Supporting youth mental health with arts-based strategies: a global perspective.

The devastating impact of youth mental health concerns is increasingly evident on a global scale. This crisis calls for innovative solutions that are sufficiently accessible, scalable, and cost-effective to support diverse communities around the world. One such solution involves engagement in the arts: incorporating and building upon existing local resources and cultural practices to bolster youth mental health. In this article, we describe the global youth mental health crisis and note major gaps in the knowledge and resources needed to address it. We then discuss the potential for arts- and culture-based strategies to help meet this challenge, review the mounting evidence regarding art's ability to support mental health, and call for action to undertake critical research and its translation into accessible community practices. Four steps are suggested: (1) elevate and prioritize youth voice, (2) develop core outcome measures, (3) identify and analyze successful models around the globe, and (4) generate clear funding pathways for research and translational efforts. Worldwide implementation of arts- and culture-based strategies to address youth mental health will provide critical resources to support the health, wellbeing and flourishing of countless youth across the globe.

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone.

Members of the DISABLED (DAB) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric CLATHRIN-associated sorting proteins that recruit cargo proteins into endocytic vesicles. Here, we report a Drosophila disabled mutant revealing a novel role for DAB proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activity-dependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila DAB and CLATHRIN were highly colocalized within two distinct classes of puncta, including relatively dim puncta that were located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including DYNAMIN, supported a general role for CLATHRIN-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool.

Activity-dependent interactions of NSF and SNAP at living synapses.

As core components of the neurotransmitter release apparatus, SNAREs, NSF and SNAPs mediate fusion of neurotransmitter-filled synaptic vesicles within specialized regions of the presynaptic plasma membrane known as active zones (AZs). The present study combines genetic approaches in Drosophila with biochemical and live-imaging methods to provide new insights into the in vivo behavior and interactions of NSF and SNAP in neurotransmitter release. This work employs a temperature-sensitive (TS) paralytic NSF mutant, comatose, to show that disruption of NSF function results in activity-dependent redistribution of NSF and SNAP to periactive zone (PAZ) regions of the presynaptic plasma membrane and accumulation of protein complexes containing SNAREs, NSF and SNAP. Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Recovery After Photobleaching (FRAP) studies in comatose revealed that NSF and SNAP exhibit activity-dependent binding to each other within living presynaptic terminals as well as distinctive interactions and mobilities. These observations extend current models describing the spatial organization of NSF, SNAP and SNARE proteins in synaptic vesicle trafficking.

Molecular mechanisms determining conserved properties of short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants.

Current models of synaptic vesicle trafficking implicate a core complex of proteins comprised of N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs in synaptic vesicle fusion and neurotransmitter release. Despite this progress, major challenges remain in establishing the in vivo functions of these proteins and their roles in determining the physiological properties of synapses. The present study employs glutamatergic adult neuromuscular synapses of Drosophila, which exhibit conserved properties of short-term synaptic plasticity with respect to mammalian glutamatergic synapses, to address these issues through genetic analysis. Our findings establish an in vivo role for SNAP-25 in synaptic vesicle priming, and support a zippering model of SNARE function in this process. Moreover, these studies define the contribution of SNAP-25-dependent vesicle priming to the detailed properties of short-term depression elicited by paired-pulse (PP) and train stimulation. In contrast, NSF is shown here not to be required for WT PP depression, but to be critical for maintaining neurotransmitter release during sustained stimulation. In keeping with this role, disruption of NSF function results in activity-dependent redistribution of the t-SNARE proteins, SYNTAXIN and SNAP-25, away from neurotransmitter release sites (active zones). These findings support a role for NSF in replenishing active zone t-SNAREs for subsequent vesicle priming, and provide new insight into the spatial organization of SNARE protein cycling during synaptic activity. Together, the results reported here establish in vivo contributions of SNAP-25 and NSF to synaptic vesicle trafficking and define molecular mechanisms determining conserved functional properties of short-term depression.

Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis.

