Ubiquitin C-terminal hydrolase L1 regulates the morphology of neural progenitor cells and modulates their differentiation.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a component of the ubiquitin system, which has a fundamental role in regulating various biological activities. However, the functional role of the ubiquitin system in neurogenesis is not known. Here we show that UCH-L1 regulates the morphology of neural progenitor cells (NPCs) and mediates neurogenesis. UCH-L1 was expressed in cultured NPCs as well as in embryonic brain. Its expression pattern in the ventricular zone (VZ) changed between embryonic day (E) 14 and E16, which corresponds to the transition from neurogenesis to gliogenesis. At E14, UCH-L1 was highly expressed in the ventricular zone, where neurogenesis actively occurs; whereas its expression was prominent in the cortical plate at E16. UCH-L1 was very weakly detected in the VZ at E16, which corresponds to the start of gliogenesis. In cultured proliferating NPCs, UCH-L1 was co-expressed with nestin, a marker of undifferentiated cells. In differentiating cells, UCH-L1 was highly co-expressed with the early neuronal marker TuJ1. Furthermore, when UCH-L1 was induced in nestin-positive progenitor cells, the number and length of cellular processes of the progenitors decreased, suggesting that the progenitor cells were differentiating. In addition, NPCs derived from gad (UCH-L1-deficient) mice had longer processes compared with controls. The ability of UCH-L1 to regulate the morphology of nestin-positive progenitors was dependent on its binding affinity for ubiquitin but not on hydrolase activity; this result was also confirmed using gad-mouse-derived NPCs. These results suggest that UCH-L1 spatially mediates and enhances neurogenesis in the embryonic brain by regulating progenitor cell morphology.

Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis.

The experimentally induced cryptorchid mouse model is useful for elucidating the in vivo molecular mechanism of germ cell apoptosis. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. Here, we analyzed the function of two closely related members of the ubiquitin C-terminal hydrolase (UCH) family in testicular germ cell apoptosis experimentally induced by cryptorchidism. The two enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. The testes of gracile axonal dystrophy (gad) mice, which lack UCH-L1, were resistant to cryptorchid stress-related injury and had reduced ubiquitin levels. The level of both anti-apoptotic (Bcl-2 family and XIAP) and prosurvival (pCREB and BDNF) proteins was significantly higher in gad mice after cryptorchid stress. In contrast, Uchl3 knockout mice showed profound testicular atrophy and apoptotic germ cell loss after cryptorchid injury. Ubiquitin level was not significantly different between wild-type and Uchl3 knockout mice, whereas the levels of Nedd8 and the apoptotic proteins p53, Bax, and caspase3 were elevated in Uchl3 knockout mice. These results demonstrate that UCH-L1 and UCH-L3 function differentially to regulate the cellular levels of anti-apoptotic, prosurvival, and apoptotic proteins during testicular germ cell apoptosis.

Accumulation of beta- and gamma-synucleins in the ubiquitin carboxyl-terminal hydrolase L1-deficient gad mouse.

The synuclein family includes three isoforms, termed alpha, beta and gamma. alpha-Synuclein accumulates in various pathological lesions resulting from neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. However, neither beta- nor gamma-synuclein has been detected in Lewy bodies, and thus it is unclear whether these isoforms contribute to neurological pathology. In the present study, we used immunohistochemistry to demonstrate accelerated accumulation of beta- and gamma-synucleins in axonal spheroids in gracile axonal dystrophy (gad) mice, which do not express ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). gamma-Synuclein immunoreactivity in the spheroids appeared in the gracile nucleus at 3 weeks of age and was maintained until 32 weeks. beta-Synuclein immunoreactivity appeared in spheroids around 12 weeks of age. In contrast, alpha-synuclein immunoreactivity was barely detectable in spheroids. Immunoreactivity for synaptophysin and ubiquitin were either faint or undetectable in spheroids. Given that UCH-L1 deficiency results in axonal degeneration and spheroid formation, our findings suggest that beta- and gamma-synuclein participate in the pathogenesis of axonal swelling in gad mice.

Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.

The ubiquitin pathway functions in the process of protein turnover in eukaryotic cells. This pathway comprises the enzymes that ubiquitinate/deubiquitinate target proteins and the proteasome that degrades ubiquitin-conjugated proteins. Ubiquitin C-terminal hydrolases (UCHs) are thought to be essential for maintaining ubiquitination activity by releasing ubiquitin (Ub) from its substrates. Mammalian UCH-L1 and UCH-L3 are small proteins that share considerable homology at the amino acid level. Both of these UCHs are highly expressed in the testis/ ovary and neuronal cells. Our previous work demonstrated that UCH-L1-deficient gracile axonal dystrophy (gad) mice exhibit progressively decreasing spermatogonial stem cell proliferation, suggesting that UCH isozymes in the testis function during spermatogenesis. To analyze the expression patterns of UCH isozymes during spermatogenesis, we isolated nearly homogeneous populations of spermatogonia, spermatocytes, spermatids, and Sertoli cells from mouse testes. Western blot analysis detected UCH-L1 in spermatogonia and Sertoli cells, whereas UCH-L3 was detected in spermatocytes and spermatids. Moreover, reverse transcription-polymerase chain reaction analysis of UCH isozymes showed that UCH-L1 and UCH-L4 mRNAs are expressed in spermatogonia, whereas UCH-L3 and UCH-L5 mRNAs are expressed mainly in spermatocytes and spermatids. These results suggest that UCH-L1 and UCH-L3 have distinct functions during spermatogenesis, namely, that UCH-L1 may act during mitotic proliferation of spermatogonial stem cells whereas UCH-L3 may function in the meiotic differentiation of spermatocytes into spermatids.

Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo.

Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. In wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after light stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed approximately 70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo.

Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.

Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.