AP1/2ß-mediated exocytosis of tapetum-specific transporters is required for pollen development in Arabidopsis thaliana.

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (ß1 and γ for AP-1 and ß2 and α for AP-2), a medium subunit µ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of ß subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2ß adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both ß adaptins is lethal in plants. We identified a critical role of ß adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, ß adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2ß adaptins in plants and their specialized roles in specific cell types.

Genome-wide association study reveals genetic variants associated with HIV-1C infection in a Botswana study population.

Although there have been many studies of gene variant association with different stages of HIV/AIDS progression in United States and European cohorts, few gene-association studies have assessed genic determinants in sub-Saharan African populations, which have the highest density of HIV infections worldwide. We carried out genome-wide association studies on 766 study participants at risk for HIV-1 subtype C (HIV-1C) infection in Botswana. Three gene associations (AP3B1, PTPRA, and NEO1) were shown to have significant association with HIV-1C acquisition. Each gene association was replicated within Botswana or in the United States-African American or United States-European American AIDS cohorts or in both. Each associated gene has a prior reported influence on HIV/AIDS pathogenesis. Thirteen previously discovered AIDS restriction genes were further replicated in the Botswana cohorts, extending our confidence in these prior AIDS restriction gene reports. This work presents an early step toward the identification of genetic variants associated with and affecting HIV acquisition or AIDS progression in the understudied HIV-1C afflicted Botswana population.

A homozygous AP3D1 missense variant in patients with sensorineural hearing loss as the leading manifestation.

Loss-of-function variants in AP3D1 have been linked to Hermansky-Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein-protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

Hereditary spastic paraplegia associated with a novel homozygous intronic noncanonical splice site variant in the AP4B1 gene.

Pathogenic variants in the AP4B1 gene lead to a rare form of hereditary spastic paraplegia (HSP) known as SPG47. We report on a patient with a clinical suspicion of complicated HSP of the lower limbs with intellectual disability, as well as a novel homozygous noncanonical splice site variant in the AP4B1 gene, in which the effect on splicing was validated by RNA analysis. We sequenced 152 genes associated with HSP using Next-Generation Sequencing (NGS). We isolated total RNA from peripheral blood and generated cDNA using reverse transcription-polymerase chain reaction (RT-PCR). A region of AP4B1 mRNA was amplified by PCR and the fragments obtained were purified from the agarose gel and sequenced. We found a homozygous variant of uncertain significance in the AP4B1 gene NM_006594.4: c.1511-6C>G in the proband. Two different AP4B1 mRNA fragments were obtained in the patient and his carrier parents. The shorter fragment was the predominant fragment in the patient and revealed a deletion with skipping of the AP4B1 exon 10. The patient's longer fragment corresponded to an insertion of the last five nucleotides of AP4B1 intron 9. We confirmed that this variant affects the normal splicing of RNA, sustaining the molecular diagnosis of SPG47 in the patient.

Recessive Mutations in AP1B1 Cause Ichthyosis, Deafness, and Photophobia.

We describe unrelated individuals with ichthyosis, failure to thrive, thrombocytopenia, photophobia, and progressive hearing loss. Each have bi-allelic mutations in AP1B1, the gene encoding the ß subunit of heterotetrameric adaptor protein 1 (AP-1) complexes, which mediate endomembrane polarization, sorting, and transport. In affected keratinocytes the AP-1 ß subunit is lost, and the γ subunit is greatly reduced, demonstrating destabilization of the AP-1 complex. Affected cells and tissue contain an abundance of abnormal vesicles and show hyperproliferation, abnormal epidermal differentiation, and derangement of intercellular junction proteins. Transduction of affected cells with wild-type AP1B1 rescues the vesicular phenotype, conclusively establishing that loss of AP1B1 function causes this disorder.

Homozygous Loss-of-Function Mutations in AP1B1, Encoding Beta-1 Subunit of Adaptor-Related Protein Complex 1, Cause MEDNIK-like Syndrome.

MEDNIK syndrome (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma) is an autosomal-recessive disorder caused by bi-allelic mutations in AP1S1, encoding the small σ subunit of the AP-1 complex. Central to the pathogenesis of MEDNIK syndrome is abnormal AP-1-mediated trafficking of copper transporters; this abnormal trafficking results in a hybrid phenotype combining the copper-deficiency-related characteristics of Menkes disease and the copper-toxicity-related characteristics of Wilson disease. We describe three individuals from two unrelated families in whom a MEDNIK-like phenotype segregates with two homozygous null variants in AP1B1, encoding the large ß subunit of the AP-1 complex. Similar to individuals with MEDNIK syndrome, the affected individuals we report display abnormal copper metabolism, evidenced by low plasma copper and ceruloplasmin, but lack evidence of copper toxicity in the liver. Functional characterization of fibroblasts derived from affected individuals closely resembles the abnormal ATP7A trafficking described in MEDNIK syndrome both at baseline and in response to copper treatment. Taken together, our results expand the list of inborn errors of copper metabolism.

Plasma lncRNA LOC338963 and mRNA AP3B2 are upregulated in paraneoplastic Lambert-Eaton myasthenic syndrome.