The ESCRT pathway is evolutionarily conserved across eukaryotes and plays key roles in a variety of membrane remodeling processes. A new Drosophila mutant recovered in our forward genetic screens for synaptic transmission mutants mapped to the vps24 gene encoding a subunit of the ESCRT-III complex. Molecular characterization indicated a loss of VPS24 function, however the mutant is viable and thus loss of VPS24 may be studied in a developed multicellular organism. The mutant exhibits deficits in locomotion and lifespan and, notably, these phenotypes are rescued by neuronal expression of wild-type VPS24. At the cellular level, neuronal and muscle cells exhibit marked expansion of a ubiquitin-positive lysosomal compartment, as well as accumulation of autophagic intermediates, and these phenotypes are rescued cell-autonomously. Moreover, VPS24 expression in glia suppressed the mutant phenotype in muscle, indicating a cell-nonautonomous function for VPS24 in protective intercellular signaling. Ultrastructural analysis of neurons and muscle indicated marked accumulation of the lysosomal compartment in the vps24 mutant. In the neuronal cell body, this included characteristic lysosomal structures associated with an expansive membrane compartment with a striking tubular network morphology. These findings further define the in vivo roles of VPS24 and the ESCRT pathway in lysosome homeostasis and their potential contributions to neurodegenerative diseases characterized by defective ESCRT or lysosome function.

MAP1 structural organization in Drosophila: in vivo analysis of FUTSCH reveals heavy- and light-chain subunits generated by proteolytic processing at a conserved cleavage site.

The MAP1 (microtubule-associated protein 1) family is a class of microtubule-binding proteins represented by mammalian MAP1A, MAP1B and the recently identified MAP1S. MAP1A and MAP1B are expressed in the nervous system and thought to mediate interactions of the microtubule-based cytoskeleton in neural development and function. The characteristic structural organization of mammalian MAP1s, which are composed of heavy- and light-chain subunits, requires proteolytic cleavage of a precursor polypeptide encoded by the corresponding map1 gene. MAP1 function in Drosophila appears to be fulfilled by a single gene, futsch. Although the futsch gene product is known to share several important functional properties with mammalian MAP1s, whether it adopts the same basic structural organization has not been addressed. Here, we report the identification of a Drosophila MAP1 light chain, LC(f), produced by proteolytic cleavage of a futsch-encoded precursor polypeptide, and confirm co-localization and co-assembly of the heavy chain and LC(f) cleavage products. Furthermore, the in vivo properties of MAP1 proteins were further defined through precise MS identification of a conserved proteolytic cleavage site within the futsch-encoded MAP1 precursor and demonstration of light-chain diversity represented by multiple LC(f) variants. Taken together, these findings establish conservation of proteolytic processing and structural organization among mammalian and Drosophila MAP1 proteins and are expected to enhance genetic analysis of conserved MAP1 functions within the neuronal cytoskeleton.

Small heat shock proteins mediate cell-autonomous and -nonautonomous protection in a Drosophila model for environmental-stress-induced degeneration.

Cell and tissue degeneration, and the development of degenerative diseases, are influenced by genetic and environmental factors that affect protein misfolding and proteotoxicity. To better understand the role of the environment in degeneration, we developed a genetic model for heat shock (HS)-stress-induced degeneration in Drosophila This model exhibits a unique combination of features that enhance genetic analysis of degeneration and protection mechanisms involving environmental stress. These include cell-type-specific failure of proteostasis and degeneration in response to global stress, cell-nonautonomous interactions within a simple and accessible network of susceptible cell types, and precise temporal control over the induction of degeneration. In wild-type flies, HS stress causes selective loss of the flight ability and degeneration of three susceptible cell types comprising the flight motor: muscle, motor neurons and associated glia. Other motor behaviors persist and, accordingly, the corresponding cell types controlling leg motor function are resistant to degeneration. Flight motor degeneration was preceded by a failure of muscle proteostasis characterized by diffuse ubiquitinated protein aggregates. Moreover, muscle-specific overexpression of a small heat shock protein (HSP), HSP23, promoted proteostasis and protected muscle from HS stress. Notably, neurons and glia were protected as well, indicating that a small HSP can mediate cell-nonautonomous protection. Cell-autonomous protection of muscle was characterized by a distinct distribution of ubiquitinated proteins, including perinuclear localization and clearance of protein aggregates associated with the perinuclear microtubule network. This network was severely disrupted in wild-type preparations prior to degeneration, suggesting that it serves an important role in muscle proteostasis and protection. Finally, studies of resistant leg muscles revealed that they sustain proteostasis and the microtubule cytoskeleton after HS stress. These findings establish a model for genetic analysis of degeneration and protection mechanisms involving contributions of environmental factors, and advance our understanding of the protective functions and therapeutic potential of small HSPs.

Synaptic calcium-channel function in Drosophila: analysis and transformation rescue of temperature-sensitive paralytic and lethal mutations of cacophony.

