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.

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.

Property of lysosomal storage disease associated with midbrain pathology in the central nervous system of Lamp-2-deficient mice.

Lysosome-associated membrane protein-2 (LAMP-2) is the gene responsible for Danon disease, which is characterized by cardiomyopathy, autophagic vacuolar myopathy, and variable mental retardation. To elucidate the function of LAMP-2 in the central nervous system, we investigated the neuropathological changes in Lamp-2-deficient mice. Immunohistochemical observations revealed that Lamp-1 and cathepsin D-positive lysosomal structures increased in the large neurons of the mouse brain. Ubiquitin-immunoreactive aggregates and concanavalin A-positive materials were detected in these neurons. By means of ultrastructural studies, we found various-shaped accumulations, including lipofuscin, glycolipid-like materials, and membranous structures, in the neurons and glial cells of Lamp-2-deficient brains. In deficient mice, glycogen granules accumulated in hepatocyte lysosomes but were not observed in neurons. These pathological features indicate lysosomal storage disease; however, the findings are unlikely a consequence of deficiency of a single lysosomal enzyme. Although previous study results have shown a large amount of autophagic vacuoles in parenchymal cells of the visceral organs, these findings were rarely detected in the brain tissue except for some axons in the substantia nigra, in which abundant activated microglial cells with increased lipid peroxidation were observed. Thus, LAMP-2 in the central nervous system has a possible role in the degradation of the various macromolecules in lysosomes and an additional function concerning protection from oxidative stress, especially in the substantia nigra.

The Sox2 promoter-driven CD63-GFP transgenic rat model allows tracking of neural stem cell-derived extracellular vesicles.

Extracellular vesicles (EVs) can modulate microenvironments by transferring biomolecules, including RNAs and proteins derived from releasing cells, to target cells. To understand the molecular mechanisms maintaining the neural stem cell (NSC) niche through EVs, a new transgenic (Tg) rat strain that can release human CD63-GFP-expressing EVs from the NSCs was established. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/human CD63-GFP), and it was expressed in undifferentiated fetal brains. GFP signals were specifically observed in in vitro cultured NSCs obtained from embryonic brains of the Tg rats. We also demonstrated that embryonic NSC (eNSC)-derived EVs were labelled by human CD63-GFP. Furthermore, when we examined the transfer of EVs, eNSC-derived EVs were found to be incorporated into astrocytes and eNSCs, thus implying an EV-mediated communication between different cell types around NSCs. This new Sox2/human CD63-GFP Tg rat strain should provide resources to analyse the cell-to-cell communication via EVs in NSC microenvironments.

Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes.

Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

Age-associated neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3-4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids.

Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum-derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These results suggested that this Tg rat model should provide significant information for understanding the intercellular transfer and/or mother-child transfer of EVs in vivo.

Discovery of a novel type of autophagy targeting RNA.

Regulated degradation of cellular components by lysosomes is essential to maintain biological homeostasis. In mammals, three forms of autophagy, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), have been identified. Here, we showed a novel type of autophagy, in which RNA is taken up directly into lysosomes for degradation. This pathway, which we term "RNautophagy," is ATP-dependent, and unlike CMA, is independent of HSPA8/Hsc70. LAMP2C, a lysosomal membrane protein, serves as a receptor for this pathway. The cytosolic tail of LAMP2C specifically binds to almost all total RNA derived from mouse brain. The cytosolic sequence of LAMP2C and its affinity for RNA are evolutionarily conserved from nematodes to humans. Our findings shed light on the mechanisms underlying RNA homeostasis in higher eukaryotes.

Direct uptake and degradation of DNA by lysosomes.

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered "RNautophagy," an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term "DNautophagy," is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.

Photoreceptor cell apoptosis in the retinal degeneration of Uchl3-deficient mice.

UCH-L3 belongs to the ubiquitin C-terminal hydrolase family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. A murine Uchl3 deletion mutant displays retinal degeneration, muscular degeneration, and mild growth retardation. To elucidate the function of UCH-L3, we investigated histopathological changes and expression of apoptosis- and oxidative stress-related proteins during retinal degeneration. In the normal retina, UCH-L3 was enriched in the photoreceptor inner segment that contains abundant mitochondria. Although the retina of Uchl3-deficient mice showed no significant morphological abnormalities during retinal development, prominent retinal degeneration became manifested after 3 weeks of age associated with photoreceptor cell apoptosis. Ultrastructurally, a decreased area of mitochondrial cristae and vacuolar changes were observed in the degenerated inner segment. Increased immunoreactivities for manganese superoxide dismutase, cytochrome c oxidase I, and apoptosis-inducing factor in the inner segment indicated mitochondrial oxidative stress. Expression of cytochrome c, caspase-1, and cleaved caspase-3 did not differ between wild-type and mutant mice; however, immunoreactivity for endonuclease G was found in the photoreceptor nuclei in the mutant retina. Hence, loss of UCH-L3 leads to mitochondrial oxidative stress-related photoreceptor cell apoptosis in a caspase-independent manner. Thus, Uchl3-deficient mice represent a model for adult-onset retinal degeneration associated with mitochondrial impairment.

A new mouse model for infantile neuroaxonal dystrophy, inad mouse, maps to mouse chromosome 1.

Infantile neuroaxonal dystrophy (INAD) is a rare autosomal recessive hereditary neurodegenerative disease of humans. So far, no responsible gene has been cloned or mapped to any chromosome. For chromosome mapping and positional cloning of the responsible gene, establishment of an animal model would be useful. Here we describe a new mouse model for INAD, named inad mouse. In this mouse, the phenotype is inherited in an autosomal recessive manner, symptoms occur in the infantile period, and the mouse dies before sexual maturity. Axonal dystrophic change appearing as spheroid bodies in central and peripheral nervous system was observed. These features more closely resembled human INAD than did those of the gad mouse, the traditional mouse model for INAD. Linkage analysis linked the inad gene to mouse Chromosome 1, with the highest LOD score (=128.6) at the D1Mit45 marker, and haplotype study localized the inad gene to a 7.5-Mb region between D1Mit84 and D1Mit25. In this linkage area some 60 genes exist: Mutation of one of these 60 genes is likely responsible for the inad mouse phenotype. Our preliminary mutation analysis in 15 genes examining the nucleotide sequence of exons of these genes did not find any sequence difference between inad mouse and C57BL/6 mouse.