GABAA receptor-positive modulators are well-known to induce sedation, sleep, and general anesthesia. Conversely, GABAA receptor negative allosteric modulators (GABAARNAMs) can increase arousal and induce seizures. Motivated by our studies with patients with hypersomnia, and our discovery that two GABAARNAMs can restore the Excitation/Inhibition (E/I) balance in vitro and arousal in vivo, we chose to screen 11 compounds that have been reported to modulate arousal, to see if they shared a GABA-related mechanism. We determined modulation with both conventional and microfluidic patch clamp methods. We found that receptor activation was variably modulated by all 11 compounds: Rifampicin (RIF), Metronidazole (MET), Minocycline (MIN), Erythromycin (ERY), Ofloxacin (OFX), Chloroquine (CQ), Hydroxychloroquine sulfate (HCQ), Flumazenil (FLZ), Pentylenetetrazol (PTZ), (-)-Epigallocatechin Gallate (EGCG), and clarithromycin (CLR). The computational modeling of modulator–receptor interactions predicted drug action at canonical binding sites and novel orphan sites on the receptor. Our findings suggest that multiple avenues of investigation are now open to investigate large and brain-penetrant molecules for the treatment of patients with diminished CNS E/I balance.

Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate genetic and biochemical interactions between ATP7 paralogs with the conserved oligomeric Golgi (COG) complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses.

This protocol describes how inductively coupled plasma mass spectrometry (ICP-MS) can quantify metals, sulfur, and phosphorus present in biological specimens. The high sensitivity of ICP-MS enables detection of these elements at very low concentrations, and absolute quantification is achieved with standard curves. Sulfur or phosphorus standardization reduces variability that arises because of slight differences in sample composition. This protocol bypasses challenges because of limited sample amounts and facilitates studies examining the biological roles of metals in health and disease. For complete details on the use and execution of this protocol, please refer to Hartwig et al. (2020).

Although the neurophysiological correlates of sleep have been thoroughly described, genetic mechanisms that control sleep architecture, long surmised from ethological studies, family histories and clinical observations, have only been investigated during the past decade. Key contributions to the molecular understanding of sleep have come from studies in Drosophila, benefitting from a strong history of circadian rhythm research. For instance, a number of recent papers have highlighted the role of the E3 ubiquitin ligase Cullin-3 in the regulation of circadian rhythm and sleep. We propose that different Cullin-3 substrate adaptors may affect specific molecular pathways and diverse aspects of circadian rhythm and sleep. We have previously shown that mutations in BTBD9, a risk factor for Restless Legs Syndrome (RLS) encoding a Cullin-3 substrate adaptor, lead to reduced dopamine, increased locomotion and sleep fragmentation. Here, we propose that Cullin-3 acts together with BTBD9 to limit the accumulation of iron regulatory proteins in conditions of iron deficiency. Our model is consistent with clinical observations implicating iron homeostasis in the pathophysiology of RLS and predicts that lack of BTBD9 leads to misregulation of cellular iron storage, inactivating the critical biosynthetic enzyme Tyrosine Hydroxylase in dopaminergic neurons, with consequent phenotypic effects on sleep.

Eukaryotic cells maintain proteostasis through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders, such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. Here we focus on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. We demonstrate that SLC25A1 and MRPL40, two genes present in the microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial ribosomal integrity and proteostasis. Our Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. We propose that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a pathogenic mechanism for neurodevelopmental disorders.

Summary Restless Legs Syndrome (RLS), first chronicled by Willis in 1672 and described in more detail by Ekbom in 1945 [1], is a prevalent sensorimotor neurological disorder (5–10% in the population) with a circadian predilection for the evening and night. Characteristic clinical features also include a compelling urge to move during periods of rest, relief with movement, involuntary movements in sleep (viz., periodic leg movements of sleep), and fragmented sleep [2,3]. While the pathophysiology of RLS is unknown, dopaminergic neurotransmission and deficits in iron availability modulate expressivity [1,4–9]. GWAS have identified a polymorphism in an intronic region of the BTBD9 gene on chromosome 6 that confers substantial risk for RLS [2,3,10–12]. Here, we report that loss of the Drosophila homolog CG1826 (dBTBD9) appreciably disrupts sleep with concomitant increases in waking and motor activity. We further show that BTBD9 regulates brain dopamine levels in flies and controls iron homeostasis through the iron regulatory protein-2 (IRP2) in human cell lines. To our knowledge, this represents the first reverse genetic analyses of a “novel” or heretofore poorly understood gene implicated in an exceedingly common and complex sleep disorder and the development of an RLS animal model that closely recapitulates all disease phenotypes.

