Takashi Matsuwaki

Proteolytic processing of TAR DNA binding protein-43 by caspases produces C-terminal fragments with disease defining properties independent of progranulin

Neuronal and glial deposition of misfolded, proteolytically processed, polyubiquitinated and abnormally phosphorylated C‐terminal fragments (CTFs) of the TAR DNA binding protein‐43 (TDP‐43) is a pathological hallmark of frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD‐U) and certain cases of amyotrophic lateral sclerosis. We demonstrate that TDP‐43 can be proteolytically processed by caspases upon induction of apoptosis to a major 35 kDa and a minor 25 kDa CTF. These fragments are initially soluble, but over time they accumulate as insoluble and pathologically phosphorylated derivatives. However, proteolytic processing appears not to be absolutely required for the deposition of insoluble TDP‐43 species, since a caspase resistant mutant of TDP‐43 is also converted into insoluble species. Phosphorylation at S409/410 apparently occurs late during the conversion of soluble to insoluble TDP‐43, suggesting that phosphorylation is not a prerequisite for aggregation. Loss of function of the progranulin (PGRN) gene causes FTLD‐U with TDP‐43 positive inclusions and has been suggested to lead to caspase activation and subsequent TDP‐43 processing. However, siRNA‐mediated knockdown of PGRN in cell culture as well as a PGRN gene knockout in mice failed to cause the formation of the disease characterizing CTFs of TDP‐43. Our findings therefore suggest that caspase‐mediated processing generates CTFs of similar biochemical properties as those occurring in nuclear and cytoplasmic deposits of FTLD‐U patients independent of PGRN levels.

Alteration of behavioural phenotype in mice by targeted disruption of the progranulin gene.

Sexual differentiation of the brain in rodents is achieved by estrogens, which are converted from androgens in the brain, during the perinatal period. We have identified the progranulin (PGRN) gene as one of the sex steroid-inducible genes that may be involved in masculinization of the rat brain. In the present study, we generated a line of mice with targeted disruption of the PGRN gene, and investigated male sexual behaviour, aggression and anxiety. PGRN-deficient mice exhibited a decrease in ejaculation incidence, while the latency and frequency of both mount and intromission were unchanged. For the aggressive behaviour test, the resident-intruder paradigm was used, and PGRN-deficient mice exhibited enhanced aggressiveness. In wild-type mice, males exhibited lower levels of anxiety than females by the open field test, while male PGRN-deficient mice exhibited an elevated level of anxiety and sex difference in anxiety was not observed. In addition, mRNA expression of the serotonergic receptor 5-HT1A, which could be related to the inhibition of aggression and anxiety, was significantly reduced in the hippocampus of PGRN-deficient mice after aggressive encounters. On the other hand, deficiency of the PGRN gene did not affect serum testosterone concentrations. These results suggest that PGRN gene plays a role in establishing sexual dimorphic behaviours at least partially by modulating the brain serotonergic system.

Possible involvement of lysosomal dysfunction in pathological changes of the brain in aged progranulin-deficient mice.

IntroductionIt has been shown that progranulin (PGRN) deficiency causes age-related neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Previous studies also suggested that PGRN is involved in modulating lysosomal function. To elucidate the pathophysiological role of PGRN in the aged brain, in the present study, lysosomal function and pathological changes of the brain were investigated using 10- and 90-week-old wild-type and PGRN-deficient mice.ResultsWe showed that PGRN deficiency caused enhanced CD68 expression in activated microglia and astrogliosis in the cortex and thalamus, especially in the ventral posteromedial nucleus/ventral posterolateral nucleus (VPM/VPL), in the aged brain. Immunoreactivity for Lamp1 (lysosome marker) in the VPM/VPL and expression of lysosome-related genes, i.e. cathepsin D, V-type proton ATPase subunit d2, and transcription factor EB genes, were also increased by PGRN deficiency. Aggregates of p62, which is selectively degraded by the autophagy-lysosomal system, were observed in neuronal and glial cells in the VPM/VPL of aged PGRN-deficient mice. TAR DNA binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons were also observed in aged PGRN-deficient mice. PGRN deficiency caused enhanced expression of glial cell-derived cytotoxic factors such as macrophage expressed gene 1, cytochrome b-245 light chain, cytochrome b-245 heavy chain, complement C4, tumor necrosis factor-α and lipocalin 2. In addition, neuronal loss and lipofuscinosis in the VPM/VPL and disrupted myelination in the cerebral cortex were observed in aged PGRN-deficient mice.ConclusionsThe present study shows that aged PGRN-deficient mice present with NCL-like pathology as well as TDP-43 aggregates in the VPM/VPL, where a particular vulnerability has been reported in NCL model mice. The present results also suggest that these pathological changes in the VPM/VPL are likely a result of lysosomal dysfunction. How PGRN prevents lysosomal dysfunction with aging remains to be elucidated.

Increased lysosomal biogenesis in activated microglia and exacerbated neuronal damage after traumatic brain injury in progranulin-deficient mice.

Progranulin (PGRN) is known to play a role in the pathogenesis of neurodegenerative diseases. Recently, it has been demonstrated that patients with the homozygous mutation in the GRN gene present with neuronal ceroid lipofuscinosis, and there is growing evidence that PGRN is related to lysosomal function. In the present study, we investigated the possible role of PGRN in the lysosomes of activated microglia in the cerebral cortex after traumatic brain injury (TBI). We showed that the mouse GRN gene has two possible coordinated lysosomal expression and regulation (CLEAR) sequences that bind to transcription factor EB (TFEB), a master regulator of lysosomal genes. PGRN was colocalized with Lamp1, a lysosomal marker, and Lamp1-positive areas in GRN-deficient (KO) mice were significantly expanded compared with wild-type (WT) mice after TBI. Expression of all the lysosome-related genes examined in KO mice was significantly higher than that in WT mice. The number of activated microglia with TFEB localized to the nucleus was also significantly increased in KO as compared with WT mice. Since the TFEB translocation is regulated by the mammalian target of rapamycin complex 1 (mTORC1) activity in the lysosome, we compared ribosomal S6 kinase 1 (S6K1) phosphorylation that reflects mTORC1 activity. S6K1 phosphorylation in KO mice was significantly lower than that in WT mice. In addition, the number of nissl-positive and fluoro-jade B-positive cells around the injury was significantly decreased and increased, respectively, in KO as compared with WT mice. These results suggest that PGRN localized in the lysosome is involved in the activation of mTORC1, and its deficiency leads to increased TFEB nuclear translocation with a resultant increase in lysosomal biogenesis in activated microglia and exacerbated neuronal damage in the cerebral cortex after TBI.

Exacerbated inflammatory responses related to activated microglia after traumatic brain injury in progranulin-deficient mice.

Progranulin (PGRN), a multifunctional growth factor, appears to play a role in neurodegenerative diseases accompanied by neuroinflammation. In this study, we investigated the role of PGRN in neuroinflammation, especially in the activation of microglia, by means of experimental traumatic brain injury (TBI) in the cerebral cortex of mice. The expression of GRN mRNA was increased in association with neuroinflammation after TBI. Double-immunohistochemical study showed that PGRN-immunoreactive (-IR) cells were mainly overlapped with CD68-IR cells, suggesting that the main source of PGRN was CD68-positive activated microglia. To investigate the role of PGRN in inflammatory responses related to activated microglia, we compared the immunoreactivity and expression of ionized calcium-binding adaptor molecule 1 (Iba1), CD68, and CD11b as markers for activated microglia between wild-type (WT) and GRN-deficient (KO) mice. The number of Iba1- and CD11b-IR cells and gene expression of Iba1 and CD11b were not significantly different between WT and KO mice, while the number of CD68-IR cells and CD68 expression in KO mice were significantly greater than those in WT mice. Double-immunohistochemical study showed that CD68-IR microglia were also IR for TGFβ1, and TGFβ1 expression and Smad3 phosphorylation in KO mice were elevated compared to WT mice. Moreover, double-immunostaining between phospho-Smad3 and glial fibrillary acidic protein suggested increased TGFβ1-Smad3 signal mainly by astrocytes. The levels of protein carbonyl groups, which reflect protein oxidation, and laminin immunoreactivity, which is associated with angiogenesis, were also significantly increased in KO mice compared to WT mice. These results suggest that PGRN is produced in CD68-positive microglia and suppresses excessive inflammatory responses related to activated microglia after TBI in mice.

Deletion of progranulin exacerbates atherosclerosis in ApoE knockout mice

AIMS Progranulin (PGRN) is a multifunctional protein known to be involved in inflammation. However, the relation between PGRN and atherosclerosis remains elusive. The aim of this study was to define the role of PGRN in the development of atherosclerosis. METHODS AND RESULTS First, we checked the expression levels of PGRN in human atherosclerotic plaques. Immunohistochemical analysis showed that PGRN is strongly expressed in foam cells of atherosclerotic plaques. We also found that PGRN is expressed more abundantly in macrophages than in the smooth muscle cells of atherosclerotic lesions in ApoE(-/-) mice fed a high-fat diet for 12 weeks. Next, PGRN(-/-)ApoE(-/-) mice were generated to investigate the effect of PGRN on the development of atherosclerosis. PGRN(-/-)ApoE(-/-) mice exhibited severe atherosclerotic lesions compared with PGRN(+/+)ApoE(-/-) mice, despite their anti-atherogenic lipid profile. These results are partly due to enhanced expression of inflammatory cytokines, adhesion molecules, and decreased expression of endothelial nitric oxide synthase. In addition, lack of PGRN leads to accumulate excessive cholesterol in the macrophages and alter HDL-associated proteins. CONCLUSION PGRN seems to be involved in the pathogenesis of atherosclerosis, possibly by various anti-atherogenic effects, including modulation of local and/or systemic inflammation.

Progranulin enhances neural progenitor cell proliferation through glycogen synthase kinase 3β phosphorylation.

