively regulates autophagy and this was shown to be associated with the decreased neurological function and increased edema in ICH and SAH rats (Guo et al., 2018; Jadaun et al., 2021; Wang and Zhang, 2017). A recent study showed that miR-126-3p mimic attenuated neurobehavioral deficits by maintaining BBB integrity via inhibition of PIK3R2/Akt pathway after ICH in rats induced by intracerebral infusion of collagenase (Xi et al., 2017). The neuroprotective potential of miR-126-3p might be due to its activity on the upstream autophagy signaling PIK3R2/Akt that potentiates mTOR-dependent suppression of autophagy. In addition to miRNAs, other classes of ncRNAs also participate in autophagy after hemorrhagic stroke. In a rat model of collagenase-induced ICH, inhibition of lncRNA NKILA reduced levels of LC3-II and beclin-1 and decreased autophagosome density leading to attenuated autophagy that improved neurological functions and reduced hematoma size (Jia et al., 2018). In a rat model of ICH induced by intracranial injection of autologous blood, differential expression of many circRNAs was observed in the cerebral cortex around hematoma. Some of them like rno_circRNA_012556 and rno_circRNA_004782 that were down-regulated sponge the mRNAs associated with PI3K/Akt signaling, which is essential for autophagy activation (Dou et al., 2020). This indicates that autophagy-mediated cell death in ICH can be modified by specific circRNA-miRNA-mRNA interactions.

Traumatic brain injury

TBI induces physical damage to brain parenchyma due to external mechanical forces, including shearing, tearing and stretching which initiate and propagate complex pathological events that finally impair the normal function of the brain (Davis and Vemuganti, 2020). The primary insult-induced cellular and molecular aberrations in impacted brain areas due to direct mechanical damage are insensitive to therapeutic measures. While the delayed secondary response can be modulated by a variety of interventions to restore neurological function (Chandran et al., 2018; Werner and Engelhard, 2007). TBI unleashes a variety of molecular mechanisms, including those related to oxidative stress, excitotoxicity, neuroinflammation, altered BBB integrity, autophagy and apoptosis (Akamatsu and Hanafy, 2020; Nakka et al., 2016; Sulhan et al., 2020). Converging lines of evidence suggest a dual role of autophagy in the pathogenesis of TBI. Levels of Beclin-1 were observed to be markedly upregulated near the injury site after closed head injury in mice induced by weight drop method (Diskin et al., 2005). Importantly, neuronal expression of Belcin-1 at the injury area was predominantly high for 3 weeks post-TBI and showed increased TUNEL positivity indicating continuous activation of autophagy following TBI (Diskin et al., 2005). This study indicated a possible link between elimination of damaged neurons via enhanced autophagy and a reduction in secondary brain damage after TBI. Neurons in the injured temporal cortex of humans after TBI showed increased expression of LC3-II and Beclin-1, suggesting accelerated autophagosome formation (Clark et al., 2008). Moreover, treatment with rapamycin (a potent autophagy inducer) at 4h after closed head injury in mice resulted in inhibition of p70S6K phosphorylation and enhanced Beclin-1 levels, thereby promoting neuronal survival and improved behavior (Erlich et al., 2007). Furthermore, treatment with autophagy inhibitors such as 3-MA and bafilomycin A1 conferred neuroprotection after weight drop induced TBI in mice (Luo et al., 2011). Using a GFP-Lc3 transgenic mouse, temporal progression of autophagy was evaluated from day 1 to 7 after controlled cortical impact (CCI) induced TBI in mice (Sarkar et al., 2014). In this study, accumulation of autophagosomes in ipsilateral cortex and hippocampus at day 1 after TBI was observed to be due to decreased lysosomal proteins, cathepsin-D activity and increased p62/SQSTM1 aggregates leading to defective lysosomal clearance of autophagosomes that was resolved by day 7 after injury (Sarkar et al., 2014). Several other reports highlighted the beneficial effects of negative regulation of autophagy in attenuation of secondary brain damage and neurological deficits associated with TBI (Feng et al., 2017; Wang et al., 2017b; Yang et al., 2020a).

The interplay between ncRNAs and autophagy was also implicated in the pathophysiology of TBI. Overexpression of brain-specific miR-27a before TBI induction in rats suppressed neuronal autophagy-related genes in FoxO3a dependent manner that conferred neurological recovery (Sun et al., 2017). Furthermore, miRNA-23b was observed to be downregulated in TBI patients and in a rat model of weight drop induced TBI. Treatment with a miRNA-23b overexpressing vector resulted in significant improvement of cognitive function in rats subjected to TBI and this effect was mediated through repression of miR-23b target ATG12 mRNA leading to inhibition of autophagy in neurons (Sun et al., 2018). When brain extracts from mice subjected to TBI were added to HT22 cells, there was an increased exosomal expression of miR-21 (Dai Li et al., 2019). Furthermore, transfection of miR-21 suppressed Rab11a and prevented scratch injury-induced autophagy in HT22 cells (Dai Li et al., 2019). miR-21 was also shown to counteract autophagy by inhibition of Rab11a-mediated canonical autophagosome formation in a rodent model of renal ischemic injury (Liu et al., 2015)

