Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

Fast neurogenesis from carotid body quiescent neuroblasts accelerates adaptation to hypoxia.

Unlike other neural peripheral organs, the adult carotid body (CB) has a remarkable structural plasticity, as it grows during acclimatization to hypoxia. The CB contains neural stem cells that can differentiate into oxygen-sensitive glomus cells. However, an extended view is that, unlike other catecholaminergic cells of the same lineage (sympathetic neurons or chromaffin cells), glomus cells can divide and thus contribute to CB hypertrophy. Here, we show that O2-sensitive mature glomus cells are post-mitotic. However, we describe an unexpected population of pre-differentiated, immature neuroblasts that express catecholaminergic markers and contain voltage-dependent ion channels, but are unresponsive to hypoxia. Neuroblasts are quiescent in normoxic conditions, but rapidly proliferate and differentiate into mature glomus cells during hypoxia. This unprecedented "fast neurogenesis" is stimulated by ATP and acetylcholine released from mature glomus cells. CB neuroblasts, which may have evolved to facilitate acclimatization to hypoxia, could contribute to the CB oversensitivity observed in highly prevalent human diseases.

Neurotransmitter Modulation of Carotid Body Germinal Niche.

The carotid body (CB), a neural-crest-derived organ and the main arterial chemoreceptor in mammals, is composed of clusters of cells called glomeruli. Each glomerulus contains neuron-like, O2-sensing glomus cells, which are innervated by sensory fibers of the petrosal ganglion and are located in close contact with a dense network of fenestrated capillaries. In response to hypoxia, glomus cells release transmitters to activate afferent fibers impinging on the respiratory and autonomic centers to induce hyperventilation and sympathetic activation. Glomus cells are embraced by interdigitating processes of sustentacular, glia-like, type II cells. The CB has an extraordinary structural plasticity, unusual for a neural tissue, as it can grow several folds its size in subjects exposed to sustained hypoxia (as for example in high altitude dwellers or in patients with cardiopulmonary diseases). CB growth in hypoxia is mainly due to the generation of new glomeruli and blood vessels. In recent years it has been shown that the adult CB contains a collection of quiescent multipotent stem cells, as well as immature progenitors committed to the neurogenic or the angiogenic lineages. Herein, we review the main properties of the different cell types in the CB germinal niche. We also summarize experimental data suggesting that O2-sensitive glomus cells are the master regulators of CB plasticity. Upon exposure to hypoxia, neurotransmitters and neuromodulators released by glomus cells act as paracrine signals that induce proliferation and differentiation of multipotent stem cells and progenitors, thus causing CB hypertrophy and an increased sensory output. Pharmacological modulation of glomus cell activity might constitute a useful clinical tool to fight pathologies associated with exaggerated sympathetic outflow due to CB overactivation.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703.

Orai1 and TRPC1 Proteins Co-localize with CaV1.2 Channels to Form a Signal Complex in Vascular Smooth Muscle Cells.

Voltage-dependent CaV1.2 L-type Ca2+ channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca2+ channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and CaV1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2+ mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2+ concentration ([Ca2+]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that CaV1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2+ entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.

Oxygen sensing by the carotid body: mechanisms and role in adaptation to hypoxia.

Oxygen (O2) is fundamental for cell and whole-body homeostasis. Our understanding of the adaptive processes that take place in response to a lack of O2(hypoxia) has progressed significantly in recent years. The carotid body (CB) is the main arterial chemoreceptor that mediates the acute cardiorespiratory reflexes (hyperventilation and sympathetic activation) triggered by hypoxia. The CB is composed of clusters of cells (glomeruli) in close contact with blood vessels and nerve fibers. Glomus cells, the O2-sensitive elements in the CB, are neuron-like cells that contain O2-sensitive K(+)channels, which are inhibited by hypoxia. This leads to cell depolarization, Ca(2+)entry, and the release of transmitters to activate sensory fibers terminating at the respiratory center. The mechanism whereby O2modulates K(+)channels has remained elusive, although several appealing hypotheses have been postulated. Recent data suggest that mitochondria complex I signaling to membrane K(+)channels plays a fundamental role in acute O2sensing. CB activation during exposure to low Po2is also necessary for acclimatization to chronic hypoxia. CB growth during sustained hypoxia depends on the activation of a resident population of stem cells, which are also activated by transmitters released from the O2-sensitive glomus cells. These advances should foster further studies on the role of CB dysfunction in the pathogenesis of highly prevalent human diseases.

Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

KEY POINTS: Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. ABSTRACT: Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells.

Disease mechanisms as Subtypes: Mitochondrial and bioenergetic dysfunction.

Parkinson disease (PD) is the second most common neurodegenerative disease in the world. Despite its enormous human and societal cost, there is no disease-modifying therapy for PD. This unmet medical need reflects our limited understanding of PD pathogenesis. One of the most important clues comes from the recognition that PD motor symptoms arises from the dysfunction and degeneration of a very select group of neurons in the brain. These neurons have a distinctive set of anatomic and physiologic traits that reflect their role in brain function. These traits elevate mitochondrial stress, potentially making them particularly vulnerable to age, as well as to genetic mutations and environmental toxins linked to PD incidence. In this chapter, the literature supporting this model is outlined, along with gaps in our knowledge base. The translational implications of this hypothesis are then discussed, with a focus on why disease-modification trials have failed to date and what this means for the development of new strategies for altering disease course.

The hunger strikes back: an epigenetic memory for autophagy.

Historical and demographical human cohorts of populations exposed to famine, as well as animal studies, revealed that exposure to food deprivation is associated to lasting health-related effects for the exposed individuals, as well as transgenerational effects in their offspring that affect their diseases' risk and overall longevity. Autophagy, an evolutionary conserved catabolic process, serves as cellular response to cope with nutrient starvation, allowing the mobilization of an internal source of stored nutrients and the production of energy. We review the evidence obtained in multiple model organisms that support the idea that autophagy induction, including through dietary regimes based on reduced food intake, is in fact associated to improved health span and extended lifespan. Thereafter, we expose autophagy-induced chromatin remodeling, such as DNA methylation and histone posttranslational modifications that are known heritable epigenetic marks, as a plausible mechanism for transgenerational epigenetic inheritance of hunger.

Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington's disease model mice.

Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

Transgenic NADH dehydrogenase restores oxygen regulation of breathing in mitochondrial complex I-deficient mice.

The hypoxic ventilatory response (HVR) is a life-saving reflex, triggered by the activation of chemoreceptor glomus cells in the carotid body (CB) connected with the brainstem respiratory center. The molecular mechanisms underlying glomus cell acute oxygen (O2) sensing are unclear. Genetic disruption of mitochondrial complex I (MCI) selectively abolishes the HVR and glomus cell responsiveness to hypoxia. However, it is unknown what functions of MCI (metabolic, proton transport, or signaling) are essential for O2 sensing. Here we show that transgenic mitochondrial expression of NDI1, a single-molecule yeast NADH/quinone oxidoreductase that does not directly contribute to proton pumping, fully recovers the HVR and glomus cell sensitivity to hypoxia in MCI-deficient mice. Therefore, maintenance of mitochondrial NADH dehydrogenase activity and the electron transport chain are absolutely necessary for O2-dependent regulation of breathing. NDI1 expression also rescues other systemic defects caused by MCI deficiency. These data explain the role of MCI in acute O2 sensing by arterial chemoreceptors and demonstrate the optimal recovery of complex organismal functions by gene therapy.

ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain.

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1- microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

Short communication: genetic ablation of L-type Ca2+ channels abolishes depolarization-induced Ca2+ release in arterial smooth muscle.

RATIONALE: In arterial myocytes, membrane depolarization-induced Ca(2+) release (DICR) from the sarcoplasmic reticulum (SR) occurs through a metabotropic pathway that leads to inositol trisphosphate synthesis independently of extracellular Ca(2+) influx. Despite the fundamental functional relevance of DICR, its molecular bases are not well known. OBJECTIVE: Biophysical and pharmacological data have suggested that L-type Ca(2+) channels could be the sensors coupling membrane depolarization to SR Ca(2+) release. This hypothesis was tested using smooth muscle-selective conditional Ca(v)1.2 knockout mice. METHODS AND RESULTS: In aortic myocytes, the decrease of Ca(2+) channel density was paralleled by the disappearance of SR Ca(2+) release induced by either depolarization or Ca(2+) channel agonists. Ca(v)1.2 channel deficiency resulted in almost abolition of arterial ring contraction evoked by DICR. Ca(2+) channel-null cells showed unaltered caffeine-induced Ca(2+) release and contraction. CONCLUSION: These data suggest that Ca(v)1.2 channels are indeed voltage sensors coupled to the metabolic cascade, leading to SR Ca(2+) release. These findings support a novel, ion-independent, functional role of L-type Ca(2+) channels linked to intracellular signaling pathways in vascular myocytes.

Autophagy regulation by RNA alternative splicing and implications in human diseases.

Autophagy and RNA alternative splicing are two evolutionarily conserved processes involved in overlapping physiological and pathological processes. However, the extent of functional connection is not well defined. Here, we consider the role for alternative splicing and generation of autophagy-related gene isoforms in the regulation of autophagy in recent work. The impact of changes to the RNA alternative splicing machinery and production of alternative spliced isoforms on autophagy are reviewed with particular focus on disease relevance. The use of drugs targeting both alternative splicing and autophagy as well as the selective regulation of single autophagy-related protein isoforms, are considered as therapeutic strategies.

ULK3-dependent activation of GLI1 promotes DNMT3A expression upon autophagy induction.

Macroautophagy/autophagy is a tightly regulated catabolic process, which contributes at baseline level to cellular homeostasis, and upon its stimulation to the adaptive cellular response to intra- and extracellular stress stimuli. Decrease of autophagy activity is occurring upon aging and thought to contribute to age-related-diseases. Recently, we uncovered, upon autophagy induction, the role of de novo DNMT3A (DNA methyltransferase 3 alpha)-mediated DNA methylation on expression of the MAP1LC3 (microtubule associated protein 1 light chain 3) proteins, core components of the autophagy pathway, which resulted in reduced baseline autophagy activity. Here, we report that serine/threonine kinase ULK3 (unc-51 like kinase 3)-dependent activation of GLI1 (GLI family zinc finger 1) contributes to the transcriptional upregulation of DNMT3A gene expression upon autophagy induction, thereby bringing additional understanding of the long-term effect of autophagy induction and a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: CBZ: carbamazepine; ChIP: chromatin immunoprecipitation; Clon: clonidine; DNMT3A: DNA methyltransferase 3 alpha; GLI1: GLI family zinc finger 1; GLI2: GLI family zinc finger 2; MAP1LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PLA: proximity ligation assay; RT-qPCR: quantitative reverse transcription PCR; shRNA: small hairpin RNA; siRNA: small interfering RNA; Treh: trehalose; ULK3: unc-51 like kinase 3.

SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome-lysosome fusion.

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.

Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Inhibition of microglial EZH2 leads to anti-tumoral effects in pediatric diffuse midline gliomas.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPG), within diffuse midline gliomas are aggressive pediatric brain tumors characterized by histone H3-K27M mutation. Small-molecule inhibitors for the EZH2-H3K27 histone methyltransferase have shown promise in preclinical animal models of DIPG, despite having little effect on DIPG cells in vitro. Therefore, we hypothesized that the effect of EZH2 inhibition could be mediated through targeting of this histone modifying enzyme in tumor-associated microglia. METHODS: Primary DIPG tissues, and cocultures between microglia and patient-derived DIPG or -pediatric high-grade glioma (pHGG) cell lines, were used to establish the H3-K27M status of each cell type. Antisense RNA strategies were used to target EZH2 gene expression in both microglia and glioma cells. Microglia anti-tumoral properties were assessed by gene expression profile, tumor cell invasion capacity, microglial phagocytic activity, and associated tumor cell death. RESULTS: In primary DIPG tissues, microglia do not carry the H3-K27M mutation, otherwise characteristic of the cancer cells. Activation of a microglial tumor-supportive phenotype by pHGG, independently of their H3-K27M status, is associated with a transient H3K27me3 downregulation. Repression of EZH2 in DIPG cells has no impact on tumor cell survival or their ability to activate microglia. However, repression of EZH2 in microglia induces an anti-tumor phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumor-related cell death. CONCLUSIONS: These results indicate that microglia, beyond the tumor cells, contribute to the observed response of DIPG to EZH2 inhibition. Results highlight the potential importance of microglia as a new therapeutic avenue in DIPG.

The DNA methyltransferase DNMT3A contributes to autophagy long-term memory.

Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2'-deoxycytidine; BafA1: bafilomycin A1; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle's balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.