Stress dynamically modulates neuronal autophagy to gate depression onset

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Major depressive disorder is one of the most disabling psychiatric disorders, with a global lifetime prevalence of 10.6% (ref. ³). Stress is a significant risk factor for depression and widely present in daily life. Moderate stress in the short term is beneficial for survival, whereas prolonged stress exposure can perturb emotional homeostasis and contribute to depression¹. However, it remains elusive how the brain dynamically copes with stress. Understanding the dynamic cellular mechanisms within specific neural circuits underlying stress is crucial for the development of more efficacious antidepressant treatments.

Protein turnover by degradation and synthesis allows fine-tuning of neuronal homeostasis⁴. Among various mechanisms that control protein turnover, autophagy (herein mainly concerning macroautophagy) has drawn increasing attention in recent decades². Autophagy is a major catabolic stepwise process through which intracellular proteins are engulfed in autophagosomes and subsequently transported to lysosomes for degradation⁵. Notably, though autophagy genes are extensively expressed in the nervous system, the physiological function of brain autophagy remains an open question. So far most research has focused on its role in neurological diseases^2,5,6,7, leaving an enormous opportunity for exploring its role in stress-related psychiatric diseases.

By specifically targeting the degradation of synaptic components^{8,9,10,11,12,13,14}, brain autophagy has been suggested to regulate synaptic function and various related physio- and pathological functions, including learning and memory^15,16,17,18 and psychiatric disorders^{19,20,21,22,23,24,25}. Yet, little has been uncovered regarding the precise cellular substrates and neural circuits underlying how brain autophagy regulates emotions during stress.

The clinical relevance of autophagy dysfunction in depression has been demonstrated by impaired autophagy-related signalling (for example, mTOR, p62, LC3, Atg7, Beclin-1) in blood or brain samples from patients with depression²³. Conversely, autophagy signalling is elevated in samples from patients with depression and stressed animals treated with antidepressants^24,26. Nevertheless, the precise targets to which antidepressants regulate autophagy in the brain remain elusive.

The lateral habenula (LHb), one of the most vulnerable targets of stress, is extensively activated by numerous stressors^{27,28,29,30,31,32} and therefore shows a high turnover of synaptic proteins^30,33. In particular, chronic stress potentiates excitatory inputs, alters the expression of various synaptic proteins and disrupts neuronal homeostasis in the LHb^{27,28,29,30,31,32}. Here we found that LHb autophagy is essential for emotional homeostasis through counterbalancing excessive synaptic proteins upregulated by stress. We discovered a causal role of LHb autophagy in coping with stress. Maladaptation to chronic stress disrupts LHb autophagy, the reversal of which provides exciting possibilities for new rapid and sustained antidepressant strategies.

Antidepressants activate LHb autophagy

Given the potential role of autophagy in the actions of antidepressant drugs²⁴, we evaluated whether autophagy levels in specialized brain circuits were modulated by antidepressants in mice that received chronic restraint stress (CRS), a well-established animal model of depression^29,31. We measured in parallel autophagy levels in nine stress-related brain regions under the systemic administration of either a classical selective serotonin reuptake inhibitor (SSRI) paroxetine³⁴ or a new rapid antidepressant ketamine³⁵ (Extended Data Fig. 1a,b). We observed that both paroxetine (Extended Data Fig. 1a) and ketamine (Extended Data Fig. 1b) enhanced autophagy in the LHb. This was demonstrated by decreased protein level of p62 (Extended Data Fig. 1a1,b1), a classical autophagy marker degraded by autophagic processes², and increased protein level of Beclin-1 (Extended Data Fig. 1a2,b2), which is specifically involved in autophagosome formation². Meanwhile, p62 and Beclin-1 levels in eight other brain regions were unaffected by either paroxetine or ketamine, including the ventral hippocampus (vHippo), medial prefrontal cortex (mPFC), ventral tegmental areas (VTA), lateral hypothalamus, nucleus accumbens (NAc), lateral septum, dorsal raphe nucleus and median raphe nucleus (Extended Data Fig. 1a,b), suggesting that regulatory effects of distinct antidepressants on brain autophagy take place mainly in the LHb. One day of treatment of paroxetine had no impact on depression-like behaviours (Extended Data Fig. 2a,b) and did not alter p62 or Beclin-1 levels in the LHb (Extended Data Fig. 2c). In parallel, 7 days, but not 1 day, of treatment of paroxetine significantly reduced excitatory synaptic transmission and bursting activity in LHb neurons of CRS mice (Extended Data Fig. 2d–i). These results indicate that activation of LHb autophagy is temporally coincided with the silencing of LHb neuronal activity and the onset of paroxetine’s antidepressant-like effects in chronic stressed mice³⁴.

