InteractiveFly: GeneBrief

: Biological Overview | References

The Pallidin protein is a central subunit of a multimeric complex called biogenesis of lysosome-related organelles complex 1 (BLOC1) that regulates specific endosomal functions and has been linked to schizophrenia. Downregulation of Pallidin and other members of BLOC1 in the surface glia, the Drosophila equivalent of the blood-brain barrier, reduces and delays nighttime sleep in a circadian-clock-dependent manner. In agreement with BLOC1 involvement in amino acid transport, downregulation of the large neutral amino acid transporter 1 (LAT1)-like transporters JhI-21 and mnd, as well as of TOR (target of rapamycin) amino acid signaling, phenocopy Pallidin knockdown. Furthermore, supplementing food with leucine normalizes the sleep/wake phenotypes of Pallidin downregulation, and this study identified a role for Pallidin in the subcellular trafficking of JhI-21. Finally, evidence is provided that Pallidin in surface glia is required for GABAergic neuronal activity. These data identify a BLOC1 function linking essential amino acid availability and GABAergic sleep/wake regulation (Li, 2023).

Biogenesis of lysosome-related organelles complex 1 (BLOC1) is an octameric complex linked to endosomal compartments and the cytoskeleton (Cheli, 2010). The genes coding for the 8 subunits in mice (Pallidin, dysbindin, BLOS1, BLOS2, BLOS3, cappuccino, muted, and snapin) are broadly expressed within the brain and in peripheral tissues. The complex regulates the trafficking of various receptors and transporters and appears to play a prominent role in the biogenesis of recycling endosomes. Mice bearing severe or complete loss-of-function mutations in BLOC1 genes are viable and fertile. They display common phenotypes, originating from defects in highly specialized lysosome-related organelles, such as reduced pigmentation due to impaired retinal and epidermal melanosomes or extended bleeding times resulting from the lack of dense granules in platelets. In humans, mutations in BLOC1 genes and other functionally related genes are found in the Hermansky-Pudlak syndrome (Li, 2023).

In addition, genetic studies have identified variants of the dysbindin gene and other BLOC1 genes as risk factors for developing schizophrenia. Although the latter results are debated, several postmortem studies have reported reduced levels of dysbindin mRNA and protein in the brain of schizophrenics. Furthermore, genome-wide association studies also reported a link between dysbindin genetic variants and cognitive abilities (Li, 2023).

These findings have led to investigations aiming at deciphering the role of BLOC1 in neuronal function using not only mouse models defective for individual BLOC1 gene function but also the Drosophila model, in which the complex is well conserved. These studies confirmed the involvement of BLOC1 in behavior and memory. They identified potential cellular and molecular mechanisms such as abnormal glutamatergic, GABAergic, and dopaminergic transmission. However, the vast majority of these studies have been carried out in animals with spontaneous or artificial mutations. This approach makes the interpretation of the resulting phenotypes challenging, given the broad expression of BLOC1 in many cell types. Furthermore, the complex is expressed during development, when it can be involved in neurodevelopmental diseases such as autism spectrum disorders (Li, 2023).

Apart from one recent report (Lee, 2018) the involvement of BLOC1 in sleep/wake regulation has not been investigated. Interestingly, it has been found that Pallidin is upregulated in a somnolent mouse model with defective histaminergic transmission (Seugnet, 2023). Thus, further investigation is required given the critical implication of sleep in brain function, and in particular in schizophrenia, in which sleep disruption could be in part a consequence of the pathology, or an aggravating factor as seen in human and rodent models. This study investigated the potential role of BLOC1 in sleep/wake regulation in Drosophila. Neurotransmission systems, ion channels, and glial functions in Drosophila and mammals are globally conserved (Li, 2023).

There are differences regarding sleep/wake in insects compared with mammals, such as the absence of slow wave and paradoxical sleep. Nevertheless, the evidence for conserved regulatory principles and functions is compelling. The flexibility and short generation time of the Drosophila model is an asset in molecular genetic studies, providing hypotheses that can be tested in rodents and ultimately used for therapy. A conditional knockdown strategy was used to target Pallidin, a major component of BLOC1. The pool of Pallidin in the cell is associated almost entirely with the complex, and this protein plays a central role through its interactions with dysbindin, BLOS1, and cappuccino (Cheli, 2010), with its loss leading to their degradation (Li, 2023).

This study focused on glia, as a previous results suggest a function for Pallidin in non-neuronal cells. Glial cells are increasingly proven to play very significant roles in the control of sleep/wake and circadian rhythms both in Drosophila and in rodent models. Glia-dependent neurotransmitter reuptake, neurotransmitter metabolism, and glial calcium transients critically influence neuronal networks that control sleep timing and sleep homeostasis. Neuroglia signaling pathways have been shown to modulate sleep-deprivation-induced learning impairments. Recent reports have demonstrated the prominent role of glial cells at the interface between the brain and the circulating fluids during the sleep/wake cycle (Li, 2023 and references therein).