Expression profiling studies showed altered expression of many miRNAs with functional significance in autophagy regulation such as miR-144, miR-184, miR-451 and miR-2137 (upregulated after TBI) and miR-107, miR-137, miR-190 and miR-541 (downregulated after TBI) (Meissner et al., 2016). miR-144 known to aggravate microglial autophagy following ICH in mice (Yu et al., 2017). In addition, miR-184 was shown to repress oxidative stress induced growth inhibitor 1 which modulate mTOR/p70S6K signaling mediated autophagy following arsenic exposure in human hepatocytes (Gao et al., 2018) and miR-107 inhibits Akt/mTOR signaling, and thus autophagy in chondrocytes (Zhao et al., 2019). Many other miRNAs such as miR-451, miR-137, miR-190 and miR-541 were also known to exert differential effects on autophagy in divergent disease conditions (Hu et al., 2020b; Song et al., 2014; Xu et al., 2020b; Yu et al., 2019b).

Role of lncRNAs in post-TBI autophagy is also evident. Gene ontology and KEGG pathway analysis revealed that many cellular signalling pathways like phagosome formation and PI3K/Akt signaling are modulated by a network of lncRNAs and their interacting mRNAs (Zhong et al., 2016). Previous studies also showed altered expression of many lncRNAs in rodent models of TBI as well as in brain tissue from TBI patients (Morris-Blanco et al., 2017; Yang et al., 2019). The lncRNAs Zfas1 and Gas5 were observed to be upregulated in hippocampus after fluid percussion injury in rats (Wang et al., 2017a). Positive regulation of autophagy by lncRNA Zfas1 contributes to neuronal apoptosis (Hu et al., 2020a) and importantly, Zfas1 knockout markedly quenched secondary brain injury in mice after weight drop induced TBI (Feng et al., 2021). In contrast, when 293T cells were challenged with LPS, binding of Gas5 to miR-23a resulted in increased expression of mir-23a target ATG3 that enabled ATG5-ATG12 complex formation leading to autophagy (Li et al., 2018b). However, this treatment led to increased cell survival indicating that autophagy induction by Gas5 augmented cell viability in this model. In contrast, suppression of Gas5 attenuated neuronal apoptosis after TBI in mice, and probably effect of Gas5 on autophagy might be detrimental in post-TBI brain (Dai et al., 2019). Increased serum levels of lncRNA CRNDE was observed in TBI patients which was consistent with its elevated hippocampal expression in rats subjected to TBI (Yi et al., 2019). Notably, siRNA-mediated silencing of CRNDE after TBI improved neurobehavioral function in rats through suppression of unregulated autophagy in neurons (Yi et al., 2019).

TBI in adult rodents is also known to alter the expression profiles of circRNAs in cerebral cortex (Jiang et al., 2019; Mehta et al., 2020) as well as in hippocampus (Xie et al., 2018). It has been shown that circRNAs such as circRNA_00608 and circRNA_010705 were significantly upregulated, while circRNA_001167 and circRNA_001168 were markedly downregulated after TBI (Xie et al., 2018). The target miRNAs of circRNAs like circRNA_010705 and circRNA_001167 that are upregulated after TBI include miR-27a-3p, miR-27b-3p, miR-34a-3p and miR-34a-5p, miR-146b-5p (Xie et al., 2018), which regulate autophagy in different pathological conditions (Hanwei et al., 2020; Sun et al., 2020; Tian et al., 2020; Zhang et al., 2021b). Inhibitory effects of miR-27a on autophagy in TBI were elucidated already (Sun et al., 2017), and hence manipulation of circRNA_010705 might regulate miR-27a/FoxO3a/autophagy, thereby influence molecular and functional outcomes in TBI. Furthermore, in mice subjected to CCI injury, expression of 16 circRNAs altered (5 upregulated and 11 downregulated) in cerebral cortex are known to be associated with phosphatidylinositol signaling pathway that regulates the upstream autophagy signaling (Chen et al., 2019b). Thus, studying the circRNA/miRNA/mRNA/autophagy networks offer a new therapeutic avenue in acute brain injuries.

Identification of autophagy-related genes (ATGs) and proteins provide novel therapeutic targets to minimize the secondary damage and promote the functional recovery after acute CNS injuries. Pharmacological modulation of upstream autophagy regulators such as AMP-activated protein kinase and mTOR showed promising neuroprotective effects in preclinical models of stroke and TBI (Ao et al., 2019; Jiang et al., 2015; Li et al., 2007; Li and Han, 2018; Yu et al., 2018) (Jiang et al., 2018a; Wei et al., 2016; Xu et al., 2018). Furthermore, targeting nucleation of autophagosome formation by inhibiting class III PI3K vacuolar protein sorting 34 (VPS34) by 3-methyal adenine exhibited both beneficial and harmful effects in preclinical stroke models (Zhang et al., 2021a). Restoring lysosomal proteolytic capacity, which is essential for autophagic degradation of cellular contents and damaged organelles resulted in attenuation of neuronal death in mice after transient MCAO (Hossain et al., 2021). Beneficial and detrimental effects of autophagy in stroke and TBI are yet to be precisely characterized. The variability of outcomes between different laboratories might be due to the complex role of autophagy and the heterogeneity of experimental variables (species, age, sex, type and intensity of neurotoxic stimulus) employed (Galluzzi et al., 2016). Determination of accurate autophagic flux and use of highly specific pharmacological modulators might help in understanding the dual role autophagy in acute CNS injuries (Klionsky et al., 2021; Mizushima and Murphy, 2020).

