sign in sign up
<<
Home , Protein biosynthesis, Ubiquitin C

C-terminal hydrolase L1 (UCH-L1) loss causes neurodegeneration by altering turnover in the first postnatal weeks

Anna T. Reinickea,1, Karoline Labana,1, Marlies Sachsa, Vanessa Krausb, Michael Waldena, Markus Dammec, Wiebke Sachsa, Julia Reichelta, Michaela Schweizerd, Philipp Christoph Janiesche, Kent E. Duncane, Paul Saftigc, Markus M. Rinschenf,g, Fabio Morellinib,1, and Catherine Meyer-Schwesingera,1,2

aInstitute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; bBehavioral Biology, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; cInstitute of , Christian Albrechts University Kiel, 24118 Kiel, Germany; dCenter for Molecular Neurobiology Hamburg, Morphology and Electron Microscopy, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; eNeuronal Translational Control Research Group, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; fDepartment II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany; and gCenter for Mass Spectrometry and Metabolomics, The Scripps Research Institute, La Jolla, CA 92037

Edited by Eric Klann, New York University, New York, NY, and accepted by Editorial Board Member Nancy Y. Ip March 5, 2019 (received for review July 23, 2018) Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abun- mUB is reduced in spontaneous UCH-L1 mutant mice (11, 12), dant and enigmatic of the CNS. Based on existing UCH- suggesting that UCH-L1 regulates ubiquitin by main- L1 knockout models, UCH-L1 is thought to be required for the taining a pool of ready-to-use ubiquitin for various cellular events. maintenance of axonal integrity, but not for neuronal development Genetic deletion of UCH-L1 results in gracile axonal dystro- despite its high expression in . Several lines of evidence phy (gad) and premature death with early sensory ataxia fol- suggest a role for UCH-L1 in mUB homeostasis, although the specific lowed by motor ataxia (11–13), revealing a crucial role of UCH- in vivo substrate remains elusive. Since the precise mechanisms L1 in neuronal health. In humans, five pathogenic of underlying UCH-L1–deficient neurodegeneration remain unclear, UCH-L1 have been discovered, three of which affect the hy-

we generated a transgenic mouse model of UCH-L1 deficiency. By drolysis function of the and which are associated with MEDICAL SCIENCES performing biochemical and behavioral analyses we can show that the pathogenesis of neurodegenerative diseases (14). A missense UCH-L1 deficiency causes an acceleration of sensorimotor reflex de- in the UCH-L1 leads to an I93M substitution at velopment in the first postnatal week followed by a degeneration the protein level, which is strongly associated with the develop- of motor function starting at periadolescence in the setting of nor- ment of Parkinson disease (PD) (15). A homozygous missense mal cerebral mUB levels. In the first postnatal weeks, neuronal pro- mutation within the ubiquitin of UCH-L1 (E7A) tein synthesis and proteasomal protein degradation are enhanced, results in early onset progressive neurodegeneration in humans with stress, and energy depletion, leading to similarly to the effects observed in mice with genetic UCH- proteasomal impairment and an accumulation of nondegraded L1 deletion (16). is an important hallmark ubiquitinated protein. Increased protein turnover is associated with of many neurodegenerative diseases and can arise directly as a enhanced mTORC1 activity restricted to the postnatal period in result of dysregulated protein synthesis (17). Indeed, UCH-L1 UCH-L1–deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1–deficient Significance neurons. Strikingly, rapamycin treatment in the first 8 postnatal – days ameliorates the neurological phenotype of UCH-L1 deficient Ubiquitin C-terminal hydrolase L1 (UCH-L1) is important for neu- mice up to 16 weeks, suggesting that early control of protein ho- ronal development. Mechanistically, UCH-L1 has an