This study provides evidence in Drosophila that Pallidin and other BLOC1 components regulate the initiation of sleep in the early part of the night, at the level of surface glial (SG) cells. In Drosophila, SG cells express tight junctions and molecular components in common with the mammalian BBB. As in the BBB this organization prevents the entry of macromolecules and tightly regulates the exchanges of solutes between the circulatory system (the hemolymph in Drosophila) and the brain, thus maintaining the particular interstitial fluid composition necessary for neuronal activity. This function relies on intense transporter activity. The data support a mechanism whereby BLOC1 regulates LAT1-like transporters subcellular trafficking in both perineurial and subperineurial cells, leading to adequate essential amino acid supply and GABAergic neuronal activity promoting sleep, in a circadian-clock-dependent manner. This study observed that downregulating Pallidin using a pan-neuronal driver also resulted in reduced night sleep, suggesting that SG is not the only cell type in the brain where Pallidin is required to regulate sleep (Li, 2023).

Blocking the cycling of the molecular clock by placing flies in a per⁰ mutant background completely abolishes the Pallidin knockdown sleep/wake phenotype, suggesting that the circadian clock is implicated. Accordingly, it was found that hyperpolarization of PDF-expressing clock neurons is sufficient to normalize the main Pallidin knockdown phenotype: increased sleep latency at night. This raises the possibility that Pallidin affects clock-dependent sleep/wake regulatory networks and in particular, those under the control of the large PDF-expressing neurons (lLNv). In lLNv neurons, GABAergic input is modulated in a circadian manner, resulting in higher inhibition in the early night, promoting sleep, while a disruption of this input results in delayed and fragmented sleep (Li, 2023).

The GABAergic neurons responsible for lLNv inhibition are so far unidentified. The delayed and reduced night sleep observed upon Pallidin knockdown in SG is also similar to the phenotype previously reported with pan-GABAergic neuron inhibition. Conversely, feeding flies the GABA agonist THIP65 or increasing GABAergic neuronal firing by expressing TrpA1 can potently induce sleep. The latter manipulation cannot promote sleep when Pallidin is downregulated in SG, suggesting that the gene is required to allow sustained GABAergic neuronal activity. Alternatively, Pallidin function could be regulated directly by the clock present in perineurial cells, which modulates the efflux properties of subperineurial cells in a circadian manner (Li, 2023).

Downregulation of several members of BLOC1 in SG phenocopies the knockdown of Pallidin, strongly suggesting that the whole complex is involved in this Pallidin-dependent sleep/wake regulation. This is consistent with the observation that the pool of Pallidin and dysbindin present in the cell is almost entirely associated with the complex and that the lack of one subunit leads to a destabilization of the others (Li, 2023).

The lack of sleep alteration with snapin manipulation may result from lower efficiency of the genetic tools used for downregulation. Alternatively, it may also reflect different gene dosage sensitivity and functionality for the different members of the complex, as previously reported, or the existence of multiple subunits within the complex2 or different complexes containing a subset of the subunits (Li, 2023).

In Drosophila larval neuromuscular junction, Snapin has, for example, been shown to regulate synaptic homeostasis (Dickman, 2012), while Pallidin does not seem to affect baseline neurotransmission and is required during sustained neuronal activity to replenish the pool of releasable synaptic vesicles (Chen, 2017). BLOC1 has been shown to be required for the biogenesis of recycling endosomes through its interactions with sorting endosomes and the cytoskeleton (Delevoye, 2016). These BLOC1 functions and the regulation of sleep by BLOC1 members reported in this study are in agreement with a recent report showing bidirectional interactions between sleep and endocytosis in SG (Lee, 2018). Intriguingly, the endocytosis in SG was reported to be the most intense in the early part of the night, when sleep is the deepest and the phenotype of Pallidin downregulation is the most pronounced. In addition, the activity of the recycling endosome associated small GTPase Rab11 appears to play a prominent role in this context. Consistent with these findings, extensive protein-protein interaction analyses identified rab11 as the best interacting partner for BLOC1 in both Drosophila and humans.5 Thus, BLOC1 may facilitate the high endocytic activity of SG during the early part of the night, notably the biogenesis of recycling endosomes, while having a less prominent role at other times during the day. In line with the results, Pallidin mutant mice display reduced sleep amount during the light phase of the day, the primary sleep period in rodents, and shorter average sleep-bout duration (Li, 2023).

However, BBB-specific and conditional knockout models in mice would be necessary to determine whether the model outlined here applies to mammals. In humans, the incidence of sleep/wake disorders in the rare Hermansky-Pudlak syndrome, bearing deficits in BLOC1 or other functionally related complexes, is unknown but deserves scrutiny. In contrast, sleep disruption is common among patients suffering from schizophrenia, another pathology associated with BLOC1 deficits. Interestingly, one of the prominent sleep abnormalities in schizophrenia patients is a reduction in sleep spindle density, which strongly relies on GABAergic activity, and the latter is also affected in this pathology. Accordingly, previous studies in dysbindin mutant mice have shown that BLOC1 can disrupt GABAergic activity (Li, 2023).