INTRODUCTION/AIMS: Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular junction disorder. Long noncoding RNA (lncRNA) can regulate the expression of mRNA and is involved in the development of autoimmune diseases, but few genetic studies are available. In this study we aimed to explore the lncRNA and mRNA changes of LEMS. METHODS: Plasma lncRNA and mRNA expression profiles of three LEMS patients with small cell lung cancer (SCLC) and three matched healthy controls were analyzed by microarray. Differentially expressed lncRNAs and adjacent mRNAs were jointly analyzed, and candidates were verified by quantitative real-time polymerase chain reaction (qRT-PCR). The identified genes were subsequently evaluated in 9, 8, and 4 patients with paraneoplastic LEMS, nontumor LEMS, and SCLC, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine possible functions. RESULTS: A total of 320 lncRNA and 168 mRNAs were differentially expressed in the three LEMS with SCLC and compared with healthy controls. Among these, lncRNA LOC338963 and its neighboring mRNA AP3B2 were upregulated jointly, which was confirmed by qRT-PCR. qRT-PCR revealed significant upregulation of the two genes in patients with paraneoplastic LEMS compared with nontumor LEMS or SCLC. GO analysis of AP3B2 identified the enrichment terms anterograde synaptic vesicle transport and establishment of synaptic vesicle localization. KEEG analysis showed that AP3B2 was enriched in lysosomal pathways. DISCUSSION: LOC338963 and AP3B2 were upregulated in patients with paraneoplastic LEMS, suggesting their involvement in pathogenesis. These genes could be targets for exploring the pathomechanism of paraneoplastic LEMS.

Characterization of 22q12 Microdeletions Causing Position Effect in Rare NF2 Patients with Complex Phenotypes.

Neurofibromatosis type 2 is an autosomal dominant tumor-prone disorder mainly caused by NF2 point mutations or intragenic deletions. Few individuals with a complex phenotype and 22q12 microdeletions have been described. The 22q12 microdeletions' pathogenic effects at the genetic and epigenetic levels are currently unknown. We here report on 22q12 microdeletions' characterization in three NF2 patients with different phenotype complexities. A possible effect of the position was investigated by in silico analysis of 22q12 topologically associated domains (TADs) and regulatory elements, and by expression analysis of 12 genes flanking patients' deletions. A 147 Kb microdeletion was identified in the patient with the mildest phenotype, while two large deletions of 561 Kb and 1.8 Mb were found in the other two patients, showing a more severe symptomatology. The last two patients displayed intellectual disability, possibly related to AP1B1 gene deletion. The microdeletions change from one to five TADs, and the 22q12 chromatin regulatory landscape, according to the altered expression levels of four deletion-flanking genes, including PIK3IP1, are likely associated with an early ischemic event occurring in the patient with the largest deletion. Our results suggest that the identification of the deletion extent can provide prognostic markers, predictive of NF2 phenotypes, and potential therapeutic targets, thus overall improving patient management.

mRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase.

We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry.

Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with ß-arrestin1 and that ß-arrestin1 interacted with the ß2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either ß-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the ß-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells.IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2-ß-arrestin1-AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

Hermansky-Pudlak syndrome-2 alters mitochondrial homeostasis in the alveolar epithelium of the lung.

BACKGROUND: Mitochondrial dysfunction has emerged as an important player in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a common cause of idiopathic interstitial lung disease in adults. Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder that causes a similar type of pulmonary fibrosis in younger adults, although the role of mitochondrial dysfunction in this condition is not understood. METHODS: We performed a detailed characterization of mitochondrial structure and function in lung tissues and alveolar epithelial cells deficient in the adaptor protein complex 3 beta 1 (Ap3b1) subunit, the gene responsible for causing subtype 2 of HPS (HPS-2). RESULTS: We observed widespread changes in mitochondrial homeostasis in HPS-2 cells, including the acquisition of abnormally shaped mitochondria, with reduced number of cristae, and markedly reduced activity of the electron transport chain and the tricarboxylic acid cycle. We also found that mitochondrial redox imbalance and activity of the mitochondrial unfolded protein response were dysregulated in HPS-2 cells and this associated with various other changes that appeared to be compensatory to mitochondrial dysfunction. This included an increase in glycolytic activity, an upregulation in the expression of mitochondrial biogenesis factors and enhanced activation of the energy-conserving enzyme AMP-activated protein kinase. CONCLUSION: In summary, our findings indicate that mitochondrial function is dramatically altered in HPS-2 lung tissues, suggesting dysfunction of this organelle might be a driver of HPS lung disease.

Blended phenotype of combination of HERC2 and AP3B2 deficiency and Angelman syndrome caused by paternal isodisomy of chromosome 15.

