Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor.

Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.

Reliability and head-loading effects in imaging diagnosis of vertical subluxation in patients with rheumatoid arthritis.

OBJECTIVES: The purpose of this study was to examine the reproducibility of vertical subluxation (VS) parameters using X-ray, computed tomography (CT), and tomosynthesis (TS) while comparing the head-loading effects. METHODS: The VS parameters of 26 patients (retrospective review) were evaluated. Using the intra-class correlation coefficient, we statistically examined the intra-rater and inter-rater reliabilities of the parameters. Head-loaded and -unloaded imagings were compared using a Wilcoxon signed-rank test. RESULTS: The intra-rater reliability of TS and CT showed intra-class correlation coefficients of ≥0.8 (X-ray range: 0.6-0.8), with similar results for the inter-rater reliabilities. Furthermore, in head-loading imaging, the TS had significantly higher VS scores than that of CT (P < .05). CONCLUSIONS: In comparison with the X-ray, TS and CT were more accurate and reproducible. In terms of head loading, the VS values for TS were worse than those for CT, indicating that TS was more effective than CT in diagnosing VS.

Sequential hydrolysis of FAD by ecto-5' nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B2 into cells.

Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. We determined that this two-step hydrolysis process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to the lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, we found that hydrolysis of these forms of vitamin B2 was impaired, and intracellular levels of vitamin B2 were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B2-dependent pyridoxal 5'-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.

Nucleic acid uptake occurs independent of lysosomal acidification but dependent on ATP consumption during RNautophagy/DNautophagy.

RNautophagy/DNautophagy (RDA) is an autophagic process that refers to the direct uptake of nucleic acids by lysosomes for degradation. Autophagy relies on lysosomes and lysosomal acidification is crucial for the degradation of intracellular components. However, whether lysosomal acidification interferes with nucleic acid uptake during RDA is unclear. In this study, we focused on vacuolar H+-ATPase (V-ATPase), the major proton pump responsible for maintaining an acidic pH in lysosomes. Our results show that lysosomes take up nucleic acids independently of the intralysosomal acidic pH during RDA. Isolated lysosomes treated with bafilomycin A1, a potent V-ATPase inhibitor, did not degrade, but took up RNA at similar levels as the control lysosomes. Similarly, the knockdown of Atp6v1a, the gene that encodes V-ATPase catalytic subunit A, did not affect the RNA uptake ability of isolated lysosomes. In addition, we demonstrated that nucleic acid uptake by isolated lysosomes necessitates ATP consumption, although V-ATPase is not required for the uptake process. These results broaden our understanding of the mechanisms underlying nucleic acid degradation via autophagy.

Nonresponder Considerations for Romosozumab Treatment.

Romosozumab can increase bone mineral density (BMD) in patients with osteoporosis, but some patients do not respond to it. This study aimed to identify risk factors for being a nonresponder to romosozumab treatment. This retrospective observational study included 92 patients. Romosozumab (210 mg) was subcutaneously administered to the participants every 4 weeks over 12 months. We excluded patients who previously underwent treatment for osteoporosis to assess the impact of romosozumab alone. We evaluated the proportion of patients who did not respond to romosozumab treatment to the lumbar spine and hip with increased BMD. Nonresponders were defined as those with a bone density change of < 3% after 12 months of treatment. We compared demographics and biochemical markers between responders and nonresponders. We found that 11.5% of patients were nonresponders at the lumbar spine, and 56.8% were nonresponders at the hip. A risk factor for nonresponse at the spine was low type I procollagen N-terminal propeptide (P1NP) values at 1 month. The cutoff value for P1NP at month 1 was 50 ng/ml. We found that 11.5% and 56.8% of patients experienced no significant improvement in the lumbar spine and hip BMD, respectively. Clinicians should use nonresponse risk factors to inform decisions about romosozumab treatment for patients with osteoporosis.

Synthesis and biological evaluation of coumarin derivatives as selective CYP2A6 inhibitors.

Cytochrome P450 2A6 (CYP2A6) inhibitors are expected to be suitable as smoking cessation aids and for cancer prevention. Because the typical coumarin-based CYP2A6 inhibitor methoxsalen also inhibits CYP3A4, unintended drug-drug interactions are still a concern. Therefore, the development of selective CYP2A6 inhibitors is desirable. In this study, we synthesized coumarin-based molecules, determined the IC50 values for CYP2A6 inhibition, verified the possibility of mechanism-based inhibition, and compared the selectivity for CYP2A6 versus CYP3A4. The results demonstrated that we developed CYP2A6 inhibitors that were more potent and selective than methoxsalen.