inhibitory meostasis is imperative for long-term neuronal survival. In sum- effect on mTOR-containing complexes thereby controlling protein mary, we identified a critical presymptomatic period during which synthesis and degradation. Neurodegeneration arising from UCH- – UCH-L1 dependent enhanced protein synthesis results in neuronal L1 deficiency is the result of an early increased protein turnover strain and progressive loss of neuronal function. (synthesis and decay), which strains neurons by increasing energy requirement and endoplasmic reticulum stress. Rapamycin treat- UCH-L1 | neurodegeneration | development | protein synthesis | mTOR ment during a critical developmental period partially rescues motor degeneration in adulthood. biquitin C-terminal hydrolase L1 (UCH-L1) is a small 27- UkDa protein belonging to the UCH family of deubiquiti- Author contributions: F.M. and C.M.-S. designed research; A.T.R., K.L., M. Sachs, V.K., nating enzymes (DUBs) (1) predominantly expressed in neurons M.W., M.D., W.S., J.R., M. Schweizer, P.C.J., K.E.D., M.M.R., F.M., and C.M.-S. performed research; P.S., M.M.R., F.M., and C.M.-S. analyzed data; and A.T.R., M.M.R., F.M., and where it can comprise up to 5% of total brain protein (2, 3), but C.M.-S. wrote the paper. is also in other types under specialized conditions, e.g., in- The authors declare no conflict of interest. jured kidney (4) and cancerous cells (5). At a cellular ThisarticleisaPNASDirectSubmission.E.K.isaguesteditorinvitedbythe level, UCH-L1 exhibits strong cytoplasmic staining in neurons Editorial Board. – throughout the brain. A total of 20 50% of UCH-L1 can be Published under the PNAS license. membrane associated, which is thought to occur indirectly via Data deposition: The data reported in this paper have been deposited with the macromolecular complexes in neurons (6). Specific in vivo sub- ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier strates of UCH-L1 have not been identified yet (6). UCH-L1 has PXD010449). high affinity for ubiquitin, which it effectively proteolyzes from 1A.T.R., K.L., F.M., and C.M.-S. contributed equally to this work. small C-terminal amino acids as well as by cleaving diubiquitins 2To whom correspondence should be addressed. Email: [email protected] in vitro (7). However, compared with UCH-L3, it is a poor hy- hamburg.de. drolase of ubiquitinated , due to structurally restricted This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. access to its (8, 9). UCH-L1 binds monoubiquitin (mUB) 1073/pnas.1812413116/-/DCSupplemental. in neurons, thereby elongating ubiquitin half- (10). Indeed, Published online March 28, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1812413116 PNAS | April 16, 2019 | vol. 116 | no. 16 | 7963–7972 Downloaded by guest on October 1, 2021 + has been shown to regulate the levels of proteins involved in the nm3419 mice (11, 12). Although Uch-l1 /d and Uch-l1d/d mice formation of Alzheimer disease (AD) or PD aggregates (18–20). exhibited sensorimotor degeneration in adulthood, sensorimotor In this study, we explored the effects of UCH-L1 deficiency on reflexes at postnatal days 5 and 8 exhibited an unrecognized, par- + + protein synthesis and decay in the brain at different develop- adoxical accelerated execution in comparison with Uch-l1 / lit- mental stages to identify the potential mechanisms underlying termates (Fig. 1 E and F and SI Appendix,Fig.S4). The first signs of neurodegeneration in UCH-L1–deficient mice. By means of motor impairment were detected at 5 wk in the accelerated rotarod behavioral, biochemical, and rescue experiments in newly gen- test (Fig. 1G and SI Appendix,Fig.S4), which progressed affecting erated transgenic mice we demonstrate that ablation of UCH-L1 locomotion in the open field test (Fig. 1H and SI Appendix,Fig.S4) results in a presymptomatic increase in protein turnover followed and forelimb strength at 12 wk (Fig. 1I and SI Appendix,Fig.S4). + by proteasomal impairment causing progressive neurodegeneration Notably, Uch-l1 /d displayed an intermediate postnatal phenotype, and motor deficits. highlighting the early importance of UCH-L1 for brain function. + Matching the dose-dependent early phenotype of Uch-l1 /d and Results Uch-l1d/d mice, time course analyses revealed that UCH- UCH-L1–Deficient Mice Develop Progressive Motor Degeneration. We L1 transcript, protein, and activity levels were highest in young mice generated constitutive UCH-L1–deficient mice by flanking and declined thereafter (SI Appendix,Fig.S5). 