Thus, a potential role for amino acid transport at the BBB in those contexts would deserve investigation given these results. Early studies of Pallidin function pointed to a role in amino acid import into the brain transport, as suggested by lower sensitivity to intraperitoneal injections of the LAT1 substrates l-DOPA and tryptophan. The LAT1 transporter plays a major role in the import of these amino acids and in the import of large neutral essential amino acids such as leucine, isoleucine, and histidine (Li, 2023).

A conditional knockout of LAT1 in brain endothelial cells confirmed the crucial role of LAT1 in the regulation essential amino acid abundance within the brain, its impact on GABAergic transmission, and its potential implication in autism spectrum disorders. Intriguingly, the relative abundance of amino acids in the brain of Pallidin mutant mice resembles those found in mice with a BBB specific knockout LAT1 (Li, 2023).

This study provides evidence that Pallidin function in SG is linked to essential amino acid supply and to LAT1-like transporter activity. First, downregulation of the LAT1-like transporters and of TOR signaling phenocopy knock down, to a large extent or completely, the BLOC1 genes. Second, supplementing the food with leucine can normalize the JhI-21, Pallidin, and Blos2 phenotypes. Interestingly, the phenotype of the JhI-21 transporter knockdown could be rescued by valine and tryptophan supplementation, while Pallidin knockdown could be rescued only by leucine. This suggests that Pallidin does not solely modulate LAT1-like transporter function and has complex effect on multiple transport systems, as previously suggested (Li, 2023).

JhI-21 and minidisc are the Drosophila closest homologs of LAT1 light-chain. JhI-21 is expressed broadly in the Drosophila brain, with most of the signal in perinuclear punctae that colocalizes partially with the lysosomal marker ATG8. The subcellular trafficking of JhI-21 in SG is abnormal following Pallidin downregulation, with a substantial fraction of the transporter localized in the nucleus. This abnormal trafficking is likely to reduce the functionality of the transporter, explaining the similar sleep/wake phenotypes of JhI-21 and Pallidin downregulation in SG and their normalization by essential amino acid supplementation. A nuclear localization has already been reported in glioma cell lines for the solute carrier (SLC) Eaat1 and Eaat2 transporters, and for the K+ inwardly rectifying channel. In both cases, this unusual subcellular localization was associated with an overall reduced functionality of the protein in the cell. Aside transporters, several full-length transmembrane receptors have been repeatedly reported to be localized in the nucleus where some of them may act as a transcription factors (Li, 2023).

The mechanisms underlying this phenomenon are mostly unknown. In this study, it was not possible to detect obvious changes in autophagy, suggesting that Pallidin downregulation does not induce major changes in lysosomal activity and rather affects more specifically particular cargos, as previously suggested. Interestingly, in addition to their cytoplasmic localization, a nuclear localization has previously been reported for two BLOC1 subunits: BLOS2102 and, in particular, dysbindin. ATG8 is also found both in lysosomes and in the nucleus where it can regulate gene expression in association with transcription factors. Such lysosomal-nuclear connections open the possibility of trapping a diversity of unexpected proteins in the nucleus in normal as well as in abnormal conditions (Li, 2023).

In Drosophila, protein intake, threonine intake, and D-serine levels play a role in sleep/wake regulation. Although the changes in amino acid intake presumably change global free amino acid levels within the brain, the effect on sleep/wake regulation seems to originate from specific sleep/wake regulatory networks or neurotransmission systems. In humans, amino-acid-supplemented diets have been designed to improve sleep and wakefulness, on the basis of the principle that this will increase the synthesis of monoamines. The results point to a model independent from these previously identified mechanisms: higher recycling endosome activity in the early part of the night, mediated by BLOC1, would lead to high LAT1-like activity and essential amino acid import in the brain. The TOR signaling appears also to be required and may facilitate LAT1-like transporter function. This assessment suggests that Pallidin-dependent sleep regulation does not involve a modulation of monoamine levels, in contrast with previous reports. However, in these reports the conclusions were reached using complete knock out and not by downregulation in specific cell types, making comparison with the present results difficult. For example, Drosophila Dysbindin has been suggested to regulate global brain dopamine level by modulating its recycling through glial cells (Li, 2023).

In contrast, the current data suggest that essential amino acid import to the brain facilitated by Pallidin would enhance GABAergic transmission required for sleep. This study showed that a large number of GABAergic neurons in the adult fly brain are in direct cellular contacts with SG. Although the significance and the function of these contacts remain to be determined, these observations fit the idea that a significant subset of these neurons have particular metabolic needs (Li, 2023).

