Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'.

Acetylcholine is widely believed to modulate the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors. However, evidence for this mechanism in vivo has been less forthcoming. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release, and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Our findings indicate that measurements of dopamine using the red-shifted fluorescent sensor RdLight1 should be interpreted with caution when combined with optogenetics. In light of this and other publications that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'.

Acetylcholine is widely believed to modulate the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors. However, evidence for this mechanism in vivo has been less forthcoming. A recent paper in eLife (Mohebi et al., 2023) reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release measured with the red fluorescent sensor RdLight1. Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release, and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Our findings indicate that measurements of dopamine using the red-shifted fluorescent sensor RdLight1 should be interpreted with caution when combined with optogenetics. In light of this and other publications that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

Thyroid hormone rewires cortical circuits to coordinate body-wide metabolism and exploratory drive.

Animals adapt to varying environmental conditions by modifying the function of their internal organs, including the brain. To be adaptive, alterations in behavior must be coordinated with the functional state of organs throughout the body. Here we find that thyroid hormone- a prominent regulator of metabolism in many peripheral organs- activates cell-type specific transcriptional programs in anterior regions of cortex of adult mice via direct activation of thyroid hormone receptors. These programs are enriched for axon-guidance genes in glutamatergic projection neurons, synaptic regulators across both astrocytes and neurons, and pro-myelination factors in oligodendrocytes, suggesting widespread remodeling of cortical circuits. Indeed, whole-cell electrophysiology recordings revealed that thyroid hormone induces local transcriptional programs that rewire cortical neural circuits via pre-synaptic mechanisms, resulting in increased excitatory drive with a concomitant sensitization of recruited inhibition. We find that thyroid hormone bidirectionally regulates innate exploratory behaviors and that the transcriptionally mediated circuit changes in anterior cortex causally promote exploratory decision-making. Thus, thyroid hormone acts directly on adult cerebral cortex to coordinate exploratory behaviors with whole-body metabolic state.

Dopamine and glutamate regulate striatal acetylcholine in decision-making.

Striatal dopamine and acetylcholine are essential for the selection and reinforcement of motor actions and decision-making1. In vitro studies have revealed an intrastriatal circuit in which acetylcholine, released by cholinergic interneurons (CINs), drives the release of dopamine, and dopamine, in turn, inhibits the activity of CINs through dopamine D2 receptors (D2Rs). Whether and how this circuit contributes to striatal function in vivo is largely unknown. Here, to define the role of this circuit in a living system, we monitored acetylcholine and dopamine signals in the ventrolateral striatum of mice performing a reward-based decision-making task. We establish that dopamine and acetylcholine exhibit multiphasic and anticorrelated transients that are modulated by decision history and reward outcome. Dopamine dynamics and reward encoding do not require the release of acetylcholine by CINs. However, dopamine inhibits acetylcholine transients in a D2R-dependent manner, and loss of this regulation impairs decision-making. To determine how other striatal inputs shape acetylcholine signals, we assessed the contribution of cortical and thalamic projections, and found that glutamate release from both sources is required for acetylcholine release. Altogether, we uncover a dynamic relationship between dopamine and acetylcholine during decision-making, and reveal multiple modes of CIN regulation. These findings deepen our understanding of the neurochemical basis of decision-making and behaviour.

Structure of the nutrient-sensing hub GATOR2.

Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients1,2. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively3-5. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 ß-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through ß-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes6. The scaffold orients the WD40 ß-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.

Rapid purification and metabolomic profiling of synaptic vesicles from mammalian brain.

Neurons communicate by the activity-dependent release of small-molecule neurotransmitters packaged into synaptic vesicles (SVs). Although many molecules have been identified as neurotransmitters, technical limitations have precluded a full metabolomic analysis of SV content. Here, we present a workflow to rapidly isolate SVs and to interrogate their metabolic contents at high-resolution using mass spectrometry. We validated the enrichment of glutamate in SVs of primary cortical neurons using targeted polar metabolomics. Unbiased and extensive global profiling of SVs isolated from these neurons revealed that the only detectable polar metabolites they contain are the established neurotransmitters glutamate and GABA. In addition, we adapted the approach to enable quick capture of SVs directly from brain tissue and determined the neurotransmitter profiles of diverse brain regions in a cell-type-specific manner. The speed, robustness, and precision of this method to interrogate SV contents will facilitate novel insights into the chemical basis of neurotransmission.