1–3 by loxP sites. Crossing UCH-L1 floxed mice with constitutive Cre deleter mice resulted in the generation of UCH-L1 wild-type Monoubiquitin Levels Are Reduced at Post- but Not Presymptomatic + + + (Uch-l1 / ) heterozygous (Uch-l1 /d) and knockout (Uch-l1d/d) Time Points in UCH-L1–Deficient Brains. The accelerated execution mice (SI Appendix, Fig. S1). Loss of one UCH-L1 allele led to a of postnatal sensorimotor reflexes paired with the high expres- 50% reduction of UCH-L1 transcript (Fig. 1A) and protein (SI sion levels of UCH-L1 in young mice suggests that UCH-L1 is Appendix, Fig. S2) in juvenile but not adult mice. At postnatal required for brain development during the first postnatal weeks. day 0 only 18.7% of newborn pups were Uch-l1d/d, suggesting a UCH-L1 was suggested to be critically important for maintaining developmental perinatal disadvantage due to loss of UCH- free ubiquitin levels and for the function of the ubiquitin pro- + L1 expression (Fig. 1B). Uch-l1d/d and not Uch-l1 /d mice were teasome system in neurons (2). We therefore analyzed the levels + + lighter than Uch-l1 / mice from postnatal week 8 and displayed of mUB and polyubiquitin in total brain lysates and specific brain dystonic/spastic movements of their hind feet from 10 wk, as well areas with age. In whole brain lysates of wild-type mice, levels of as femoral/gluteal muscle wasting at 20 wk (Fig. 1 C and D). As an mono- and polyubiquitin decreased with age (SI Appendix, Fig. indication of neurodegenerative processes we detected spheroid S6), suggesting a pronounced need for ubiquitination in brain bodies in the cerebellum and enhanced signs of neuronal in- development. To our surprise, mUB levels in whole brain lysates + flammation in the cerebellum and spinal cord of 25-wk-old Uch- were unchanged in 5-d-old and 3- and 20-wk-old Uch-l1 /d and + + l1d/d mice compared with Uch-l1 / littermates (SI Appendix,Fig. Uch-l1d/d mice; however, levels of polyubiquitinated proteins were S3). Thus, Uch-l1d/d mice develop a similar neurological phenotype drastically decreased in 3-wk-old Uch-l1d/d brains compared with + + + + to spontaneous UCH-L1–deficient mice, namely the gad mice and Uch-l1 / mice and remained lower compared with Uch-l1 / mice

+ Fig. 1. Following a postnatal period of enhanced reflexes, Uch-l1 /d and Uch-l1d/d mice develop neurologic impairment. (A) qPCR for quantification of total + + brain UCH-L1 mRNA levels in 3- to 5-wk-old and 20- to 28-wk-old littermates; n = 5–7; **P < 0.01; n.d., not detected; n.s., not significant to / weeks 20–28. (B) + + + Frequency of genotypes resulting from heterozygous breeding; *P < 0.05 to Uch-l1 / .(C) Development of body weight in Uch-l1 /d and Uch-l1d/d females and males; n = 16–20; ***P < 0.001 to Uch-l1+/+.(D) Micrographs of Uch-l1d/d at 25 wk, demonstrating muscle wasting of the hind legs in comparison with Uch-l1+/+. Postnatal reflexes such as (E) the cliff avoidance reflex postnatal day 5 (P5); n = 21–40 and (F) the righting reflex postnatal day 8 (P8) are enhanced in Uch-l1d/d and Uch-l1+/d mice; n = 21–40. Thereafter, neurological impairment is visible in the (G) accelerated rotarod at weeks 5 and 12 (W5, W12); n = 8–23, (H)openfieldtestat + + week7(W7);n = 18–19, and (I) grip strength test at week 12 (W12); n = 18–19. *P < 0.05; **P < 0.01; ***P < 0.001 to age-matched Uch-l1 / ; §§P < 0.01; §§§P < + 0.001 to Uch-l1 /d.