The ncRNAs serve as both biomarkers and therapeutic candidates to modulate autophagy in stroke and TBI (Chandran et al., 2017; Tiedt and Dichgans, 2018). For instance, a population-based prospective Framingham Heart Study revealed that those with altered circulating levels of miR-941 were at increased risk of stroke over 5 years (Mick et al., 2017). Similarly, frontotemporal brain tissue from TBI patients was characterized by elevated levels of lncRNAs such as n333955, n332943, and ENST00000384390 (Yang et al., 2019). Knockdown of lncRNA MEG3 significantly decreased the infarct volume in mice following transient MCAO (Li et al., 2022). Furthermore, lncRNA MEG3 knockdown enhanced levels of its target miR-181c-5p, which in turn repressed ATG7 leading to inhibition of autophagy (Li et al., 2022). RNA-based therapeutics are gaining importance owing to their limited off-target effects and ability to act on targets that are generally not druggable with small molecules (Anthony, 2022). For example, Nusinersen is the first antisense oligonucleotide approved by the FDA to treat spinal muscular dystrophy (MacCannell et al., 2022). Similarly, a recombinant adeno-associated virus serotype 5, huntingtin microRNA (rAAV5-miHTT), is currently in phase II clinical trial for Huntington’s disease (NCT04120493) (Vallès et al., 2021). Autophagy modulation at different stages by exogenous agents and endogenous proteins regulate many outcomes after stroke and TBI (Fig. 3). The potential of different ncRNAs in manipulating autophagy from initiation to lysosomal degradation or recycling (Fig. 4), suggests the possibility of developing ncRNAs as therapeutic candidates for the treatment of stroke and TBI. Combination of different interventions, including natural induction of autophagy by intermittent fasting, caloric restriction, and physical exercise with pharmacological modulators of autophagy might provide efficient neuroprotection against acute brain injuries (Ajoolabady et al., 2021; de Cabo and Mattson, 2019; Vemuganti and Arumugam, 2021).

Fig. 3.

Potential autophagy-related targets manipulated by exogenous agents and endogenous regulators to affect outcomes of stroke and TBI. AMPK, 5’ adenosine monophosphate-activated protein kinase; mTOR, mechanistic target of rapamycin; cPKCγ, conventional protein kinase C-gamma; NaHS, sodium hydrosulfide; ATGs, autophagy related proteins; VPS34, class III PI3K vacuolar protein sorting 34; FOXO-3a, forkhead box protein O3a; LC3, microtubule-associated protein 1A/1B-light chain 3; BNIP3, BCL2 Interacting Protein 3; LAMP2, lysosome-associated membrane protein 2.

Fig. 4.

Regulation of autophagy by different ncRNAs in acute brain injuries. Differential expression of ncRNAs following stroke and TBI substantially intervenes at multiple stages of autophagy from initiation to lysosomal degradation of cargo.

Conclusion and future perspectives

The bidirectional role of autophagy in cell death due to internal and external challenges is still a topic of debate. While three decades of autophagy research disentangled core autophagy machinery that aids in cargo trafficking, autophagosome formation and molecular networks involved in lysosomal degradative process, recent studies indicated a new role of autophagy in shaping the transcriptome abundance in the cell. This intricate network of autophagy/transcriptome ensures proper RNA trafficking essential for spatiotemporal expression of proteins and compensates nonsense-mediated RNA decay. RNautophagy is also important for RNA quality control, specifically to eliminate faulty mRNAs to curb the translation of dysfunctional or toxic proteins and to digest retrotransposon RNAs to prevent their insertion into the genome. Furthermore, ribophagy is important for proper ribosomal turnover that impacts cellular homeostasis under metabolic stress. Together RNautophagy and ribophagy substantially alter the cellular proteome under stressful conditions. Conversely, many RNAs can modulate autophagy. Control of autophagic proteins by miRNAs is a promising avenue to decrease brain damage and promote neurological recovery after acute brain injury. It is essential to understand the role of autophagy based on the context. For example, hypoxic preconditioning confers protection against further hypoxia in SH-SY5Y cells by activation of HIF-1α/Beclin-1 driven autophagy (Lu et al., 2018). However, autophagy induction promotes neuronal death after in vivo ischemia-reperfusion and TBI in rodent models (Ajoolabady et al., 2021; Clark et al., 2008).

There are many unanswered questions in autophagy that need future research. For example, it is not known if ribophagy is responsible for total ribosome turnover, what makes the appearance of a specific ribosomal populations in autophagosomes and if that shapes the total proteome of neurons/glia after CNS insults. It is also not known if acute brain injuries like stroke and TBI alters RNautophagy and if that influences translational fidelity and outcomes.