How could amino acid transport regulate GABAergic transmission? Amino acids are at the interplay among many processes, being involved in protein synthesis, energy metabolism, neurotransmitter synthesis, and degradation. Amino acid transporters may further intertwine these processes: as a prime example, LAT1 is an antiporter that can associate the import of leucine to the export of glutamine, two amino acids that are crucial in the glutamate/GABA/glutamine cycle in the brain (Li, 2023).

Pharmacological experiments have indeed suggested that LAT1 in the BBB regulates GABA homeostasis in the interstitial fluid. Branched-chain amino acids (BCAA) and leucine in particular are thought to be critical providers of nitrogen for glutamate synthesis through transamination, affecting glutamate/GABA/glutamine cycling. Enzymes for BCAA metabolism are conserved in Drosophila, suggesting similar metabolic regulation in this model organism. An interesting example is Drosophila mutants for GABA transaminase (GABAT), which sleep 2-3 h longer than their genetic controls because of impaired GABA degradation. For survival, these mutants require that food include glutamate or BCAAs (leucine and valine), whose transamination can provide glutamate in the cells. These data indicate that GABA metabolism and BCAAs as a source of glutamate play a substantial role in sleep/wake regulation and brain energy metabolism. Interestingly, this study found that the GABA/glutamate ratio tends to be decreased in Pallidin knockdown flies, while the glutamate/aspartate ratio is increased compared with controls. These results suggest that BLOC1 function in SG modulates neurotransmitter and brain amino acid metabolism on a global level and may control the inhibitory/excitatory balance (Li, 2023).

The data presented in this study emphasize the implication of circulating amino acids, in particular BCAA, in sleep/wake regulation, corroborating several recent metabolomics studies in mammalian models and in humans, in which branched-chain amino acid (BCAA) levels have been repeatedly shown to be modulated by the circadian clock and/or the sleep homeostat. For example, a recent study identified in insomniac patients prominent changes in the levels of several circulating BCAA, including increased levels of leucine during the night. Food supplemented with BCAA can correct sleep disorders in a mouse model with chronic sleep disruption. It is worth noting that genes involved in amino acid transport are among those commonly disrupted in autism and schizophrenia. In conclusion, this study provides potential mechanisms at the blood-brain interface that may be relevant to both sleep disruption and psychosis and emphasizes the possibility of diet-based therapies (Li, 2023).

One limitation of this study has been the inability to monitor Pallidin protein expression using immunofluorescence. A previously published antibody produced a non-specific signal in the adult brain, and attempts to generate a new antibody were unsuccessful. This precluded determining the subcellular localization of the protein and assessing the local efficiency of the knockdown constructs. The lack of appropriate drivers prevented more precise manipulation of the neuronal targets affected by Pallidin function: the GABAergic neurons presynaptic to lLNv neurons are unknown, and therefore there is no identified driver making it possible to monitor or control their activity, and in addition, there are no available LexA construct specifically expressed in lLNv neurons. For future investigations, it will be important to develop an efficient conditional system, independent of temperature, to limit the knockdown of Pallidin to the adult stage. Finally, further work is required to elucidate the involvement of the TOR signaling pathway in this context and the Pallidin-JhI-21-dependent interactions between SG and GABAergic neurons (Li, 2023).

Drosophila Arl8 is a general positive regulator of lysosomal fusion events

The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. This study shows that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, this study demonstrates that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor (Boda, 2019).

The BLOC-1 Subunit Pallidin Facilitates Activity-Dependent Synaptic Vesicle Recycling

Membrane trafficking pathways must be exquisitely coordinated at synaptic terminals to maintain functionality, particularly during conditions of high activity. This study has generated null mutations in the Drosophila homolog of pallidin, a central subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), to determine its role in synaptic development and physiology. Pallidin localizes to presynaptic microtubules and cytoskeletal structures, and that the stability of Pallidin protein is highly dependent on the BLOC-1 components Dysbindin and Blos1. This study demonstrate dthat the rapidly recycling vesicle pool is not sustained during high synaptic activity in pallidin mutants, leading to accelerated rundown and slowed recovery. Following intense activity, A loss was observed of early endosomes and a concomitant increase in tubular endosomal structures in synapses without Pallidin. Together, these data reveal that Pallidin subserves a key role in promoting efficient synaptic vesicle recycling and re-formation through early endosomes during sustained activity (Chen, 2017).

The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse

Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. This idea was tested by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. Quantitative mass spectrometry was applied to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). 491 proteins sensitive to dysbindin and BLOC-1 loss of function were found. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, it was demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. These results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects (Gokhale, 2016).

The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity

Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. This study consisted of a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. Components of the vesicle fusion machinery were identified as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, the role for NSF was studied in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, this study found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. These results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity (Gokhale, 2015).