Surgical outcomes of intraoperative O-arm versus C-arm fluoroscopy in occipitocervical fixation: a retrospective analysis.

PURPOSE: This study aims to compare the effect of using O-arm and C-arm fluoroscopy on the surgical outcomes of occipitocervical fixation. METHODS: The study included patients who underwent occipitocervical fixation using O-arm or C-arm between 2005 and 2021. Of 56 patients, 34 underwent O-arm-assisted surgery (O-group) and 22 underwent C-arm-assisted surgery (C-group). We assessed surgical outcomes, including operative time, intraoperative blood loss, perioperative complications, and bone union. RESULTS: Almost half of the patients had rheumatoid arthritis-related disorders in both groups. Sixteen cases (47.1%) in the O-group and 12 cases (54.5%) in the C-group were fixed from occipito (Oc) to C3, 12 cases (38.2%) in the O-group and 7 cases (31.8%) in the C-group from Oc to C4-7, 5 cases (14.7%) in the O-group, and 3 cases (13.6%) in the C-group from Oc to T2 (p = 0.929). There was no significant difference in operative time (p = 0.239) and intraoperative blood loss (p = 0.595) between the two groups. Dysphagia was the most common complication in both groups (O-group vs. C-group, 11.7% vs. 9.1%). Regarding implant-related complications, occipital plate dislodgement was observed in four cases (18.2%) in the C-group (p = 0.02). The bone union rate was 96.3% in the O-group and 93.3% in the C-group (P = 1). CONCLUSIONS: O-arm use is associated with a reduced rate of occipital plate dislodgment and has a similar complication incidence compared with C-arm-assisted surgery and does not prolong operative time despite the time needed for setting and scanning. Accordingly, an O-arm is safe and useful for occipitocervical fixation surgery.

Arginine is a disease modifier for polyQ disease models that stabilizes polyQ protein conformation.

The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal ß-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. Here we identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. We first screened representative chemical chaperones using an in vitro polyQ aggregation assay, and identified arginine as a potent polyQ aggregation inhibitor. Our in vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic ß-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases.

Early life stress from allergic dermatitis causes depressive-like behaviors in adolescent male mice through neuroinflammatory priming.

Allergic dermatitis (AD), associated with pruritus and itchiness, is one of the major stressful conditions early in life. AD also influences the incidence of neuropsychiatric disorders and developmental disorders through neuro-immune interactions. To the best of our knowledge, there is no report that assesses the effects of early childhood dermatitis on psychiatric disorders later in life using an animal model. Here, we developed an oxazolone (Ox)-induced AD model in the early life of male C57BL/6J mice whose ears were challenged by Ox repeatedly from postnatal days (PD) 2 to PD30. On PD30, the Ox-treated ears were remarkably thickened and showed epidermal hyperplasia coupled with increased expression of T helper 2 cytokines, interleukin (IL)-4, and IL-13 in the ear tissue. Additionally, serum immunoglobulin E levels and serum corticosterone levels were higher in the Ox-treated mice than those in the control mice. Although Ox-treated PD40 mice showed neither behavioral abnormalities nor increases in pro-inflammatory cytokine expression in the brain, this study revealed that they experienced downregulation of CD200R1 expression in the amygdala under basal conditions and that additional lipopolysaccharide (LPS) administration induced enhanced neuroinflammatory reaction as the priming effect was accompanied by an increase of Iba-1-positive microglia in the amygdala and hippocampus. Furthermore, the Ox-treated PD40 mice showed depressive-like behaviors 24 h after LPS administration, whereas the control mice did not. Interestingly, the expression of indoleamine 2,3-dioxygenase and kynurenine 3-monooxygenase, key rate-limiting enzymes of the kynurenine metabolism pathway, was upregulated in the hippocampus, prefrontal cortex, and amygdala of the Ox-treated mice 4 h after LPS administration. Based on these results, we suggest that early life stress from AD aggravates susceptibility to systemic inflammation in the adolescent brain, leading to depressive behaviors with abnormal kynurenine metabolism.

Deficiency of AMPAR-Palmitoylation Aggravates Seizure Susceptibility.

