Spontaneously Occurring Formation of Intranuclear and Cytoplasmic Inclusions in Renal Proximal Epithelium Due to Accumulation of D-Amino Acid Oxidase in Wistar Hannover Rats.

Intranuclear and cytoplasmic inclusions in the renal proximal tubular epithelium were observed in nontreated male and female Wistar Hannover rats in a 26-week study (32 weeks of age) and a 104-week study (110 weeks of age). The incidence rates were less than 5% in these two studies. In affected animals, the inclusions were observed in more than 60% of proximal tubular epithelium as various sized (approximately 1-8 µm in diameter) round and eosinophilic materials, but not in distal tubules, Henle's loop, or collecting ducts. Ultrastructurally, inclusions appeared finely granular, homogenous with middle-electron density, and without a limiting membrane. These inclusions were determined to be protein histochemically stained by Azan-Mallory and immunoreactive with an antibody against D-amino acid oxidase (DAO). There was no abnormality in in-life observations or in clinical test values suggestive of renal dysfunction. There were no associated degenerative or inflammatory changes in the kidneys, and no similar inclusions were observed in the other organs. These inclusions are very similar to propiverine hydrochloride (propiverine) and norepinephreine/serotonin reuptake inhibitor-induced inclusions. This is the first report of accumulation of DAO and formation of inclusions occurring spontaneously in rat kidneys. The data are important for toxicological studies using Wistar Hannover rats.

Calpain 5 is highly expressed in the central nervous system (CNS), carries dual nuclear localization signals, and is associated with nuclear promyelocytic leukemia protein bodies.

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF hand motif characteristic of classical calpains but retains catalytic and Ca(2+) binding domains, and it contains a unique C-terminal domain. TRA-3, an ortholog of CAPN5, has been shown to be involved in necrotic cell death in Caenorhabditis elegans. CAPN5 is expressed throughout the CNS, but its expression relative to other calpains and subcellular distribution has not been investigated previously. Based on relative mRNA levels, Capn5 is the second most highly expressed calpain in the rat CNS, with Capn2 mRNA being the most abundant. Unlike classical calpains, CAPN5 is a non-cytosolic protein localized to the nucleus and extra-nuclear locations. CAPN5 possesses two nuclear localization signals (NLS): an N-terminal monopartite NLS and a unique bipartite NLS closer to the C terminus. The C-terminal NLS contains a SUMO-interacting motif that contributes to nuclear localization, and mutation or deletion of both NLS renders CAPN5 exclusively cytosolic. Dual NLS motifs are common among transcription factors. Interestingly, CAPN5 is found in punctate domains associated with promyelocytic leukemia (PML) protein within the nucleus. PML nuclear bodies are implicated in transcriptional regulation, cell differentiation, cellular response to stress, viral defense, apoptosis, and cell senescence as well as protein sequestration, modification, and degradation. The roles of nuclear CAPN5 remain to be determined.

ALS-associated protein FIG4 is localized in Pick and Lewy bodies, and also neuronal nuclear inclusions, in polyglutamine and intranuclear inclusion body diseases.

FIG4 is a phosphatase that regulates intracellular vesicle trafficking along the endosomal-lysosomal pathway. Mutations of FIG4 lead to the development of Charcot-Marie-Tooth disease type 4J and amyotrophic lateral sclerosis (ALS). Moreover, ALS-associated proteins (transactivation response DNA protein 43 (TDP-43), fused in sarcoma (FUS), optineurin, ubiquilin-2, charged mutivesicular body protein 2b (CHMP2B) and valosin-containing protein) are involved in inclusion body formation in several neurodegenerative diseases. Using immunohistochemistry, we examined the brains and spinal cords of patients with various neurodegenerative diseases, including sporadic TDP-43 proteinopathy (ALS and frontotemporal lobar degeneration). TDP-43 proteinopathy demonstrated no FIG4 immunoreactivity in neuronal inclusions. However, FIG4 immunoreactivity was present in Pick bodies in Pick's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, neuronal nuclear inclusions in polyglutamine and intranuclear inclusion body diseases, and Marinesco and Hirano bodies in aged control subjects. These findings suggest that FIG4 is not incorporated in TDP-43 inclusions and that it may have a common role in the formation or degradation of neuronal cytoplasmic and nuclear inclusions in several neurodegenerative diseases.

RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53.

The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis.

The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS, we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from nine ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1 species was used. Misfolded SOD1 in the form of granular aggregates was regularly detected in the nuclei of ventral horn astrocytes, microglia, and oligodendrocytes in ALS patients carrying or lacking SOD1 mutations. There was negligible staining in neurodegenerative and non-neurological controls. Misfolded SOD1 appeared occasionally also in nuclei of motoneurons of ALS patients. The results suggest that misfolded SOD1 present in glial and motoneuron nuclei may generally be involved in ALS pathogenesis.

Ubiquitin-conjugating enzyme E2-25K increases aggregate formation and cell death in polyglutamine diseases.

Polyglutamine diseases are characterized by neuronal intranuclear inclusions of expanded polyglutamine proteins, which are also ubiquitinated, indicating impairment of the ubiquitin-proteasome system. E2-25K (Hip2), an ubiquitin-conjugating enzyme, interacts directly with huntingtin and may mediate ubiquitination of the neuronal intranuclear inclusions in Huntington disease. E2-25K could thus modulate aggregation and toxicity of expanded huntingtin. Here we show that E2-25K is involved in aggregate formation of expanded polyglutamine proteins and polyglutamine-induced cell death. Both a truncated mutant, lacking the catalytic tail domain, as well as a full antisense sequence, reduce aggregate formation. Strikingly, both E2-25K mutants also reduced polyglutamine-induced cell death. In postmortem brain material of both Huntington disease and SCA3, E2-25K staining of polyglutamine aggregates was observed in a subset of neurons bearing intranuclear neuronal inclusions. These results demonstrate that targeting by ubiquitination plays an important role in the pathology of polyglutamine diseases.

Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by genomic expansions of CTG or CCTG repeats. When transcribed, these mutations give rise to repeat expansion RNAs that form nuclear inclusions and compromise the function of myonuclei. Here, we have used in situ hybridization and immunofluorescence to compare DM1 and DM2 and search for proteins that associate with the RNA nuclear (ribonuclear) inclusions. Although muscle disease is generally more severe in DM1, the ribonuclear inclusions were 8- to 13-fold more intense in DM2, implying greater amounts of repeat expansion RNA. Expression of repeat expansion RNA in myoblasts has been implicated in the pathogenesis of congenital DM1. However, we found that repeat expansion RNA is also expressed in myoblasts in DM2, a disorder that has not been associated with a congenital phenotype. Of 10 putative CUG binding proteins tested for colocalization with mutant RNA, only proteins in the muscleblind family were recruited into ribonuclear inclusions. Previous studies have shown activation of the protein kinase, PKR, by expanded CUG repeats in vitro. However, breeding experiments utilizing PKR knockout mice indicate that this kinase is not required for disease pathogenesis in a transgenic mouse model of DM1. We conclude that ribonuclear inclusions are a key feature of the muscle pathology in DM and that sequestration of muscleblind proteins may have a direct role in the disease process.