A marked enhancement of a BLOC‐1 gene, pallidin, associated with somnolent mouse models deficient in histamine transmission

Histamine (HA) and orexin (Ox, or hypocretin) neurons act distinctly and synergistically in wake control. A double knock out mouse genotype lacking both HA and Ox shows all sleep disorders of human narcolepsy. We identified in this mouse brain a sharp upregulation of a BLOC-1 gene, pallidin that is associated with dramatic changes in the balance of cholinergic and aminergic systems in mice and an enhanced sleep in drosophila. This study demonstrates potential sleep disorders-associated compensatory mechanisms with pallid as a novel biomarker.


A marked enhancement of a BLOC-1 gene, pallidin, associated with somnolent mouse models deficient in histamine transmission
Histamine and orexin (or hypocretin) neurons act distinctly and synergistically in wake control. A double knockout mouse genotype lacking both histamine and orexins shows all sleep disorders of human narcolepsy. We identified in this mouse brain a sharp upregulation of a BLOC-1 gene, pallidin, that is selectively associated with a deficient histamine neurotransmission and dramatic changes in the balance of cholinergic and aminergic systems in mice as well as an enhanced sleep in drosophila. This study demonstrates potential sleep disorders-associated compensatory mechanisms with pallidin as a novel biomarker.
The maintenance of wakefulness requires a complex brain arousal network made up of various neurotransmitters and neuropeptides. Using knockout (KO) mouse models we have previously shown that histamine (HA) and orexin (Ox, also called hypocretin) neurons act distinctly and synergistically in terms of wake control. 1,2 An impaired histaminergic neurotransmission is associated with sleepiness in animal models and human sleep disorders while the lack of Ox neuropeptides constitutes a direct cause of narcolepsy, a neurological disease characterized by sleepiness and cataplexy. 1,2 We have generated a double KO mouse genotype lacking both HA and Ox [Histidine decarboxylase (hdc, HA-synthetizing enzyme)-Orexin KO, referred to as HO-KO] which shows all phenotypes of human narcolepsy such as sleepiness, hypersomnia, sleep-onset rapid eye movement and cataplexy-like episodes, EEG hypersynchronization and marked obesity. 3 This mouse strain constitutes therefore a complete murine model of narcolepsy. In order to identify the consequences on gene expression of sleep disorders and to uncover novel molecular and cellular processes involved in sleep-wake control, we performed transcriptomic profiling in the frontal cortex of HO-KO mice and their wild-type littermates.
We identified differentially expressed genes in this double mutant mouse that potentially reflect unidentified mechanisms controlling sleep and wakefulness (Appendix S1 and Table S1). Figure 1A shows a subset of these genes confirmed by independent quantitative PCR (QPCR). The non-protein coding Hdc transcript was highly upregulated likely because of a negative feedback regulation between HA release and Hdc expression 4 ( Figure 1B). We obtained similar findings for the Ox non-protein coding transcript ( Figure 1B, middle). Interestingly this regulation was not uniform in the brain, indicating local compensatory regulatory mechanisms on neurotransmission ( Figure 1B). The expression of genes not previously associated with sleep-wake regulation was also affected, notably pallidin (also called BLOC1S6), a gene coding a major subunit of the Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1), that displayed a massively enhanced expression (>900%), in the frontal cortex as well as in the hypothalamus and thalamus ( Figure 1B).
To determine whether the upregulation of pallidin results from a HA or Ox deficiency or both, we performed QPCR in single KO mice lacking either Hdc or Ox. We found that enhanced pallidin expression The BLOC-1 complex is involved in protein trafficking among different endosomal compartments and has been linked to schizophrenia and cognitive performance. 5 Indeed, BLOC-1 genes play important roles in neuronal functions and in particular neurotransmission. 5 In addition, it has been reported that pallidin could regulate transport at the blood-brain barrier and thus impact monoamine synthesis, and in particular that of serotonin. 6 In line with this hypothesis, we found a marked increase in serotonin levels ( Figure 1D are present in non-neuronal cell types of the brain, notably in the different blood-brain interfaces, including the choroid plexuses which form the blood-CSF barrier, where HA affects gene expression. 7,8 We thus evaluated pallidin mRNA expression in the choroid plexuses of Hdc-KO mice, and found that it was upregulated in these nonneuronal cells ( Figure 1E) as in the frontal cortex ( Figure 1B).
A study based on locomotor activity suggested that pallidin could be involved in sleep-wake regulation in the mouse, 9 yet, direct evidence is lacking. The role of pallidin in sleep-wake regulation remains, therefore, to be explored, in particular using conditional, cell type-specific KO approaches. Yet, such attempt is currently highly challenging in mammals given the broad expression of pallidin in numerous cells types in the brain and periphery and currently the lack of flexible tools to selectively vary pallidin expression. We thus turned to the Drosophila model, which is intensively used in sleep-wake research. All the genes involved in BLOC-1 function are conserved in Drosophila, with similar interactions and functions as those identified in mammals. 10 We tested neuronal and non-neuronal function for pallidin using pharmacological or heatinducible transgenes Gal4-UAS systems to overexpress pallidin ( Figure 1F,G). In Drosophila, glial cells regulate neurotransmission and control the exchanges between the brain and circulating fluid, the hemolymph, fulfilling a function similar to the blood-brain barrier in mammals. We found that overexpression of pallidin in neurons did not produce any detectable effect ( Figure 1F) while that in the glia using a heat-inducible system significantly enhanced sleep ( Figure 1G). In contrast, in a separate report on Drosophila, we found a wake-promoting effect following downregulation of

pallidin. 11
Pallidin appeared among the most dramatically upregulated genes in the brain of mice deficient for HA transmission and characterized by a somnolent phenotype. Moreover, we found that overexpression of pallidin in Drosophila glia results in increased sleep time.
Therefore, pallidin upregulation in HO-KO and Hdc-KO is likely linked to the altered control of sleep-wake in these somnolent mice. Gene expression dosage appears to be a critical factor in the stability of the BLOC-1 complex 5 both in mammals and Drosophila. If such is the case in the mammalian blood-brain interfaces, the upregulation of pallidin may significantly modulate the functionality of BLOC-1. The signaling pathway leading to this transcriptional response together with its potential impact on sleep-wake regulation remain to be identified. HA transmission has been shown to regulate the bloodbrain interfaces 7 and ependymal cells, the circumventricular organs, and the choroid plexuses are in close proximity with HA terminals.
Interestingly, all of the latter cells display dramatically enhanced cfos expression in response to acute pain induced by formalin injection, and in some cases even to saline injection, in Hdc- KO

Dr. Lin is an Editorial Board member of CNS Neuroscience and
Therapeutics and a coauthor of this article. To exclude any bias, he was not involved in any editorial decision-making related to the acceptance of this article for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be made availabe on request.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.