Mikako Sakurai

Facilitation of Extinction Learning for Contextual Fear Memory by PEPA: A Potentiator of AMPA Receptors

Contextual fear memory is attenuated by the re-exposure of mice to the context without aversive stimulus. This phenomenon is called extinction. Here, we report that a potentiator of AMPA receptors, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluorophenoxyacetamide (PEPA), potently facilitates extinction learning in mice. C57BL/6J mice were exposed to novel context and stimulated by electrical footshock. After 24 h (extinction training) and 72 h (extinction test), the mice were repeatedly exposed to the context without footshock and the duration of their freezing response was measured. The duration of freezing response in the extinction test was consistently shorter than the value in extinction training. Intraperitoneal injection of PEPA 15 min before extinction training remarkably reduced the duration of freezing responses during the extinction training and test, compared with the vehicle-injected control mice. This action of PEPA on extinction was dose-dependent and inhibited by NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide), an AMPA receptor antagonist. PEPA had no effect on acquisition and consolidation of fear memory itself. Electrophysiological studies suggested that PEPA activates the neural network much more potently in the medial prefrontal cortex (mPFC) than in the basolateral amygdala and hippocampal CA1 field. Quantitative PCR studies suggested the pronounced expression of PEPA-preferring AMPA receptor subunits (GluR3 and GluR4) and a splice variant (flop) in the mPFC. An intra-mPFC injection of PEPA facilitated the extinction much more potently than an intra-amygdala injection of PEPA did. These results suggest that PEPA facilitates extinction learning through AMPA receptor activation mainly in the mPFC.

Developmental Regulation of Ubiquitin C-Terminal Hydrolase Isozyme Expression During Spermatogenesis in Mice

Abstract 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.

Aberrant molecular properties shared by familial Parkinson’s disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1

Parkinsons disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD, and we have previously shown that the I93M UCH-L1-transgenic mice exhibit dopaminergic cell loss. Over 90% of neurodegenerative diseases, including PD, occur sporadically. However, the molecular mechanisms underlying sporadic PD as well as PD associated with I93M UCH-L1 are largely unknown. UCH-L1 is abundant (1-5% of total soluble protein) in the brain and is a major target of oxidative/carbonyl damage associated with sporadic PD. As well, abnormal microtubule dynamics and tubulin polymerization are associated with several neurodegenerative diseases including frontotemporal dementia and parkinsonism linked to chromosome 17. Here we show that familial PD-associated mutant UCH-L1 and carbonyl-modified UCH-L1 display shared aberrant properties: compared with wild-type UCH-L1, they exhibit increased insolubility and elevated interactions with multiple proteins, which are characteristics of several neurodegenerative diseases-linked mutants. Circular dichroism analyses suggest similar structural changes in both UCH-L1 variants. We further report that one of the proteins interacting with UCH-L1 is tubulin, and that aberrant interaction of mutant or carbonyl-modified UCH-L1 with tubulin modulates tubulin polymerization. These findings may underlie the toxic gain of function by mutant UCH-L1 in familial PD. Our results also suggest that the carbonyl modification of UCH-L1 and subsequent abnormal interactions of carbonyl-modified UCH-L1 with multiple proteins, including tubulin, constitute one of the causes of sporadic PD.

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.

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.

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.

Reduction in memory in passive avoidance learning, exploratory behaviour and synaptic plasticity in mice with a spontaneous deletion in the ubiquitin C-terminal hydrolase L1 gene

Overexpression of ubiquitin C‐terminal hydrolase L1 (UCH‐L1) in mice rescues amyloid β‐protein‐induced decreases in synaptic plasticity and memory. However, the physiological role of UCH‐L1 in the brain is not fully understood. In the present study, we investigated the role of UCH‐L1 in the brain by utilizing gracile axonal dystrophy (gad) mice with a spontaneous deletion in the gene Uch‐l1 as a loss‐of‐function model. Although gad mice exhibit motor paresis beginning at ∼ 12 weeks of age, it is possible to analyse their brain phenotypes at a younger age when no motor paresis is evident. Maintenance of memory in a passive avoidance test and exploratory behaviour in an open field test were reduced in 6‐week‐old gad mice. The maintenance of theta‐burst stimulation‐induced long‐term potentiation (LTP) of field synaptic responses from Schaffer collaterals to CA1 pyramidal cells in hippocampal slices was also impaired in gad mice. The LTP in gad mice was insensitive to actinomycin D, suggesting that a transcription‐dependent component of the LTP is impaired. Phosphorylation of cyclic AMP response element binding protein (CREB) in the CA1 region of hippocampal slices from gad mice occurred earlier than in the slices from wild‐type mice and was transient, suggesting that CREB phosphorylation is altered in gad mice. These results suggest that memory in passive avoidance learning, exploratory behaviour and hippocampal CA1 LTP are reduced in gad mice. We propose that UCH‐L1‐mediated maintenance of the temporal integrity and persistence of CREB phosphorylation underlies these impairments.

Clock genes regulate neurogenic transcription factors, including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells.

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.

Accumulation of β- and γ-synucleins in the ubiquitin carboxyl-terminal hydrolase L1-deficient gad mouse

Abstract The synuclein family includes three isoforms, termed α, β and γ. α-Synuclein accumulates in various pathological lesions resulting from neurodegenerative disorders including Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. However, neither β- nor γ-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 β- and γ-synucleins in axonal spheroids in gracile axonal dystrophy ( gad ) mice, which do not express ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). γ-Synuclein immunoreactivity in the spheroids appeared in the gracile nucleus at 3 weeks of age and was maintained until 32 weeks. β-Synuclein immunoreactivity appeared in spheroids around 12 weeks of age. In contrast, α-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 β- and γ-synuclein participate in the pathogenesis of axonal swelling in gad mice.

Ubiquitin dimers control the hydrolase activity of UCH-L3

Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.