Background Interferon (IFN)-alpha is an innate immune cytokine that causes high rates of depression in humans and therefore has been used to study the impact of cytokines on the brain and behavior. To establish a non-human primate model of cytokine-induced depression, we examined the effects of IFN-alpha on rhesus monkeys. Methods Eight rhesus monkeys were administered recombinant human (rHu)-IFN-alpha (20 MIU/m2) or saline for 4 weeks in counterbalanced fashion, and videotaped behavior, as well as plasma and cerebrospinal fluid (CSF), were obtained at regular intervals to assess behavioral, neuroendocrine, immune and neurotransmitter parameters. Additionally, expression and activity of IFN-alpha/beta receptors in monkey peripheral blood mononuclear cells (PBMCs) were assessed. Results Compared to saline treatment, IFN-alpha administration was associated with persistent increases in anxiety-like behaviors and decreases in environmental exploration. In addition, IFN-alpha induced significant increases in plasma concentrations of ACTH, cortisol, and interleukin-6 that tended to diminish after chronic administration, especially in dominant animals. Interestingly, in 3 animals, depressive-like, huddling behavior was observed. Monkeys that displayed huddling behavior exhibited significantly higher plasma concentrations of ACTH and lower CSF concentrations of the dopamine metabolite, homovanillic acid. Rhesus monkey PBMCs were found to express mRNA and protein for the IFN-alpha/beta receptor. Moreover, treatment of PBMCs with rHu-IFN-alpha led to induction of STAT1, one of the primary IFN-alpha-induced signaling molecules. Conclusions IFN-alpha evoked behavioral, neuroendocrine and immune responses in rhesus monkeys that are similar to humans. Moreover, alterations in hypothalamic-pituitary-adrenal axis responses and dopamine metabolism may contribute to IFN-alpha-induced depressive-like huddling behavior.

The calcium-regulated transcription factor NFAT is emerging as a key regulator of neuronal development and plasticity but precise cellular consequences of NFAT function remain poorly understood. Here, we report that the single Drosophila NFAT homolog is widely expressed in the nervous system including motor neurons and unexpectedly controls neural excitability. Likely due to this effect on excitability, NFAT regulates overall larval locomotion and both chronic and acute forms of activity-dependent plasticity at the larval glutamatergic neuro-muscular synapse. Specifically, NFAT-dependent synaptic phenotypes include changes in the number of pre-synaptic boutons, stable modifications in synaptic microtubule architecture and pre-synaptic transmitter release, while no evidence is found for synaptic retraction or alterations in the level of the synaptic cell adhesion molecule FasII. We propose that NFAT regulates pre-synaptic development and constraints long-term plasticity by dampening neuronal excitability.

Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. Here, we demonstrate activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (a SERCA mutant), and present an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. all of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from our genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila.

Objective: Some central hypersomnolence syndromes are associated with a positive allosteric modulator of γ-aminobutyric acid (GABA)-A receptors in cerebrospinal fluid. Negative allosteric modulators of GABA-A receptors, including clarithromycin, have been reported to reduce sleepiness in these patients. We sought to systematically assess the effects of clarithromycin on objective vigilance and subjective sleepiness. Methods: This was a 5-week, randomized, placebo-controlled, double-blind, crossover trial of clarithromycin 500mg with breakfast and lunch, in patients with hypersomnolence syndromes (excluding narcolepsy with cataplexy) and evidence for abnormal cerebrospinal fluid potentiation of GABA-A receptors. The study occurred at a university-affiliated medical center. The primary outcome measure was median reaction time on the psychomotor vigilance task (PVT) at week 2 in each condition. Secondary outcomes included the Epworth Sleepiness Scale, Stanford Sleepiness Scale, Functional Outcomes of Sleep Questionnaire, Pittsburgh Sleep Quality Index, SF-36, and additional PVT measures. Results: Twenty-three patients began treatment. Three patients dropped out, and final analyses were performed on 20 complete cases. Median reaction time was not significantly different between clarithromycin and placebo. Subjective measures of sleepiness were significantly improved on clarithromycin versus placebo. Altered taste perception occurred, but was the only side effect more common on clarithromycin than placebo. No serious adverse events occurred. Interpretation: Subjective sleepiness, but not psychomotor vigilance, improved during a 2-week course of clarithromycin. Although additional studies are needed, this suggests that clarithromycin may be a reasonable treatment option in patients with treatment-refractory hypersomnolence.