Synaptic AMPAR expression controls the strength of excitatory synaptic transmission and plasticity. An excess of synaptic AMPARs leads to epilepsy in response to seizure-inducible stimulation. The appropriate regulation of AMPARs plays a crucial role in the maintenance of the excitatory/inhibitory synaptic balance; however, the detailed mechanisms underlying epilepsy remain unclear. Our previous studies have revealed that a key modification of AMPAR trafficking to and from postsynaptic membranes is the reversible, posttranslational S-palmitoylation at the C-termini of receptors. To clarify the role of palmitoylation-dependent regulation of AMPARs in vivo, we generated GluA1 palmitoylation-deficient (Cys811 to Ser substitution) knock-in mice. These mutant male mice showed elevated seizure susceptibility and seizure-induced neuronal activity without impairments in synaptic transmission, gross brain structure, or behavior at the basal level. Disruption of the palmitoylation site was accompanied by upregulated GluA1 phosphorylation at Ser831, but not at Ser845, in the hippocampus and increased GluA1 protein expression in the cortex. Furthermore, GluA1 palmitoylation suppressed excessive spine enlargement above a certain size after LTP. Our findings indicate that an abnormality in GluA1 palmitoylation can lead to hyperexcitability in the cerebrum, which negatively affects the maintenance of network stability, resulting in epileptic seizures.SIGNIFICANCE STATEMENT AMPARs predominantly mediate excitatory synaptic transmission. AMPARs are regulated in a posttranslational, palmitoylation-dependent manner in excitatory synapses of the mammalian brain. Reversible palmitoylation dynamically controls synaptic expression and intracellular trafficking of the receptors. Here, we generated GluA1 palmitoylation-deficient knock-in mice to clarify the role of AMPAR palmitoylation in vivo We showed that an abnormality in GluA1 palmitoylation led to hyperexcitability, resulting in epileptic seizure. This is the first identification of a specific palmitoylated protein critical for the seizure-suppressing process. Our data also provide insight into how predicted receptors such as AMPARs can effectively preserve network stability in the brain. Furthermore, these findings help to define novel key targets for developing anti-epileptic drugs.

Lysosomal targeting of SIDT2 via multiple YxxΦ motifs is required for SIDT2 function in the process of RNautophagy.

RNA degradation is an essential process for maintaining cellular homeostasis. Previously, we discovered a novel RNA degradation system, RNautophagy, during which direct import of RNA into lysosomes in an ATP-dependent manner followed by degradation takes place. The putative nucleic acid transporter SID-1 transmembrane family member 2 (SIDT2) predominantly localizes to lysosomes and mediates the translocation of RNA into lysosomes during RNautophagy. However, little is known about the mechanisms of sorting SIDT2 to lysosomes. Here, we show that three cytosolic YxxΦ motifs (in which x is any amino acid and Φ is an amino acid with a bulky hydrophobic side chain) are required for the lysosomal localization of SIDT2, and that SIDT2 interacts with adaptor protein complexes AP-1 and AP-2. We also find that localization to lysosomes by these three motifs is necessary for SIDT2 function in the process of RNautophagy, and that SIDT2 strikingly increases endogenous RNA degradation at the cellular level. To our knowledge, this is the first study to report an endogenous intracellular protein for which overexpression substantially increased intracellular RNA degradation. This study provides new insight into lysosomal targeting of proteins and intracellular RNA degradation, and further confirms the critical function of SIDT2 in RNautophagy.This article has an associated First Person interview with the first author of the paper.

Characterization of soy-deprestatin, a novel orally active decapeptide that exerts antidepressant-like effects via gut-brain communication.

We found that the orally administered thermolysin digest of ß-conglycinin exhibits antidepressant-like effects in tail suspension and forced swim tests in mice. A comprehensive peptide analysis of the digest using liquid chromatography/mass spectrometry was performed, and LSSTQAQQSY emerged as a candidate antidepressant-like peptide. Orally administered synthetic LSSTQAQQSY exhibited antidepressant-like effects at a dose of 0.3 mg/kg; therefore, we named the decapeptide soy-deprestatin. In contrast, intraperitoneally administered soy-deprestatin was ineffective. We then hypothesized that it acted on the gut, and its signal was transferred to the brain. Indeed, orally administered soy-deprestatin exhibited antidepressant-like activity in sham-treated, but not vagotomized, mice. Oral administration of soy-deprestatin also increased the c-Fos expression in the nucleus of the solitary tract, which receives inputs from the vagus nerve. These results suggested that the antidepressant-like effects were mediated by the vagus nerve. Thermolysin digest- and soy-deprestatin-induced antidepressant-like effects were also blocked by antagonists of serotonin 5-HT1A, dopamine D1, or GABAA receptors. We also clarified the order of receptor activation as 5-HT1A, D1, and GABAA, using selective agonists and antagonists. Taken together, soy-deprestatin may exhibit antidepressant-like effects after oral administration via a novel pathway mediated by 5-HT1A, followed by D1 and GABAA systems. This is the first orally active peptide demonstrating antidepressant-like effects via gut-brain communication.-Mori, Y., Asakura, S., Yamamoto, A., Odagiri, S., Yamada, D., Sekiguchi, M., Wada, K., Sato, M., Kurabayashi, A., Suzuki, H., Kanamoto, R., Ohinata, K. Characterization of soy-deprestatin, a novel orally active decapeptide that exerts antidepressant-like effects via gut-brain communication.