Abnormal Striatal Development Underlies the Early Onset of Behavioral Deficits in Shank3B-/- Mice.

The neural substrates and pathophysiological mechanisms underlying the onset of cognitive and motor deficits in autism spectrum disorders (ASDs) remain unclear. Mutations in ASD-associated SHANK3 in mice (Shank3B-/-) result in the accelerated maturation of corticostriatal circuits during the second and third postnatal weeks. Here, we show that during this period, there is extensive remodeling of the striatal synaptic proteome and a developmental switch in glutamatergic synaptic plasticity induced by cortical hyperactivity in striatal spiny projection neurons (SPNs). Behavioral abnormalities in Shank3B-/- mice emerge during this stage and are ameliorated by normalizing excitatory synapse connectivity in medial striatal regions by the downregulation of PKA activity. These results suggest that the abnormal postnatal development of striatal circuits is implicated in the onset of behavioral deficits in Shank3B-/- mice and that modulation of postsynaptic PKA activity can be used to regulate corticostriatal drive in developing SPNs of mouse models of ASDs and other neurodevelopmental disorders.

Sunlight Brightens Learning and Memory.

Sunlight can alter mood, behavior, and cognition, but the cellular basis of this phenomenon remains to be fully elucidated. In this issue of Cell, Zhu et al. shed light on a UV-dependent metabolic pathway that leads to increased synaptic release of glutamate and enhanced motor learning and memory in mice.

Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes.

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.

KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease.

Mechanism of arginine sensing by CASTOR1 upstream of mTORC1.

The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids. In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORC1 (refs 4, 5, 6, 7). Arginine activates mTORC1 upstream of the Rag family of GTPases, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1). However, the mechanism by which the mTORC1 pathway detects and transmits this arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient, amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor.

The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway.

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 µM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway.

Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway.

Eukaryotic cells coordinate growth with the availability of nutrients through the mechanistic target of rapamycin complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag guanosine triphosphatases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. Here we present the 2.7 angstrom crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway.

Sestrin2 is a leucine sensor for the mTORC1 pathway.

Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanosine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, aGTPase-activating protein; GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a dissociation constant of 20 micromolar, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway.

Nutrient-sensing mechanisms across evolution.

For organisms to coordinate their growth and development with nutrient availability, they must be able to sense nutrient levels in their environment. Here, we review select nutrient-sensing mechanisms in a few diverse organisms. We discuss how these mechanisms reflect the nutrient requirements of specific species and how they have adapted to the emergence of multicellularity in eukaryotes.

Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

The Sestrins interact with GATOR2 to negatively regulate the amino-acid-sensing pathway upstream of mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a major regulator of cell growth that responds to numerous environmental cues. A key input is amino acids, which act through the heterodimeric Rag GTPases (RagA or RagB bound to RagC or RagD) in order to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. GATOR2 is a complex of unknown function that positively regulates mTORC1 signaling by acting upstream of or in parallel to GATOR1, which is a GTPase-activating protein (GAP) for RagA or RagB and an inhibitor of the amino-acid-sensing pathway. Here, we find that the Sestrins, a family of poorly understood growth regulators (Sestrin1-Sestrin3), interact with GATOR2 in an amino-acid-sensitive fashion. Sestrin2-mediated inhibition of mTORC1 signaling requires GATOR1 and the Rag GTPases, and the Sestrins regulate the localization of mTORC1 in response to amino acids. Thus, we identify the Sestrins as GATOR2-interacting proteins that regulate the amino-acid-sensing branch of the mTORC1 pathway.

The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1.

The mTORC1 kinase is a master growth regulator that senses numerous environmental cues, including amino acids. The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by promoting the translocation of mTORC1 to the lysosomal surface, its site of activation. The Rags are unusual GTPases in that they function as obligate heterodimers, which consist of RagA or B bound to RagC or D. While the loading of RagA/B with GTP initiates amino acid signaling to mTORC1, the role of RagC/D is unknown. Here, we show that RagC/D is a key regulator of the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D must be GDP bound for the interaction to occur. We identify FLCN and its binding partners, FNIP1/2, as Rag-interacting proteins with GAP activity for RagC/D, but not RagA/B. Thus, we reveal a role for RagC/D in mTORC1 activation and a molecular function for the FLCN tumor suppressor.

A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1.

The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist. Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2. Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation. In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5. GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer. In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor. Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer.