Mature erythrocytes in mammals have no nuclei, although they differentiate from nucleated precursor cells. The mechanism by which enucleation occurs is not well understood. Here we show that deoxyribonuclease II (DNase II) is indispensable for definitive erythropoiesis in mouse fetal liver. No live DNase II-null mice were born, owing to severe anemia. When mutant fetal liver cells were transferred into lethally irradiated wild-type mice, mature red blood cells were generated from the mutant cells, suggesting that DNase II functions in a non-cell-autonomous manner. Histochemical analyses indicated that the critical cellular sources of DNase II are macrophages present at the site of definitive erythropoiesis in the fetal liver. Thus, DNase II in macrophages appears to be responsible for destroying the nuclear DNA expelled from erythroid precursor cells.

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/ p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders. Cell Death and Differentiation (2001) 8, 977 ± 984.

Cathepsin D-deficient (CDϪ/Ϫ) mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/ autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demonstrated in the neuronal perikarya of CDϪ/Ϫ mouse brains after P20. Autophagosomes and granular osmiophilic deposits were detected in neurons at P0 but were few in number, whereas they increased in the neuronal perikarya within days after birth. Some large-sized neurons having autophagosome/autolysosome-like bodies in the perikarya appeared in the CNS tissues, especially in the thalamic region and the cerebral cortex, at P17. These lysosomal bodies occupied the perikarya of almost all neurons in CDϪ/Ϫ mouse brains obtained from P23 until the terminal stage. Because these neurons exhibited autofluorescence, it was considered that ceroid lipofuscin may accumulate in lysosomal structures of CDϪ/Ϫ neurons. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brain. Moreover, neurons near the terminal stage were often shrunken and possessed irregular nuclei through which small dense chromatin masses were scattered. These results suggest that the CNS neurons in CDϪ/Ϫ mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis.

Apoptosis is often accompanied by the degradation of chromosomal DNA. Caspase-activated DNase (CAD) is an endonuclease that is activated in dying cells, whereas DNase II is present in the lysosomes of macrophages. Here, we show that CAD(-/-) thymocytes did not undergo apoptotic DNA degradation. But, when apoptotic cells were phagocytosed by macrophages, their DNA was degraded by DNase II. The thymus of DNase II(-/-)CAD(-/-) embryos contained many foci carrying undigested DNA and the cellularity was severely reduced due to a block in T cell development. The interferon-beta gene was strongly up-regulated in the thymus of DNase II(-/-)CAD(-/-) embryos, suggesting that when the DNA of apoptotic cells is left undigested, it can activate innate immunity leading to defects in thymic development.

Growth, differentiation, and programmed cell death (apoptosis) are mainly controlled by cytokines. The Janus kinase-signal transducers and activators of transcription (JAK-STAT) signal pathway is an important component of cytokine signaling. We have previously shown that STAT3 induces a molecule designated as SSI-1, which inhibits STAT3 functions. To clarify the physiological roles of SSI-1 in vivo, we generated, here, mice lacking SSI-1. These SSI-1؊͞؊ mice displayed growth retardation and died within 3 weeks after birth. Lymphocytes in the thymus and spleen of the SSI-1؊͞؊ mice exhibited accelerated apoptosis with aging, and their number was 20-25% of that in SSI-1؉͞؉ mice at 10 days of age. However, the differentiation of lymphocytes lacking SSI-1 appeared to be normal. Among various pro-and anti-apoptotic molecules examined, an up-regulation of Bax was found in lymphocytes of the spleen and thymus of SSI-1؊͞؊ mice. These findings suggest that SSI-1 prevents apoptosis by inhibiting the expression of Bax.The homeostatic regulation of cell populations is controlled by a balance among proliferation, growth arrest, and apoptosis, and this balance is mainly controlled by cytokines and growth factors. Cytokines act by binding to receptors expressed on the surfaces of responsive cells, which are associated with one or more members of the Janus kinase (JAK) family of cytoplasmic tyrosine kinases. The JAK-signal transducers and activators of transcription (STAT) signal pathway plays an important role in cytokine signaling (1-3), and is unique in that it features a direct linkage of receptor-ligand interaction on the cell surface to gene expression in the nucleus (4-6). However, the mechanism of negative control of cytokine actions involved in limiting their signal transductions is comparatively less well characterized. In 1997, the molecules that were expressed by stimulation of cytokine such as interleukin 6 (IL-6) and inhibited cytokine signal transmission by binding to JAK were isolated [STAT-induced STAT inhibitor-1 (SSI-1), suppressor of cytokine signaling (SOCS-1), Jak-binding protein (JAB)] (7-9). Subsequently, SSI-1 was found to form a family consisting of at least eight molecules, which were structurally characterized by an SH2 domain and a C-terminal conserved region (SC-motif͞SOCS-box͞CH-domain) (10-12), and it was recently known that SSI-1 inhibits not only IL-6 signaling but also interferon (IFN)-␥, IL-2, IL-3, and growth hormone signaling in vitro (13). It is expected that further study of SSI family molecules engaged in the negative feedback mechanism of cytokines will clarify the control mechanism of cytokines, which have remained obscure. MATERIALS AND METHODS Generation of SSI-1-Deficient Mice.A 129͞G mouse genomic library (Stratagene) containing the SSI-1 gene was screened, subcloned into the pBluescript vector, and characterized by restriction endonuclease mapping and DNA sequencing. A targeting vector was designed to replace the SSI-1 intron with phosphoglycerate kinase neo. This targeting...