Sleep Disturbance as a Potential Modifiable Risk Factor for Alzheimer's Disease.

Sleep disturbance is a common symptom in patients with various neurodegenerative diseases, including Alzheimer's disease (AD), and it can manifest in the early stages of the disease. Impaired sleep in patients with AD has been attributed to AD pathology that affects brain regions regulating the sleep⁻wake or circadian rhythm. However, recent epidemiological and experimental studies have demonstrated an association between impaired sleep and an increased risk of AD. These studies have led to the idea of a bidirectional relationship between AD and impaired sleep; in addition to the conventional concept that impaired sleep is a consequence of AD pathology, various evidence strongly suggests that impaired sleep is a risk factor for the initiation and progression of AD. Despite this recent progress, much remains to be elucidated in order to establish the benefit of therapeutic interventions against impaired sleep to prevent or alleviate the disease course of AD. In this review, we provide an overview of previous studies that have linked AD and sleep. We then highlight the studies that have tested the causal relationship between impaired sleep and AD and will discuss the molecular and cellular mechanisms underlying this link. We also propose future works that will aid the development of a novel disease-modifying therapy and prevention of AD via targeting impaired sleep through non-pharmacological and pharmacological interventions.

Intercellular chaperone transmission via exosomes contributes to maintenance of protein homeostasis at the organismal level.

The heat shock response (HSR), a transcriptional response that up-regulates molecular chaperones upon heat shock, is necessary for cell survival in a stressful environment to maintain protein homeostasis (proteostasis). However, there is accumulating evidence that the HSR does not ubiquitously occur under stress conditions, but largely depends on the cell types. Despite such imbalanced HSR among different cells and tissues, molecular mechanisms by which multicellular organisms maintain their global proteostasis have remained poorly understood. Here, we report that proteostasis can be maintained by molecular chaperones not only in a cell-autonomous manner but also in a non-cell-autonomous manner. We found that elevated expression of molecular chaperones, such as Hsp40 and Hsp70, in a group of cells improves proteostasis in other groups of cells, both in cultured cells and in Drosophila expressing aggregation-prone polyglutamine proteins. We also found that Hsp40, as well as Hsp70 and Hsp90, is physiologically secreted from cells via exosomes, and that the J domain at the N terminus is responsible for its exosome-mediated secretion. Addition of Hsp40/Hsp70-containing exosomes to the culture medium of the polyglutamine-expressing cells results in efficient suppression of inclusion body formation, indicating that molecular chaperones non-cell autonomously improve the protein-folding environment via exosome-mediated transmission. Our study reveals that intercellular chaperone transmission mediated by exosomes is a novel molecular mechanism for non-cell-autonomous maintenance of organismal proteostasis that could functionally compensate for the imbalanced state of the HSR among different cells, and also provides a novel physiological role of exosomes that contributes to maintenance of organismal proteostasis.

Glucocerebrosidase deficiency accelerates the accumulation of proteinase K-resistant α-synuclein and aggravates neurodegeneration in a Drosophila model of Parkinson's disease.

Alpha-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recent multicenter genetic studies have revealed that mutations in the glucocerebrosidase 1 (GBA1) gene, which are responsible for Gaucher's disease, are strong risk factors for PD and DLB. However, the mechanistic link between the functional loss of glucocerebrosidase (GCase) and the toxicity of αSyn in vivo is not fully understood. In this study, we employed Drosophila models to examine the effect of GCase deficiency on the neurotoxicity of αSyn and its molecular mechanism. Behavioral and histological analyses showed that knockdown of the Drosophila homolog of GBA1 (dGBA1) exacerbates the locomotor dysfunction, loss of dopaminergic neurons and retinal degeneration of αSyn-expressing flies. This phenotypic aggravation was associated with the accumulation of proteinase K (PK)-resistant αSyn, rather than with changes in the total amount of αSyn, raising the possibility that glucosylceramide (GlcCer), a substrate of GCase, accelerates the misfolding of αSyn. Indeed, in vitro experiments revealed that GlcCer directly promotes the conversion of recombinant αSyn into the PK-resistant form, representing a toxic conformational change. Similar to dGBA1 knockdown, knockdown of the Drosophila homolog of ß-galactosidase (ß-Gal) also aggravated locomotor dysfunction of the αSyn flies, and its substrate GM1 ganglioside accelerated the formation of PK-resistant αSyn. Our findings suggest that the functional loss of GCase or ß-Gal promotes the toxic conversion of αSyn via aberrant interactions between αSyn and their substrate glycolipids, leading to the aggravation of αSyn-mediated neurodegeneration.