Progranulin (PGRN) is an estrogen-inducible growth factor thought to affect multiple processes in the CNS, including brain sexual differentiation, adult neurogenesis in the hippocampus, and development of neurodegenerative diseases. However, the precise physiological functions of PGRN in individual nerve cells are not fully understood. The aim of the present study was to enhance the understanding of PGRN function in the CNS by investigating the effects of PGRN on neural progenitor cells (NPCs). We found that significant amounts of endogenous PGRN were secreted from isolated NPCs in cultures. To assess the bioactivities of endogenous and exogenous PGRN, we studied NPCs derived from wild-type mice (WT-NPCs) and PGRN-deficient mice (KO-NPCs). We found that proliferation of KO-NPCs was significantly enhanced by PGRN treatment; however, PGRN treatment apparently did not affect proliferation of WT-NPCs perhaps because of the high levels of endogenous PGRN expression. NPC death and asymmetric cellular division of KO-NPCs and WT-NPCs, which results in production of neural stem cells, astrocytes, or oligodendrocytes, were not affected by PGRN treatment. We also investigated the signaling mechanism(s) that mediate PGRN-induced NPC proliferation and found that phosphorylation of serine 9 (S9) of glycogen synthase kinase 3-beta (GSK3β), which was dependent on phosphatidylinositol 3-kinase (PI3K) activity, was induced by PGRN treatment. In addition, a GSK3β-specific inhibitor enhanced NPC proliferation. Taken together, our observations indicate that PGRN enhanced NPC proliferation, at least in part, via inducing GSK3β phosphorylation.

Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes

Abstract Progranulin (PGRN) haploinsufficiency resulting from loss‐of‐function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP‐43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down‐regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation‐independent mannose 6‐phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat. Moreover, the change of PGRN levels led to a cell‐type‐specific increase of insoluble TDP‐43. In the brain tissue of FTLD‐TDP patients with PGRN deficiency, CTSD and phosphorylated TDP‐43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases.

Cyclooxygenase-2-related signaling in the hypothalamus plays differential roles in response to various acute stresses

We previously suggested that cyclooxygenase (COX)-2 plays a role as a common mediator of stresses in the brain. In the present study, we evaluated the possible involvement of COX-2-related signaling in the activation of the hypothalamic-pituitary-adrenal (HPA) axis under three different stress conditions, namely infectious (lipopolysaccharide, LPS), hypoglycemic (2-deoxy-d-glucose, 2DG) and restraint (1h) stresses in rats. Both an unselective COX inhibitor (indomethacin) and a selective COX-2 inhibitor (NS-398) significantly attenuated the increase of serum corticosterone levels after LPS and restraint stresses, but not after 2DG injection. COX-2 and microsomal prostaglandin E synthase (mPGES)-1 mRNA levels in the hypothalamus were significantly increased after LPS injection in intact rats. In adrenalectomized (ADX) rats, the expression of both genes was significantly increased after 2DG and restraint stresses, which was blocked by treatment with corticosterone. Interleukin-1β (IL-1β) mRNA levels in the hypothalamus in intact rats were increased only by LPS injection, though those in ADX rats were increased by all three stress stimuli. These results suggest that the relationship between COX-2-related signaling and activation of the HPA axis is stress-specific, and that COX-2-related signaling preferably mediates infectious and restraint stresses. Furthermore, the expression of COX-2 and mPGES-1 mRNA under the infectious stress condition was not negatively regulated by endogenous glucocorticoids, likely due to an increase in IL-1β levels.

Discoidin domain receptor 2 (DDR2) is required for maintenance of spermatogenesis in male mice.

Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK). We recently identified homozygous smallie mutant mice (BKS.HRS. Ddr2slie/slie/J, Ddr2slie/slie mutants), which lack a functional DDR2. Ddr2slie/slie mutant mice are dwarfed and infertile due to peripheral dysregulation of the endocrine system. To understand the role of DDR2 signaling in spermatogenesis, we studied the expression of several receptors, enzymes, and proteins related to spermatogenesis in wild‐type and Ddr2slie/slie mutant mice at 10 weeks and 5 months of age. DDR2 were expressed in adult wild‐type male mice in Leydig cells. The number of differentiated spermatozoa in the seminal fluid was significantly lower in the Ddr2slie/slie mutant mice than in the wild‐type mice. The number of TUNEL‐positive cells was significantly greater in 5‐month‐old Ddr2slie/slie mutants. Testosterone was significantly reduced at 5 months of age, but LH was similar in both types of mice at both 10 weeks and 5 months of age. The expression levels of LH receptors (Lhcgr), StAR, P450scc, and Hsd3β6 were not significantly different between the two types of mice at 10 weeks of age, but they were significantly reduced in 5‐month‐old Ddr2slie/slie mutants compared to wild‐type mice of the same age. DDR2 was expressed in the Leydig cells of adult wild‐type male mice. In conclusion, our results indicated that DDR2 signaling plays a critical role in the maintenance of male spermatogenesis. Mol. Reprod. Dev. 77: 29–37, 2010.