7964 | www.pnas.org/cgi/doi/10.1073/pnas.1812413116 Reinicke et al. Downloaded by guest on October 1, 2021 + upon aging (Fig. 2A). Uch-l1 /d brains showed a significant re- processes in Uch-l1d/d mice. On the other hand, with advanced + + duction of pUB only at 20–28 wk of age compared with Uch-l1 / neurodegeneration, UCH-L1 deficiency resulted in an accumula- mice. Investigation of specific brain areas showed that mUB levels tion of polyubiquitinated proteins in 25-wk-old cerebella pre- were slightly reduced only in the brainstem of 3-wk-old Uch-l1d/d dominantly in the white matter, as well as in cultured Uch-l1d/d + + mice in comparison with Uch-l1 / littermates (Fig. 2B). With neurons (SI Appendix,Fig.S10), indicating a complex time- and aging, mUB levels additionally decreased in cerebrum and spinal brain area-dependent perturbation of protein homeostasis in UCH- cord of 25-wk-old Uch-l1d/d mice compared with wild-type litter- L1 deficiency. mates (Fig. 2B), correlating with the magnitude of UCH-L1 expression in the five brain areas studied (SI Appendix,Fig.S7A). Proteasomal Activity Is Enhanced in Young UCH-L1–Deficient Mice + + + In Uch-l1 /d mice, mUB levels were comparable to Uch-l1 / mice Followed by an Age-Dependent Decline. UCH-L1 deficiency resul- at 3–5 wk and decreased at 20–28 wk in the cerebrum compared ted in accelerated postnatal reflexes with decreased levels of + + with Uch-l1 / mice (SI Appendix,Fig.S7B). Further, mUB levels polyubiquitinated proteins in juvenile mice, followed by motor + + did not differ between primary cultured neurons of Uch-l1 / , Uch- degeneration and an abnormal accumulation of polyubiquitinated + l1 /d,andUch-l1d/d littermates (Fig. 2C), suggesting that UCH-L1 is proteins in old adult mice. The is integral to protein not required for the initial maintenance of the mUB pool in young homeostasis since it degrades misfolded, damaged, or aggregated brains. Measurement of overall UCH hydrolase activity exhibited proteins. Also, proteasome function declines with age and neu- only a 5% reduction of UCH activity in 3-wk-old Uch-l1d/d brains rodegeneration (15). We therefore hypothesized that the abnor- (Fig. 3A). Since other UCH enzymes could be up-regulated in mal levels of polyubiquitinated proteins in UCH-L1–deficient young brains to stabilize the mUB pool in the situation of UCH- brains might indicate altered proteasome activity, with decreased L1 loss, we searched for a compensatory up-regulation of other levels of polyubiquitin relating to enhanced proteasomal degra- DUBs. However, no increase of the other main UCH family dation of polyubiquitinated proteins. To examine whether this was members UCH-L3, UCH-L5, or UCH-X4 transcripts was observed the case, we quantified levels, composition, and overall activity of (SI Appendix,Fig.S8), suggesting either functional compensation by the 26S proteasome to evaluate how the proteasome is affected by other UCH enzymes or that UCH activity is not the predominant loss of UCH-L1 with age. Protein levels of the 20S core compo- function of UCH-L1 in the brain at the analyzed time points. We nent β5, which harbors the -like activity, were ele- thus analyzed the activity of other DUBs using the UB-VME vated up to 3 wk of age in Uch-l1d/d mice (Fig. 4A). Levels of the activity-based probe. No strong activation of other UCH enzymes 19S regulatory cap protein RPT1 and PA28α, a constituent of the was noted in 4- or 20-wk-old Uch-l1d/d brains, except for a slight alternative PA28 regulatory cap, were also up-regulated in Uch- enhancement of USP14 activity (Fig. 3B). Furthermore, proteomic l1d/d brains (Fig. 4A). These data suggest that UCH-L1 deficiency analyses of postnatal day 5 brains exhibited down-regulation or no results in an up-regulation of proteasomal levels. Chymotrypsin- MEDICAL