Review of Ginkgo biloba-induced toxicity, from experimental studies to human case reports.

Ginkgo biloba seeds and leaves have been used as a traditional herbal remedy for thousands of years, and its leaf extract has been consumed as a botanical dietary supplement for decades. Ginkgo biloba extract is a complex mixture with numerous components, including flavonol glycosides and terpene lactones, and is one of the most widely sold botanical dietary supplements worldwide. Concerns about potential health risks for the general population have been raised because of the widespread human exposure to Ginkgo biloba and its potential toxic and carcinogenic activities in rodents. The National Toxicology Program conducted 2-year gavage studies on one Ginkgo biloba leaf extract and concluded that there was clear evidence of carcinogenic activity of this extract in mice based on an increased incidence of hepatocellular carcinoma and hepatoblastoma. Recently, Ginkgo biloba leaf extract has been classified as a possible human carcinogen (Group 2B) by the International Agency for Research on Cancer. This review presents updated information on the toxicological effects from experimental studies both in vitro and in vivo to human case reports (caused by ginkgo seeds or leaves), and also summarizes the negative results from relatively large clinical trials.

Evaluation of synthesized coumarin derivatives on aromatase inhibitory activity.

In women across the world, the most common type of cancer is breast cancer. Among medical treatments, endocrine therapy based on aromatase inhibitors (AI) is expected to be effective against not only post-menopausal but also pre-menopausal breast cancer. In this study, we examined the structure-activity relationship between the aromatase inhibitory effects of 7-diethylaminocoumarin derivatives with a substituent at position 3 and coumarin derivatives with a substituent at position 7. Consequently, we found that 7-(pyridin-3-yl)coumarin (IC50 values 30.3nM) and 7,7'-diethylamino-3,3'-biscoumarin (28.7nM) are the most potent inhibitors of aromatase. These inhibitors were found to be comparable to the existing CYP19 inhibitor exemestane (42.5nM).

SIDT2 mediates gymnosis, the uptake of naked single-stranded oligonucleotides into living cells.

Single-stranded oligonucleotides (ssOligos) are efficiently taken up by living cells without the use of transfection reagents. This phenomenon called 'gymnosis' enables the sequence-specific silencing of target genes in various types of cells. Several antisense ssOligos are used for the treatment of human diseases. However, the molecular mechanism underlying the uptake of naked ssOligos into cells remains to be elucidated. Here, we show that systemic RNA interference deficient-1 (SID-1) transmembrane family 2 (SIDT2), a mammalian ortholog of the Caenorhabditis elegans double-stranded RNA channel SID-1, mediates gymnosis. We show that the uptake of naked ssOligos into cells is significantly downregulated by knockdown of SIDT2. Furthermore, knockdown of SIDT2 inhibited the effect of antisense RNA mediated by gymnosis. Overexpression of SIDT2 enhanced the uptake of naked ssOligos into cells, while a single amino acid mutation in SIDT2 abolished this effect. Our findings highlight the mechanism of extra- and intracellular RNA transport and may contribute to the further development of nucleic acid-based therapies.

RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates.

Lysosomes can degrade various biological macromolecules, including nucleic acids, proteins and lipids. Recently, we identified novel nucleic acid-degradation systems termed RNautophagy/DNautophagy (abbreviated as RDA), in which RNA and DNA are directly taken up by lysosomes in an ATP-dependent manner and degraded. We also found that a lysosomal membrane protein, LAMP2C, the cytoplasmic region of which binds to RNA and DNA, functions, at least in part, as an RNA/DNA receptor in the process of RDA. However, it has been unclear whether RDA possesses selectivity for RNA/DNA substrates and the RNA/DNA sequences that are recognized by LAMP2C have not been determined. In the present study, we found that the cytosolic region of LAMP2C binds to poly-G/dG, but not to poly-A/dA, poly-C/dC, poly-dT or poly-U. Consistent with this binding activity, poly-G/dG was transported into isolated lysosomes via RDA, while poly-A/dA, poly-C/dC, poly-dT and poly-U were not. GGGGGG or d(GGGG) sequences are essential for the interaction between poly-G/dG and LAMP2C. In addition to poly-G/dG, G/dG-rich sequences, such as a repeated GGGGCC sequence, interacted with the cytosolic region of LAMP2C. Our findings indicate that RDA does possess selectivity for RNA/DNA substrates and that at least some consecutive G/dG sequence(s) can mediate RDA.