CRS impairs LHb autophagy

To further investigate the clinical relevance of LHb autophagy in depression, we analysed in parallel autophagy-related gene expression levels by bulk RNA sequencing (RNA-seq) in six emotion-related brain regions. Transcriptomic profiles were compared between naive and CRS mice after completing a depression-like behavioural test, the forced swim test (FST) (Fig. 1a). Using KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis, we found that genes engaged in autophagy-related signalling (Fig. 1b), especially macroautophagy (Fig. 1b(i)), showed the highest absolute value of enrichment scores (z-scores) in the LHb among the many brain regions analysed in CRS mice. In addition, autophagy-related genes in the LHb indicate downregulation of autophagy compared to the naive controls. Furthermore, single-nucleus RNA-seq (snRNA-seq) in the LHb showed that autophagy-related genes show selective downregulation of autophagy in neurons rather than glial cells (Extended Data Fig. 3a). By contrast, enrichment of macroautophagy genes in the vHippo, VTA and NAc was undetectable, and significant enrichment of autophagy upregulation-related genes was detected in the lateral hypothalamus but not significant in the mPFC (Fig. 1b(i)). Moreover, at the single gene level, we observed significantly decreased mRNA expression levels of genes involved in the autophagy activation processes (Extended Data Fig. 3b,c1,2) (for example, Becn1 (encoding Beclin-1) and autophagy-related gene Atg10), as well as tendencies towards decreased Atg4d, Atg12, Atg7, Atg5 and LC3 (also known as Map1lc3a) (Extended Data Fig. 3c). We also found a tendency towards increased mRNA expression level of Mtor, whose activation inhibits autophagy processes (Extended Data Fig. 3c). These transcriptomic results were further validated using quantitative PCR (qPCR) (Extended Data Fig. 3d). Notably, negative correlations were found between the mRNA levels of core autophagy genes in the LHb of each mouse and the corresponding individual immobility durations in the FST (Fig. 1c,d), indicating a direct link between impaired LHb autophagy function and depression-like status. In addition, mRNA levels of ubiquitin–proteasome system, another main intracellular protein degradation system⁴, were unaffected in the LHb by CRS (Extended Data Fig. 3e–g), indicating the functional specificity of altered LHb autophagy in depression.

Bidirectional effects of stress

To decipher how stress affects and eventually dampens LHb autophagy, we measured LHb autophagy function in mice treated with many stressors (restraint, social defeat or footshock), which engage distinct sensory modalities (physical, psychosocial or noxious), either acutely or chronically (Fig. 1e). These chronic stress protocols are all well-accepted animal models of depression^29,30,36. Consistent with the transcriptome data, protein levels in the LHb also indicate impaired autophagy in various chronically stressed mouse models. By contrast, acute stressors all significantly enhanced LHb autophagy compared with naive mice (Fig. 1f,g). To investigate whether stress-induced neuronal activity directly enhances LHb autophagy, we applied 40-Hz phasic photostimulation (4 pulses per second) at lateral hypothalamus-LHb synaptic terminals, which mimics the neuronal firing pattern evoked by various acute stressors^29,31, and found that it also enhanced LHb autophagy (Extended Data Fig. 4a,b), suggesting an activity-dependent enhancement of LHb autophagy during acute stress.