SCIENCES change of most deubiquitinating enzymes, as opposed to the strong like activity, representing the main proteolytic activity of the regulation of UCH-L1 by knockout (Fig. 3C and SI Appendix,Fig. proteasomal 20S core unit was increased in 5- to 8-d-old and 3- to + + S9). Some E1, E2, and E3 enzymes such as UbE2r2, UBE2h, 5-wk-old Uch-l1d/d brains compared with Uch-l1 / littermates RNF40, and Smurf1 were up-regulated in Uch-l1d/d mice in com- (Fig. 4B). In contrast, chymotrypsin-like activity was significantly + + parison with Uch-l1 / littermates. Taken together, the data in- reduced in 20- to 28-wk-old Uch-l1d/d brains, suggesting that the dicate that UCH-L1 loss is not compensated by other DUBs, and primary effect of early increases in proteasome levels and activity that the loss in brain mUB occurs when neurodegeneration is al- is followed by secondary late onset decline of proteasome function ready advanced, suggesting that the reduced mUB level in neurons with age under UCH-L1 deficiency. To assess whether the ob- is one of the effects and not the cause of the neurodegenerative served decrease in chymotrypsin-like activity of the proteasome in

Fig. 2. UCH-L1–deficient mice exhibit an age- and brain area-dependent reduction in monoubiquitin. (A) Western blot (WB) for monoubiquitin (mUB) and + + + polyubiquitin (pUB) in Uch-l1 /d, Uch-l1d/d,andUch-l1 / brain lysates of postnatal day 5 (P5), 3-wk-old (W3), and 20-wk-old (W20) mice. Densitometric quantifica- + + tion; 4 exp. n = 8–17; *P < 0.05, **P < 0.01 to Uch-l1 / . (B)WBformUBinfivedistinctbrainareasof3-and25- wk-old mice. Graphs exhibit densitometric analysis; 3exp.n = 5–8; *P < 0.05. (C)WBformUBandUCH-L1in cultured neurons from Uch-l1+/d, Uch-l1d/d,andUch-l1+/+ littermates. Densitometric quantification; 4 exp. n = 7–8.

Reinicke et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 7965 Downloaded by guest on October 1, 2021 teractions of proteins from synthesis to degradation. We ob- served that proteasomal degradation was enhanced in young mutant brains and wondered whether this was necessary to bal- ance for enhanced protein synthesis. We established that S6 (as a measure of global protein synthesis activity) is highest in postnatal brains of wild- type mice, significantly decreasing with age (SI Appendix, Fig. S12A) as expected (22, 23). To assess whether protein synthesis was enhanced in the postnatal period of Uch-l1d/d mice, we in- terrogated the newly generated proteomics dataset for signifi- cantly changed proteins defined as log2 fold change >1(+/+ versus d/d) and a false discovery rate (FDR) of 0.2 after cor- rection for multiple testing. We found an increase of several activating eIF members, of Eif2 and -3, a key complex in acti- vation of . In addition, Eif4e2, a described repressor of translational activation (24), was most strongly decreased (Fig. 5A). Time course analyses of the involved in transcrip- tional activation such as the p70 ribosomal S6 protein kinases 1 and 2 (S6K1/2), (S6), which is phos- phorylated by S6K1/2 (25), and the eukaryotic 4E-binding proteins (4E-BPs) demonstrated enhanced phos- phorylation levels of all three, especially in postnatal Uch-l1d/d brains in comparison with wild-type littermates (Fig. 5B). Of note, phosphorylated levels of S6K and S6 were significantly + elevated in both Uch-l1 /d and Uch-l1d/d postnatal brains com- + + pared with Uch-l1 / mice (SI Appendix, Fig. S12B). However, 20- to 25-wk-old UCH-L1–deficient brains did not show increased Fig. 3. UCH-L1 deficiency is not significantly compensated by other DUBs. (A) pS6K/S6K, pS6/S6, or p4E-BP/4E-BP levels in comparison with +/+ UCH-based activity assay in brain lysatesof3-to5-,7-to9-,and20-to25-wk-old Uch-l1 littermates (Fig. 5B), suggesting that UCH-L1 deficiency Uch-l1d/d measures the of AMC from the synthetic substrate Ub-AMC profoundly affects protein synthesis in the postnatal period when + + by UCH deubiquitinating enzymes; n = 4–10; *P > 0.05 to Uch-l1 / .