Trafficking on the road to neutropenia Free

In this issue of Blood, Borbaran Bravo et al report 2 unrelated families with symptomatic severe neutropenia with homozygous truncating or missense mutations in COPZ1 (located on chromosome 12), which encodes the coatomer protein complex I (COPI) ζ subunit.¹ Although the precise pathogenic mechanisms may be elusive, this discovery adds to a growing list of neutropenia-associated genes involved in cytoskeletal organization and intracellular trafficking.

Unlike other inherited bone marrow failure syndromes, such as Fanconi anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, and dyskeratosis congenita (short telomere syndrome) where there is a common genetic pathway, severe congenital neutropenia defies such reductionism in pathogenesis. Several roads end in neutropenia: metabolic stress,² granulopoietic signaling,³ cytoskeletal disorganization, and impaired endomembrane transport.⁴ Genetic causes of severe symptomatic neutropenia include cytoskeleton-associated proteins WAS, HAX1, LCP1, CDC42, and SEPT6. Another group of genes participate in endomembrane dynamics: VPS45, RBSN, JAGN1, AP3B1, and STK4. With the addition of COPZ1 to this list, the role of protein sorting and intracellular trafficking of its cargo becomes more established as a shared pathway for the development of neutropenia.

Clinically, the cases involved 3 girls who suffered recurrent bacterial infections beginning in infancy. The 2 sisters with truncating mutations (COPZ1 p.Q141Ter) had neutropenia requiring a high dose of filgrastim (10-30 μg/kg per day), anemia, lymphopenia, and thrombocytopenia as well as skeletal anomalies involving the spine and foot. The girl with the missense mutation (COPZ1 p.G132R) required less filgrastim. Unlike other forms of severe congenital neutropenia, no maturation arrest in granulopoiesis was observed. Through computer simulations, genetically engineered induced pluripotent stem cell or human hematopoietic progenitor cells to mimic the mutations, CRISPR genome-edited zebrafish, and fibroblasts expressing the truncated COPZ1 mutant, the investigators found decreased retrograde transport and impaired granulocytic differentiation. Based initially on RNA sequencing and connectivity mapping, they found that HIF-1α pathways were perturbed and that a small molecule inhibitor of HIF-1α restored granulopoiesis in CD34⁺ cells.

Protein synthesis begins in the ribosomes of the cytoplasm and endoplasmic reticulum (ER). The nascent polypeptides are processed by the signal recognition particle (SRP) complex. These 2 sites are important for normal granulocyte production, as attested by neutropenia due to pathogenic variants in ribosomal genes SBDS, EFL1, and DNAJC21 or in SRP genes SRP54, SRP7, SRPA, and SRP19.^5-7 Newly synthesized proteins enter the endomembrane system and reach their destinations via a complex tubule-vesicular interorganelle network. Like cargo, the proteins are first shipped from the ER and the Golgi apparatus packed into specialized vesicles covered with different coat proteins. Some components of these vesicles recycle back to ER to keep continuous vesicle flow between these major membrane compartments (see figure). COPs facilitate trafficking of vesicles among the Golgi apparatus as well as retrograde transport from the Golgi to the ER. The COPI is composed of 7 subunits: α, β, β′, γ, δ, ε, and ζ. These subunits assemble a complex that forms the coat of a budding vesicle.⁴ 

The study provided interesting mechanistic insights to explain functional defects of the cells expressing COPZ1 mutants. Structural modeling analysis predicted structural instability of the truncated COPZ1 protein and diminished interactions with its major binding partner, COPG1. This could result in the impaired assembly of COPI vesicles and diminished retrograde vesicle trafficking from Golgi to ER. Indeed, the authors directly demonstrated the impaired Golgi to ER transport in fibroblasts expressing the truncated COPZ1 mutant. Despite such impairment, the cellular defects caused by COPZ1 mutation appear surprisingly mild. Expression of the truncated COPZ1 mutant did not significantly affect ER and Golgi morphology nor induce ER stress.

As anyone who has had to ship a reagent from one locale to another has learned: many things can go wrong. Mislabeling, failed pickup, misprocessing at the sorting facility, a plane or truck mishap, customs’ bureaucracy, poor storage conditions, inaccurate delivery, confusion in the receiving dock, and a failure to receive and sign off by the right person. Why would a defect in a coatomer protein lead to symptomatic neutropenia? This study suggested that abnormal Golgi ER trafficking resulted in aberrant cell signaling and gene expression, leading to failure in myelopoiesis. The identified abnormalities were far-reaching from activation of the interferon signaling pathway, downregulation of HIF-1α-dependent transcription, and induction of the oxidative stress. Among these events only activation of interferon signaling has a clear link to the abnormal Golgi-ER retrograde trafficking. Indeed, the interferon pathway is activated by Golgi localization of the stimulator of interferon genes (STING) protein that shuttles between the ER and the Golgi.⁸ Since retrograde Golgi-ER transport is required for STING silencing, disruption of this trafficking by COPZ1 mutations is expected to retain active STING in the Golgi, leading to stimulation of the interferon signaling.

How COPZ1 mutations downregulate HIF-1α activity remains unknown. The authors speculated that this is a consequence of defective granulocyte colony-stimulating factor signaling linked to the abnormal trafficking of its receptor. Another intriguing possibility could be the Golgi’s autonomous effects on HIF-1α signaling. Though no previous studies reported regulation of HIF-1α by either COP1Z or COPI-dependent trafficking, a pool of Golgi-associated HIF-1α was observed in melanoma cells and chondrocytes.^9,10 Interestingly, disruption of the ER-Golgi trafficking by brefeldin inhibited HIF-1α-regulated transcription.⁹ It is tempting to speculate that the Golgi serves as a depot for HIF-1α, which can be delivered to the nucleus by the COPI-dependent retrograde transport. Inhibition of such transport by COPZ1 mutants could sequester HIF-1α in the Golgi, thereby inhibiting its transcriptional activity.

Lastly, neutrophil number alone is not an absolute predictor of recurrent life-threatening infections. A functional defect must also be present. Since neutrophil granules are specialized vesicles that must receive, sort, and package diverse types of proteins, one can hypothesize that defects in vesicle trafficking will lead to an innate immunodeficiency condition. Perhaps mutated forms of ELANE, the most common cause of severe congenital neutropenia, also cause traffic problems on the road to neutropenia.

Conflict-of-interest disclosure: The authors declare no competing financial interests.