Angelman syndrome is a neurodevelopmental disorder characterized by intellectual disability (ID), a distinctive gait pattern, abnormal behaviors, severe impairment in language development, and characteristic facial features. Most cases are caused by the absence of a maternal contribution to the imprinted region on chromosome 15q11-q13. Here, we present the first reported case of a 3-year-old boy with an atypical phenotype of Angelman syndrome due to uniparental isodisomy with two recessive homozygous pathogenic variants: in HERC2 and AP3B2. Known phenotypes related to HERC2 and AP3B2 include ID and early infantile epileptic encephalopathy, respectively. The patient had severe global developmental delay and profound ID and showed a happy demeanor, stereotypic laughter, and hand-flapping movements, but also irritability. Craniofacial dysmorphic features, including brachycephaly, strabismus, wide ala nasi, short philtrum, wide open mouth, and slight hypopigmentation were seen. Progressive microcephaly was noted. Magnetic resonance imaging of the brain showed delayed myelination and cerebral atrophy. Trio whole exome sequencing and CGH-SNP array analysis revealed paternal uniparental isodisomy of chromosome 15 and two coexisting recessive diseases resulting from homozygous HERC2 and AP3B2 pathogenic variants. The pathogenic variant in HERC2 was inherited from his heterozygous-carrier father, and the variant in AP3B2 was de novo. We suppose that these unusual features were the combination of the effect of three concomitant disorders.

Novel variants in AP4B1 cause spastic tetraplegia, moderate psychomotor development delay and febrile seizures in a Chinese patient: a case report.

INTRODUCTION: The AP4B1 gene encodes a subunit of adaptor protein complex-4 (AP4), a component of intracellular transportation of proteins which plays important roles in neurons. Bi-allelic mutations in AP4B1 cause autosomal recessive spastic paraplegia-47(SPG47). CASE PRESENTATION: Here we present a Chinese patient with spastic tetraplegia, moderate psychomotor development delay and febrile seizures plus. Brain MRIs showed dilated supratentorial ventricle, thin posterior and splenium part of corpus callosum. The patient had little progress through medical treatments and rehabilitating regimens. Whole exome sequencing identified novel compound heterozygous truncating variants c.1207C > T (p.Gln403*) and c.52_53delAC (p.Cys18Glnfs*7) in AP4B1 gene. Causal mutations in AP4B1 have been reported in 29 individuals from 22 families so far, most of which are homozygous mutations. CONCLUSIONS: Our study enriched the genetic and phenotypic spectrum of SPG47. Early discovery, diagnosis and proper treatment on the conditions generally increase chances of improvement on the quality of life for patients.

PTENα regulates endocytosis and modulates olfactory function.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) α is the first identified isoform of the well-known tumor suppressor PTEN. PTENα has an evolutionarily conserved 173-aa N terminus compared with canonical PTEN. Recently, PTENα has been shown to play roles in multiple biologic processes including learning and memory, cardiac homeostasis, and antiviral immunity. Here, we report that PTENα maintains mitral cells in olfactory bulb (OB), regulates endocytosis in OB neurons, and controls olfactory behaviors in mice. We show that PTENα directly dephosphorylates the endocytic protein amphiphysin and promotes its binding to adaptor-related protein complex 2 subunit ß1 (Ap2b1). In addition, we identified mutations in the N terminus of PTENα in patients with Parkinson disease and Lewy-body dementia, which are neurodegenerative disorders with early olfactory loss. Overexpression of PTENα mutant H169N in mice OB reduces odor sensitivity. Our data demonstrate a role of PTENα in olfactory function and provide insight into the mechanism of olfactory dysfunction in neurologic disorders.-Yuan, Y., Zhao, X., Wang, P., Mei, F., Zhou, J., Jin, Y., McNutt, M. A., Yin, Y. PTENα regulates endocytosis and modulates olfactory function.

The Zic family homologue Odd-paired regulates Alk expression in Drosophila.

The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.

Autosomal-Recessive Mutations in AP3B2, Adaptor-Related Protein Complex 3 Beta 2 Subunit, Cause an Early-Onset Epileptic Encephalopathy with Optic Atrophy.

Early-onset epileptic encephalopathy (EOEE) represents a heterogeneous group of severe disorders characterized by seizures, interictal epileptiform activity with a disorganized electroencephalography background, developmental regression or retardation, and onset before 1 year of age. Among a cohort of 57 individuals with epileptic encephalopathy, we ascertained two unrelated affected individuals with EOEE associated with developmental impairment and autosomal-recessive variants in AP3B2 by means of whole-exome sequencing. The targeted sequencing of AP3B2 in 86 unrelated individuals with EOEE led to the identification of an additional family. We gathered five additional families with eight affected individuals through the Matchmaker Exchange initiative by matching autosomal-recessive mutations in AP3B2. Reverse phenotyping of 12 affected individuals from eight families revealed a homogeneous EOEE phenotype characterized by severe developmental delay, poor visual contact with optic atrophy, and postnatal microcephaly. No spasticity, albinism, or hematological symptoms were reported. AP3B2 encodes the neuron-specific subunit of the AP-3 complex. Autosomal-recessive variations of AP3B1, the ubiquitous isoform, cause Hermansky-Pudlak syndrome type 2. The only isoform for the δ subunit of the AP-3 complex is encoded by AP3D1. Autosomal-recessive mutations in AP3D1 cause a severe disorder cumulating the symptoms of the AP3B1 and AP3B2 defects.

Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 ß1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1-/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 ß1, also the µ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8-/-endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.