Dysbindin associates with seven other polypeptides to form the biogenesis of lysosome-related organelles complex 1. Null mutations in mouse dysbindin reduce the expression of other BLOC-1 subunit mRNAs and polypeptides. This suggests that dysbindin genetic downregulation could elicit multiple alterations of protein content in cells. This study identified 224 proteins whose content was modified by dysbindin/BLOC-1 partial loss of function using unbiased quantitative mass spectrometry. The screen prominently identified components of the N-ethylmaleimide-sensitive factor (NSF)-dependent vesicle fusion machinery. Focus was placed on NSF, a catalytic component of the fusion machinery, and it was asked whether NSF participates in dysbindin/BLOC-1-dependent synaptic mechanisms. Drosophila presynaptic plasticity produced by the inhibition of postsynaptic receptors was used as an assay. As previously, it was observed that mutations in fly dysbindin precluded the establishment of homeostatic synaptic plasticity, a phenotype that is rescued by presynaptic expression of dysbindin. Neuron-specific expression of dNSF1, the gene encoding Drosophila NSF, by itself does not modulate this form of plasticity, yet NSF1 expression at the synapse of dysbindin mutants rescued homeostatic synaptic plasticity defects to the same extent as dysbindin re-expression in the presynaptic compartment. These results demonstrate that partial dysbindin/BLOC-1 loss of function alters the cellular content of proteins that specifically have roles in synaptic mechanisms (Gokhale, 2015).

Genetic polymorphisms associated with schizophrenia mostly reside in noncoding regions modifying gene and/or protein levels rather protein sequence. The question of how widespread the effects are of a single mutation or polymorphism across the proteome has been poorly explored. This study addressed this question by modeling a partial reduction in the cellular content of dysbindin/BLOC-1 using shRNAs against BLOC-1 complex subunits. 224 proteins were whose content is affected by a partial loss of function of dysbindin/BLOC-1 and focused on an interactome centered around a schizophrenia susceptibility gene, dysbindin, and NSF, a component of the membrane fusion machinery that localizes to the synapse and was previously implicated in schizophrenia mechanisms. Functional outcomes of the dysbindin/BLOC-1 and NSF association were confirmed using a Drosophila synaptic adaptive response. The results demonstrate that dysbindin/BLOC-1 expression defects induce multiple downstream quantitative protein expression traits associated with the vesicle fusion apparatus, which influence synaptic plasticity in an invertebrate model synapse (Gokhale, 2015).

A proteomic search prominently highlights the following components of the vesicle fusion apparatus: munc18, tomosyn, NSF; and the SNAREs syntaxin 7, syntaxin 17, SNAP23, SNAP25, SNAP 29, and VAMP7. Importantly, most of the aforementioned vesicle fusion machinery components have been implicated by genomic and postmortem studies in several neurodevelopmental disorders, including schizophrenia, intellectual disability, and autism spectrum disorder. The current strategy is validated by the identification of proteins previously known to be downregulated in null alleles of BLOC-1 subunits and/or known to interact with BLOC-1. These proteins include subunits of the BLOC-1 complex and the SNARE VAMP7. This study further authenticated these fusion machinery components as part of a dysbindin/BLOC-1 network by (1) coimmunoprecipitation of a fusion machinery component with dysbindin/BLOC-1 subunits and/or (2) downregulation of a fusion machinery component after genetic or shRNA-mediated reduction of dysbindin/BLOC-1 subunits. NSF was studied since it is a hub of protein-protein interactions with components of the fusion machinery, and is a catalytic activity that is required for the resolution of fusion reaction products and other protein-protein complexes. NSF was found to associate with dysbindin and BLOC-1 subunits in neuroblastoma cells in culture. However, efforts to document the association of NSF and dysbindin-BLOC-1 by immunoprecipitation with NSF antibodies were unsuccessful in brain. This outcome occurred regardless of whether NSF was immunoprecipitated from brain homogenates or cross-linked synaptosomal lysates from adult mouse brain. This negative result is attributed to the high abundance of NSF in brain compared with dysbindin/BLOC-1. Reverse immunoprecipitations with dysbindin/BLOC-1 antibodies were not possible, as none of the available antibodies were suitable for immunoprecipitation. Since most of the associations between NSF and dysbindin/BLOC-1 are detected in the presence of the cross-linker DSP in cell lines, it is likely that the biochemical interactions between NSF and dysbindin/BLOC-1 are indirect. However, NSF cellular levels are decreased following shRNA-mediated or genomic reduction of BLOC-1 complex members, arguing in favor of a functional outcome of this association. No NSF downregulation phenotype was detected in hippocampal extracts of Bloc1s8sdy/sdy mice at days 7 or 50 of postnatal development. This suggests that NSF phenotypes may be anatomically restricted either to a region of the hippocampus or to an earlier and transient developmental stage. However, this reduced NSF trait is robustly and reversibly induced by genetic disruption of the dysbindin/BLOC-1 complex or by downregulation of dysbindin/BLOC-1 subunits in neuroblastoma and human embryonic kidney cells, neuroectodermal cells, and iPSC-derived human neurons (Gokhale, 2015).

