Yoshiyuki Ohsawa

Cathepsin D Deficiency Induces Lysosomal Storage with Ceroid Lipofuscin in Mouse CNS Neurons

Cathepsin D-deficient (CD−/−) 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 CD−/− 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 CD−/− 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 CD−/− 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 CD−/− mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis.

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration.

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

Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation

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-β 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.

Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases.

PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.

Apoptotic nuclear morphological change without DNA fragmentation

Apoptosis is characterized morphologically by condensation and fragmentation of nuclei and cells and biochemically by fragmentation of chromosomal DNA into nucleosomal units [1]. CAD, also known as CPAN or DFF-40, is a DNase that can be activated by caspases [2] [3] [4] [5] [6]. CAD is complexed with its inhibitor, ICAD, in growing, non-apoptotic cells [2] [7]. Caspases that are activated by apoptotic stimuli [8] cleave ICAD. CAD, thus released from ICAD, digests chromosomal DNA into nucleosomal units [2] [3]. Here, we examine whether nuclear morphological changes induced by apoptotic stimuli are caused by the degradation of chromosomal DNA. Human T-cell lymphoma Jurkat cells, as well as their transformants expressing caspase-resistant ICAD, were treated with staurosporine. The chromosomal DNA in Jurkat cells underwent fragmentation into nucleosomal units, which was preceded by large-scale chromatin fragmentation (50-200 kb). The chromosomal DNA in cells expressing caspase-resistant ICAD remained intact after treatment with staurosporine but their chromatin condensed as found in parental Jurkat cells. These results indicate that large-scale chromatin fragmentation and nucleosomal DNA fragmentation are caused by an ICAD-inhibitable DNase, most probably CAD, whereas chromatin condensation during apoptosis is controlled, at least in part, independently from the degradation of chromosomal DNA.

Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens

The eye lens is composed of fibre cells, which develop from the epithelial cells on the anterior surface of the lens. Differentiation into a lens fibre cell is accompanied by changes in cell shape, the expression of crystallins and the degradation of cellular organelles. The loss of organelles is believed to ensure the transparency of the lens, but the molecular mechanism behind this process is not known. Here we show that DLAD (‘DNase II-like acid DNase’, also called DNase IIβ) is expressed in human and murine lens cells, and that mice deficient in the DLAD gene are incapable of degrading DNA during lens cell differentiation—the undigested DNA accumulates in the fibre cells. The DLAD-/- mice develop cataracts of the nucleus lentis, and their response to light on electroretinograms is severely reduced. These results indicate that DLAD is responsible for the degradation of nuclear DNA during lens cell differentiation, and that if DNA is left undigested in the lens, it causes cataracts of the nucleus lentis, blocking the light path.

Involvement of two different cell death pathways in retinal atrophy of cathepsin D-deficient mice

To understand the mechanisms of retinal atrophy in cathepsin D-deficient mice, the postnatal development of their retinae was analyzed. TUNEL-positive cells appeared abundantly in the outer nuclear layer (ONL) and slightly in the inner nuclear layer (INL). Nitric oxide synthase (NOS) was induced in microglial cells which invaded retinal layers and phagocytosed dead cell debris, while NOS inhibitors prevented cell death in the INL but not in the ONL. Caspases 9 and 3 were activated only in the ONL after P15. Moreover, no atrophic change was detected in the retina of mice deficient in cathepsin B or L. These results suggest that cathepsin D is essential for the metabolic maintenance of retinal photoreceptor cells and that its deficiency induces apoptosis of the cells, while the loss of INL neurons is mediated by NO from microglial cells.

Lysosomal cysteine and aspartic proteinases, acid phosphatase, and an endogenous cysteine proteinase inhibitor, cystatin-beta, in rat osteoclasts.

To understand the bone resorption and lysosomal proteinases in osteoclasts, we examined by immunohistochemistry the localization of lysosomal cysteine and aspartic proteinases, acid phosphatase, and cystatin-beta in the rat tibial bone. Immunoreactivity for cathepsins B, C, H, and L, cathepsin D, acid phosphatase, and cystatin-beta was demonstrated in various cells of the bone tissue; in particular, large multinucleated osteoclasts attached to the bone surface and chondroclasts in the proximal growth plate. These cells showed intense immunoreactivity for these lysosomal enzymes and cystatin-beta. Bone surface-lining osteoblasts displayed distinct immunoreactivity for cathepsins B, C, D, H, and acid phosphatase, while osteocytes often exhibited that for cathepsins D, H and acid phosphatase. Chondrocytes in the growth plate demonstrated intense immunoreactivity for cathepsins B, D, and acid phosphatase. Immunoreactivity for cystatin-beta was detected in osteoclasts and chondroclasts only. Large, round multinucleated cells free from the bone surface exhibited weak, faint, or no immunoreactivity for the lysosomal enzymes and cystatin-beta. These results suggest that lysosomal cysteine and aspartic proteinases may play a role in the degradation of organic constituents of the bone matrix. Moreover, cystatin-beta can serve as an excellent marker protein for osteoclasts.

Characterization of Cln3p, the gene product responsible for juvenile neuronal ceroid lipofuscinosis, as a lysosomal integral membrane glycoprotein

Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited lysosomal storage disease involving a mutation in the CLN3 gene. The sequence of CLN3 was determined in 1995; however, the localization of the CLN3 gene product (Cln3p) was not confirmed. In this study, we investigated endogenous Cln3p using two peptide antibodies raised against two distinct epitopes of murine Cln3p. Identification of the liver 60 kDa protein as Cln3p was ascertained by amino acid sequence analysis using tandem mass spectrometry. Liver Cln3p was predominantly localized in the lysosomal membranes, not in endoplasmic reticulum (ER) or Golgi apparatus. As the tissue concentration of brain Cln3p was much lower than that of liver Cln3p, it could be detected only after purification from brain extract using anti‐Cln3p IgG Sepharose. The apparent molecular masses of liver Cln3p and brain Cln3p were determined to be about 60 kDa and 55 kDa, respectively. Both brain and liver Cln3p were deglycosylated by PNGase F treatment to form polypeptides with almost the same molecular mass (45 kDa). However, they were not affected by Endo h treatment. In addition, it was also elucidated that the amino terminal region of Cln3p faces the cytosol.

Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells.

Bcl-2, an anti-apoptotic protein, is believed to be localized in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope. However, Bcl-2 has also been suggested as playing a role in the maintenance of mitochondrial membrane potential, indicating its possible association with the inner mitochondrial membrane. We therefore further examined the exact localization of Bcl-2 in mitochondria purified from wild-type and bcl-2-transfected PC12 cells and pre- and postnatal rat brains. Double immunostaining demonstrated that Bcl-2 was co-localized with subunit β of F1F0ATPase in the inner mitochondrial membrane. Biochemical analysis of isolated mitochondria using digitonin and trypsin suggests an association of Bcl-2 with the inner mitochondrial membrane. More interestingly, the majority of Bcl-2 disappeared from the inner membrane of mitochondria when cultured under serum deprivation. These results suggest that Bcl-2 acts as an anti-apoptotic regulator by localizing mainly to the inner mitochondrial and smooth ER membranes. Cell Death and Differentiation (2000) 7, 666–674