Tan A. Nguyen

SIDT2 is a lysosomal membrane protein that—together with LAMP2—has been implicated in a recently described process known as RNAutophagy. Mutations in LAMP2 have long been known to cause Danon disease, a rare lysosomal storage disorder that affects multiple organ systems including brain and skeletal muscle. Recently, we have shown that loss of SIDT2 function in both humans and mice causes brain dysfunction, and it has been previously observed that loss of SIDT2 in mice also leads to muscle damage and reduced muscle strength. Here, we show that changes in the skeletal muscle of Sidt2^−/− mice are reminiscent of those seen in Danon disease as evidenced by accumulation of autophagic vacuoles with sarcolemmal features, selective atrophy of fast twitch fibers, and a shared alteration in gene expression. Moreover, we find that loss of SIDT2 not only leads to impaired autophagy in skeletal muscle as previously described but also stimulates a type I interferon response and increased apoptosis.

SIDT2 (Systemic Interference Deficient 1 Transmembrane Family Member 2) is a lysosomal membrane protein involved in RNA degradation via RNAutophagy. While animal models have indicated a link between SIDT2 deficiency and lysosomal storage disorders, no human cases have been reported. Here, we report a child with biallelic SIDT2 missense variants (p.Arg529Trp, p.Arg678Trp), who developed progressive neurological decline with cerebellar atrophy and Parkinsonian features. Functional studies revealed that the affected individual’s variants disrupted the ability of SIDT2 to interact with RNA. Fibroblasts from the affected individual showed impaired autophagy, characterised by abnormal accumulation of autophagy markers. In mouse models, Sidt2 was found to be highly expressed in the brain, particularly in the hippocampus and cerebellum. Sidt2 loss-of-function in mice resulted in not only impaired autophagy in the brain but also neurological dysfunction, including motor incoordination and eventual seizures. These findings suggest that SIDT2 deficiency contributes to both autophagic dysfunction and neurodegenerative processes, providing insight into a potential role in human neurological disease.

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.

SUMMARY: RNautophagy is a newly-described type of selective autophagy whereby cellular RNAs are transported into lysosomes for degradation. This process involves the transmembrane protein SIDT2, which transports double-stranded RNA (dsRNA) across the endolysosomal membrane. We previously demonstrated that SIDT2 is a transcriptional target of p53 , but its role in tumorigenesis – if any - is unclear. Unexpectedly, we show here that Sidt2 ^−/− mice with concurrent oncogenic Kras ^G12D activation develop significantly fewer tumors than littermate controls in a mouse model of lung adenocarcinoma (LUAD). Consistent with this observation, loss of SIDT2 also leads to enhanced survival and delayed tumor development in an Apc ^{min /+} mouse model of intestinal cancer. Within the intestine, Apc ^{min /+} ; Sidt2 ^−/− mice display accumulation of dsRNA in association with increased phosphorylation of eIF2α and JNK as well as elevated rates of apoptosis. Taken together, our data demonstrate a role for SIDT2 - and by extension RNautophagy - in promoting tumor development.

DsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that Sidt1 expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to Sidt2^−/− mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to Sidt2^−/− mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo. Key Points Murine SIDT1 colocalizes with late endolysosomes and interacts with long dsRNA [poly(I:C)] but not siRNA or dsDNA. SIDT1 expression is induced following type I IFN and poly(I:C) stimulation. Loss of SIDT1 impairs IFNβ production following infection with HSV-1 but does not affect overall survival.

Somatically acquired mutations in PHF6 (plant homeodomain finger 6) frequently occur in hematopoietic malignancies and often coincide with ectopic expression of TLX3. However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that Phf6 deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and β receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.

During infection, the cytosolic detection of viral double-stranded RNA (dsRNA) leads to the oligomerization and activation of mitochondrial antiviral signaling protein (MAVS) and the subsequent production of type I interferon (IFN). Here, we describe a novel method of visualizing and quantifying the aggregation of MAVS in response to dsRNA stimulation or viral infection in vitro using confocal microscopy.