These studies indicate that the functional outcome of NSF reduction in BLOC-1 loss of function become evident only when the synapse is challenged. Constitutive secretion in Drosophila or mammalian non-neuronal cells is unaffected, as are spontaneous and evoked neurotransmission at the Drosophila neuromuscular junction. However, a requirement for NSF in BLOC-1 loss-of-function phenotypes can be localized to a presynaptic homeostatic mechanism, which is engaged when postsynaptic receptors are blocked with philanthotoxin. After a brief incubation with philanthotoxin, the resultant reduction in postsynaptic signal transduction rapidly induces a compensatory increase in quantal content, a response known as homeostatic synaptic plasticity. This adaptive compensatory mechanism is precluded by dysbindin mutations, and can be rescued by presynaptic expression of dysbindin. However, it was possible to rescue this phenotype in the dysbindin mutants to the exact same extent through presynaptic expression NSF. The observation that RNAi downregulation or overexpression of NSF in the neuromuscular junction does not interfere with homeostatic synaptic plasticity argues that the NSF is not an obligate component downstream of dysbindin/BLOC-1 in a linear pathway, but rather is an adaptive response to network perturbation induced by a dysbindin mutant allele. This hypothesis predicts that transheterozygotic reduction of NSF and Dysbindin should impair plasticity, a result that is at odds with the finding that plasticity is normal in dysb1-/+;UAS-NSF RNAi. It is believe that this may be a consequence of a modest reduction of dysbindin polypeptide in dysb1-/+ animals, which was predicted to be ~25% (Gokhale, 2015).

How does the BLOC-1-NSF interaction affect synaptic mechanisms? A model integrating these findings has to consider three key elements. First, BLOC-1 subunits reside at endosomes as well as on synaptic vesicles in presynaptic terminals in neurons. Second, BLOC-1 binds monomeric SNAREs rather than tetrahelical SNARE bundles in vitro. Finally, NSF and SNAREs bind to dysbindin/BLOC-1, yet they do not seem to form a ternary complex. Thus, it is proposed that BLOC-1 bound to a single SNARE (perhaps for SNARE sorting into vesicles) is resolved by NSF, making SNAREs permissive for vesicle fusion. Therefore, when dysbindin and NSF levels are reduced by hypomorphic mutations in the fly or as a quantitative expression trait in humans, SNARE-dependent mechanisms might be impaired due to defective SNARE sorting, a consequence of the reduced levels of BLOC-1 complex and, additionally, by decreased NSF content that would impair the resolution of remaining SNARE-BLOC-1 complexes. Thus, noncoding polymorphisms in several genes and their quantitative expression traits may converge to impair synaptic mechanisms. It is proposed that unbiased identification of quantitative traits across the proteome of neurodevelopmental deficiency models is a simple approach to unravel mechanisms of complex neurodevelopmental disorders (Gokhale, 2015).

Genetic modifiers of abnormal organelle biogenesis in a Drosophila model of BLOC-1 deficiency

Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky-Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. This study has generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes (Cheli, 2010).

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. This study identified in the double knockout 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 (Seugnet, 2023).

Mechanical stimulation promotes enthesis injury repair by mobilizing Prrx1(+) cells via ciliary TGF-beta signaling

Proper mechanical stimulation can improve rotator cuff enthesis injury repair. However, the underlying mechanism of mechanical stimulation promoting injury repair is still unknown. This study found that Prrx1(+) cell was essential for murine rotator cuff enthesis development identified by single-cell RNA sequence and involved in the injury repair. Proper mechanical stimulation could promote the migration of Prrx1(+) cells to enhance enthesis injury repair. Meantime, TGF-beta signaling and primary cilia played an essential role in mediating mechanical stimulation signaling transmission. Proper mechanical stimulation enhanced the release of active TGF-beta1 to promote migration of Prrx1(+) cells. Inhibition of TGF-beta signaling eliminated the stimulatory effect of mechanical stimulation on Prrx1(+) cell migration and enthesis injury repair. In addition, knockdown of Pallidin to inhibit TGF-betaR2 translocation to the primary cilia or deletion of Ift88 in Prrx1(+) cells also restrained the mechanics-induced Prrx1(+) cells migration. These findings suggested that mechanical stimulation could increase the release of active TGF-beta1 and enhance the mobilization of Prrx1(+) cells to promote enthesis injury repair via ciliary TGF-beta signaling (Xiao, 2022).

A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. This study shows that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3delta subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3delta and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1-dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3-BLOC-1 super-complex (Bowman, 2021).