Voltage-gated calcium channels play a key role in chemical synaptic transmission by providing the calcium trigger for regulated neurotransmitter release. Genes encoding the primary structural subunit, alpha1, as well as accessory subunits of presynaptic calcium channels have now been identified in a variety of organisms. The cacophony (cac) gene in Drosophila, also known as nightblind A, encodes a voltage-gated calcium-channel alpha1 subunit homologous to vertebrate alpha1 subunits implicated in neurotransmitter release. A recent genetic screen in our laboratory isolated cac(TS2), a conditional cac mutant exhibiting rapid paralysis at elevated temperatures. This mutant has allowed synaptic electrophysiology after acute perturbation of a specific calcium-channel gene product, demonstrating that cac encodes a primary calcium channel functioning in neurotransmitter release. Here we report the molecular lesion in cac(TS2), a missense mutation within a calcium-dependent regulatory domain of the alpha1 subunit, as well as phenotypic rescue of temperature-sensitive and lethal cac mutations by transgenic expression of a wild-type cac cDNA. Notably, rescue of rapid, calcium-triggered neurotransmitter release was achieved by neural expression of a single cDNA containing a subset of alternative exons and lacking any conserved synaptic-protein interaction sequence. Possible implications of these findings are discussed in the context of structure-function studies of synaptic calcium channels, as well as alternative splicing and mRNA editing of the cac transcript.

Active zone localization of presynaptic calcium channels encoded by the cacophony locus of Drosophila.

Presynaptic calcium channels play a central role in chemical synaptic transmission by providing the calcium trigger for evoked neurotransmitter release. These voltage-gated calcium channels are composed of a primary structural subunit, alpha1, as well as auxiliary beta and alpha2delta subunits. Our previous genetic, molecular, and functional analysis has shown that the cacophony (cac) gene encodes a primary presynaptic calcium channel alpha1 subunit in Drosophila. Here we report that transgenic expression of a cac-encoded alpha1 subunit fused with enhanced green fluorescent protein efficiently rescues cac lethal mutations and allows in vivo analysis of calcium channel localization at active zones. The results reported here further characterize the primary role of cac-encoded calcium channels in neurotransmitter release. In addition, these studies provide a unique genetic tool for live imaging of functional presynaptic calcium channels in vivo and define a molecular marker for immunolocalization of other presynaptic proteins relative to active zones. These findings are expected to facilitate additional analysis of synaptic development and function in this important model system.

A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult.

Previous studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. Here we extend these findings by defining a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission.

A tripartite synapse model in Drosophila.

Tripartite (three-part) synapses are defined by physical and functional interactions of glia with pre- and post-synaptic elements. Although tripartite synapses are thought to be of widespread importance in neurological health and disease, we are only beginning to develop an understanding of glial contributions to synaptic function. In contrast to studies of neuronal mechanisms, a significant limitation has been the lack of an invertebrate genetic model system in which conserved mechanisms of tripartite synapse function may be examined through large-scale application of forward genetics and genome-wide genetic tools. Here we report a Drosophila tripartite synapse model which exhibits morphological and functional properties similar to those of mammalian synapses, including glial regulation of extracellular glutamate, synaptically-induced glial calcium transients and glial coupling of synapses with tracheal structures mediating gas exchange. In combination with classical and cell-type specific genetic approaches in Drosophila, this model is expected to provide new insights into the molecular and cellular mechanisms of tripartite synapse function.

Properties of short-term synaptic depression at larval neuromuscular synapses in wild-type and temperature-sensitive paralytic mutants of Drosophila.

The larval neuromuscular synapse of Drosophila serves as an important model for genetic and molecular analysis of synaptic development and function. Further functional characterization of this synapse, as well as adult neuromuscular synapses, will greatly enhance the impact of this model system on our understanding of synaptic transmission. Here we describe a form of short-term synaptic depression observed at larval, but not adult, neuromuscular synapses and explore the underlying mechanisms. Larval neuromuscular synapses exhibited a form of short-term depression that was strongly dependent on stimulation frequency over a narrow range of low frequencies (0.1-1 Hz). This form of synaptic depression, referred to here as low-frequency short-term depression (LF-STD), results from an activity-dependent reduction in neurotransmitter release. However, in contrast to the predictions of depletion models, the degree of depression was independent of the initial level of neurotransmitter release over a range of extracellular calcium concentrations. This conclusion was confirmed in two temperature-sensitive (TS) paralytic mutants, cacophony and shibire, which exhibit reduced neurotransmitter release resulting from conditional disruption of presynaptic calcium channels and dynamin, respectively. Higher stimulation frequencies (40 or 60 Hz) produced two components of depression that appeared to include LF-STD as well as a more conventional component of short-term depression. These findings reveal novel properties of short-term synaptic depression and suggest that complementary genetic analysis of larval and adult neuromuscular synapses will further define the in vivo mechanisms of neurotransmitter release and short-term synaptic plasticity.