We further measured autophagic flux either by immunostaining LC3 puncta in wild-type (WT) mice or quantifying puncta fluorescence in LC3-GFP reporter mice. Consistently, LC3 puncta fluorescence was enhanced in the LHb of mice treated with acute restraint stress (ARS), whereas it was decreased in those treated with CRS (Fig. 1h,i and Extended Data Fig. 4c). Moreover, the number of autophagosomes detected under electron microscopy in LHb neurons was drastically increased by ARS but decreased by CRS (Extended Data Fig. 4d). Consistent with the mRNA expression levels, the protein level of ubiquitin was also unaffected by acute or chronic stress (Fig. 1j–l). Notably, neither ARS nor CRS altered autophagy function in the vHippo (Extended Data Fig. 4e). In addition, food restriction, a classical trigger of autophagy in peripheral organs³⁷, enhanced autophagy in the liver without affecting the LHb (Extended Data Fig. 4f,g).

Independent roles of AMPK and mTOR

Given that mTOR inhibition and AMP-activated protein kinase (AMPK) activation are two canonical inducers of autophagy², we determined the expression levels of phosphorylated AMPK (p-AMPK) and phosphorylated mTOR (p-mTOR) in the LHb of mice under either ARS or CRS. Western blot showed that ARS increased p-AMPK level without affecting p-mTOR or the downstream phosphorylated p70S6 kinase (p-S6K), whereas CRS augmented p-mTOR and p-S6K levels without affecting p-AMPK (Fig. 1m–p). Collectively, these results indicate the independent and opposing roles of AMPK and mTOR signalling in modulating LHb autophagy at different stages of stress. Acute stress activates AMPK to initiate LHb autophagy, whereas chronic stress activates mTOR to terminate LHb autophagy.

To determine whether AMPK is required for autophagy initiation during acute stress, SBI-0206965 (SBI), a selective inhibitor of AMPK-ULK1 (Unc51-like autophagy activating kinase 1) phosphorylation, was locally infused into the LHb before ARS, and subsequent western blot experiments of relevant signalling pathways were performed (Fig. 1q). We found that p62 level was increased, whereas Beclin-1 and p-AMPK levels were decreased by SBI, without altering p-mTOR and p-S6K levels (Fig. 1r–v), suggesting an indispensable role of AMPK activation in ARS-induced autophagy in the LHb. Excitatory synaptic transmission elevated by acute stress returned to basal levels 1–3 days after stress exposure (Extended Data Fig. 5a–d), indicating a naturally occurring process of synaptic depression. However, this reversal of synaptic potentiation was abolished under local blockade of LHb autophagy by means of SBI during the exposure to ARS (Extended Data Fig. 5a–d). Given the essential role of LHb synaptic potentiation in a depression-like state^27,28,29,31, our data indicate that a failure of naturally occurring synaptic depression may facilitate depression-like behaviours after acute stress. Indeed, local infusion of SBI facilitated an anhedonia-like phenotype in the sucrose preference test (SPT), without affecting the despair-like phenotype in the tail suspension test, as well as the locomotion and anxiety-like phenotype in the open field test (OFT) (Extended Data Fig. 5e–h). Altogether, these results indicate that AMPK signalling is required for the initiation of LHb autophagy to cope with acute stress.

Antidepressant role of LHb autophagy

Given that mTOR signalling is elevated after chronic stress, we tested whether mTOR inhibition alleviates depression-like behaviours. Therefore, rapamycin, a US Food and Drug Administration (FDA)-approved mTOR inhibitor was systemically injected in CRS mice (Fig. 2a). We found that rapamycin alleviated depression-like behaviours (Fig. 2b,c), without affecting OFT performance (Extended Data Fig. 6a). Notably, systemic treatment of rapamycin under antidepressant dosage enhanced autophagy in the LHb rather than other regions (Fig. 2d). To assess whether LHb autophagy is required for antidepressant-like actions of rapamycin, we designed a short hairpin RNA (shRNA) to knock down Atg7 mRNA by means of viral gene transfer (Fig. 2e,f). Overexpression of Atg7-targeting shRNA (shAtg7) specifically in the LHb abolished antidepressant-like effects of rapamycin in both FST and SPT (Fig. 2g,h), without altering OFT performance (Extended Data Fig. 6b). These results reveal the necessary role of LHb autophagy in the antidepressant effect of rapamycin.