Sex-dimorphic effects of biogenesis of lysosome-related organelles complex-1 deficiency on mouse perinatal brain development

The function(s) of the Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1) during brain development is to date largely unknown. This study investigated how its absence alters the trajectory of postnatal brain development using as model the pallid mouse. Most of the defects observed early postnatally in the mutant mice were more prominent in males than in females and in the hippocampus. Male mutant mice, but not females, had smaller brains as compared to sex-matching wild types at postnatal day 1 (P1), this deficit was largely recovered by P14 and P45. An abnormal cytoarchitecture of the pyramidal cell layer of the hippocampus was observed in P1 pallid male, but not female, or juvenile mice (P45), along with severely decreased expression levels of the radial glial marker Glutamate-Aspartate Transporter. Transcriptomic analyses showed that the overall response to the lack of functional BLOC-1 was more pronounced in hippocampi at P1 than at P45 or in the cerebral cortex. These observations suggest that absence of BLOC-1 renders males more susceptible to perinatal brain maldevelopment and although most abnormalities appear to have been resolved in juvenile animals, still permanent defects may be present, resulting in faulty neuronal circuits, and contribute to previously reported cognitive and behavioral phenotypes in adult BLOC-1-deficient mice (Lee, 2021).

Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice

Dysbindin, a schizophrenia susceptibility marker and an essential constituent of BLOC-1 (biogenesis of lysosome-related organelles complex-1), has recently been associated with cardiomyocyte hypertrophy through the activation of Myozap-RhoA-mediated SRF signaling. This study employed sandy mice (Dtnbp1_KO), which completely lack Dysbindin protein because of a spontaneous deletion of introns 5-7 of the Dtnbp1 gene, for pathophysiological characterization of the heart. Unlike in vitro, the loss-of-function of Dysbindin did not attenuate cardiac hypertrophy, either in response to transverse aortic constriction stress or upon phenylephrine treatment. Interestingly, however, the levels of hypertrophy-inducing interaction partner Myozap as well as the BLOC-1 partners of Dysbindin like Muted and Pallidin were dramatically reduced in Dtnbp1_KO mouse hearts. Taken together, these data suggest that Dysbindin's role in cardiomyocyte hypertrophy is redundant in vivo, yet essential to maintain the stability of its direct interaction partners like Myozap, Pallidin and Muted (Borlepawar, 2020).

Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons

Cytohesin-2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. This study first demonstrated that cytohesin-2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin-2 in neurons, yeast two-hybrid screening of brain cDNA libraries was conducted using cytohesin-2 as bait and pallidin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) involved in endosomal trafficking was isolated. Pallidin interacted specifically with cytohesin-2 among cytohesin family members. Glutathione S-transferase pull-down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin-2 and pallidin. Immunofluorescence demonstrated that cytohesin-2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin-2 reduced cytoplasmic EEA1-positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co-expression of wild-type pallidin but not a mutant lacking the ability to interact with cytohesin-2. In contrast, knockdown of cytohesin-2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin-2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons (Ito, 2018).

Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)

BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. This study shows that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, approximately 300 A long and approximately 30 A in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45 degrees. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting (Lee, 2012).

The schizophrenia susceptibility factor dysbindin and its associated complex sort cargoes from cell bodies to the synapse

Dysbindin assembles into the biogenesis of lysosome-related organelles complex 1 (BLOC-1), which interacts with the adaptor protein complex 3 (AP-3), mediating a common endosome-trafficking route. Deficiencies in AP-3 and BLOC-1 affect synaptic vesicle composition. However, whether AP-3-BLOC-1-dependent sorting events that control synapse membrane protein content take place in cell bodies upstream of nerve terminals remains unknown. This hypothesis was tested by analyzing the targeting of phosphatidylinositol-4-kinase type II alpha (PI4KIIalpha), a membrane protein present in presynaptic and postsynaptic compartments. PI4KIIalpha copurified with BLOC-1 and AP-3 in neuronal cells. These interactions translated into a decreased PI4KIIalpha content in the dentate gyrus of dysbindin-null BLOC-1 deficiency and AP-3-null mice. Reduction of PI4KIIalpha in the dentate reflects a failure to traffic from the cell body. PI4KIIalpha was targeted to processes in wild-type primary cultured cortical neurons and PC12 cells but failed to reach neurites in cells lacking either AP-3 or BLOC-1. Similarly, disruption of an AP-3-sorting motif in PI4KIIalpha impaired its sorting into processes of PC12 and primary cultured cortical neuronal cells. These findings indicate a novel vesicle transport mechanism requiring BLOC-1 and AP-3 complexes for cargo sorting from neuronal cell bodies to neurites and nerve terminals (Larimore, 2011).

Search PubMed for articles about Drosophila Pallidin

Boda, A., Lorincz, P., Takats, S., Csizmadia, T., Toth, S., Kovacs, A. L. and Juhasz, G. (2019). Drosophila Arl8 is a general positive regulator of lysosomal fusion events. Biochim Biophys Acta Mol Cell Res 1866(4): 533-544. PubMed ID: 30590083

Borlepawar, A., Schmiedel, N., Eden, M., Christen, L., Rosskopf, A., Frank, D., Lullmann-Rauch, R., Frey, N., Rangrez, A. Y. (2020). Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice. Cells, 9(11) PubMed ID: 33142804

Bowman, S. L., Le, L., Zhu, Y., Harper, D. C., Sitaram, A., Theos, A. C., Sviderskaya, E. V., Bennett, D. C., Raposo-Benedetti, G., Owen, D. J., Dennis, M. K., Marks, M. S. (2021). A BLOC-1-AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers. J Cell Biol, 220(7) PubMed ID: 33886957

Chen, X., Ma, W., Zhang, S., Paluch, J., Guo, W., Dickman, D. K. (2017). The BLOC-1 Subunit Pallidin Facilitates Activity-Dependent Synaptic Vesicle Recycling. eNeuro, 4(1) PubMed ID: 28317021

Cheli, V. T., Daniels, R. W., Godoy, R., Hoyle, D. J., Kandachar, V., Starcevic, M., Martinez-Agosto, J. A., Poole, S., DiAntonio, A., Lloyd, V. K., Chang, H. C., Krantz, D. E., Dell'Angelica, E. C. (2010). Genetic modifiers of abnormal organelle biogenesis in a Drosophila model of BLOC-1 deficiency. Hum Mol Genet, 19(5):861-878 PubMed ID: 20015953

Delevoye, C., Heiligenstein, X., Ripoll, L., Gilles-Marsens, F., Dennis, M. K., Linares, R. A., Derman, L., Gokhale, A., Morel, E., Faundez, V., Marks, M. S., Raposo, G. (2016). BLOC-1 Brings Together the Actin and Microtubule Cytoskeletons to Generate Recycling Endosomes. Curr Biol, 26(1):1-13 PubMed ID: 26725201

Dickman, D. K., Tong, A. and Davis, G. W. (2012). Snapin is critical for presynaptic homeostatic plasticity. J Neurosci 32: 8716-8724. PubMed ID: 22723711

Gokhale, A., et al. (2015). The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity. J Neurosci 35: 7643-7653. PubMed ID: 25972187

Gokhale, A., Hartwig, C., Freeman, A. H., Das, R., Zlatic, S. A., Vistein, R., Burch, A., Carrot, G., Lewis, A. F., Nelms, S., Dickman, D. K., Puthenveedu, M. A., Cox, D. N., Faundez, V. (2016). The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse. J Neurosci, 36(49):12393-12411 PubMed ID: 27927957

Ito, A., Fukaya, M., Saegusa, S., Kobayashi, E., Sugawara, T., Hara, Y., Yamauchi, J., Okamoto, H., Sakagami, H. (2018). Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons. J Neurochem, 147(2):153-177 PubMed ID: 30151872

Larimore, J., Tornieri, K., Ryder, P. V., Gokhale, A., Zlatic, S. A., Craige, B., Lee, J. D., Talbot, K., Pare, J. F., Smith, Y., Faundez, V. (2011). The schizophrenia susceptibility factor dysbindin and its associated complex sort cargoes from cell bodies to the synapse. Mol Biol Cell, 22(24):4854-4867 PubMed ID: 21998198

Lee, F.Y., Wang H.-B., Hitchcock O.N., Loh D.H., Whittaker D.S., Kim Y.-S., Aiken A., Kokikian C., Dell'Angelica E.C., Colwell C.S., and Ghiani C.A. (2018). Sleep/Wake Disruption in a Mouse Model of BLOC-1 Deficiency. Front. Neurosci. 2018; 12: 759. PubMed ID: 30498428

Lee, F. Y., Larimore, J., Faundez, V., Dell'Angelica, E. C., Ghiani, C. A. (2021). Sex-dimorphic effects of biogenesis of lysosome-related organelles complex-1 deficiency on mouse perinatal brain development. J Neurosci Res, 99(1):67-89 PubMed ID: 32436302

Lee, H. H., Nemecek, D., Schindler, C., Smith, W. J., Ghirlando, R., Steven, A. C., Bonifacino, J. S., Hurley, J. H. (2012). Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1). J Biol Chem, 287(8):5882-5890 PubMed ID: 22203680

Li, H., Aboudhiaf, S., Parrot, S., Scote-Blachon, C., Benetollo, C., Lin, J. S., Seugnet, L. (2023). Pallidin function in Drosophila surface glia regulates sleep and is dependent on amino acid availability. Cell Rep, 42(9):113025 PubMed ID: 37682712

Seugnet, L., Anaclet, C., Perier, M., Ghersi-Egea, J. F., Lin, J. S. (2023). A marked enhancement of a BLOC-1 gene, pallidin, associated with somnolent mouse models deficient in histamine transmission. CNS neuroscience & therapeutics, 29(1):483-486 PubMed ID: 36258293

Xiao, H., Zhang, T., Li, C., Cao, Y., Wang, L., Chen, H., Li, S., Guan, C., Hu, J., Chen, D., Chen, C., Lu, H. (2022). Mechanical stimulation promotes enthesis injury repair by mobilizing Prrx1(+) cells via ciliary TGF-beta signaling. Elife, 11 PubMed ID: 35475783

date revised: 17 May 2024

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