(B) Ubiquitin- pS6/S6 expression is at its highest. Corroborating the finding of + + derived activity-based assay in brain lysates of 4- or 20-wk-old Uch-l1d/d or Uch-l1 / enhanced activation of proteins regulating protein translation, the using the ubiquitin-vinylmethylester probe (HA-Ub-VME), which covalently ribosomal protein RPL7 associated with enhanced ribogenesis was binds to the active site (Cys) of DUBs. WB for the HA-tag exhibits active UCH-L1 increased in Uch-l1d/d brains (Fig. 5B), especially in neurons, a with bound HA-Ub-VME probe at 38 kDa, which is absent in the Uch-l1d/d ly- finding confirmed by Western blotting for RPL7 in UCH-L1– sates. The other bands correspond to other active deubiquitinating enzymes as + + deficient cultured primary neurons (Fig. 5D) and by staining for published (57); n = 3–5. (C) analyses of postnatal day 5 Uch-l1 / , d/d +/d d/d RPL7 in forebrain neurons of 3-wk-old Uch-l1 mice (Fig. 5C) Uch-l1 ,andUch-l1 brains. Heat map shows extracted normalized label-free compared with wild-type littermates. To examine the impact of quantification (LFQ) intensities of all proteins assigned with deubiquitinase (DUB) UCH-L1 deficiency on translation in the brain in vivo, we per- activity or protein-ubiquitin ligase activity. The mean of each genotype is pre- +/d d/d sented; n = 3. Significant alterations (FDR < 0.05, after correction for multiple formed polysome profiling of neocortices from Uch-l1 , Uch-l1 testing) were UCH-L1 and HECTD1. The majority of DUB showed a decreased mice and littermate controls (Fig. 5 E and F). Quantitative analysis of the polysome to monosome ratio (P/M) revealed a mild but trend with knockout. + significant increase in Uch-l1 /d and Uch-l1d/d (Fig. 5E). Moreover, we also detected a significant shift of from lighter to + d/d heavier polysomal regions in Uch-l1 /d and Uch-l1d/d brains (Fig. old Uch-l1 brains reflected a reduction in protein degradation by + G76V 5F). Thus, more ribosomes are engaged with mRNAs in Uch-l1 /d the ubiquitin/proteasome system (UPS), we used UB -green d/d fluorescent protein (GFP) transgenic mice (21) in which GFP is and Uch-l1 neocortices, consistent with increased mRNA tagged to a constitutively active proteasomal degradation signal translation in brains deficient for UCH-L1 function. To further (UBG76V), leading to polyubiquitination and proteasomal degrada- assess the rate of protein synthesis in neurons, we monitored in- + + tion of GFP (21). We crossed this line to the Uch-l1d/d or Uch-l1 / corporation of the analog 4-azido-L-homoalanine HCl background to assess the UPS status in Uch-l1d/d brains. Our results (L-AHA) in a Click-iT-SPAAC (strain-promoted azide alkyne G76V click chemistry) reaction with primary neurons. Following a pulse revealed an accumulation of UB -GFP protein in isolated pri- d/d +/d d/d of 1 h with L-AHA, both Uch-l1 and Uch-l1 neurons incor- mary culture neurons of Uch-l1 mice, despite an increase in porated significantly more L-AHA than neurons derived from proteasome levels as indicated by β5levels(Fig.4C),andinthe +/+ Uch-l1 littermates, showing that the absence of UCH-L1 cerebrum, cerebellum, hippocampus, and brainstem of 20- to 28-wk- promoted increased protein synthesis (Fig. 5G). old UCH-L1–deficient mice (Fig. 4 D and E). Immunofluorescence G76V for GFP showed that UB -GFP accumulation was confined to Alteration of Protein Homeostasis Strains UCH-L1–Deficient Neurons. neurons in respective brain areas (Fig. 4D and SI Appendix,Fig. G76V Accumulation of misfolded proteins in the endoplasmic reticu- S11). Of note, UB -GFP accumulation was not apparent in 3- to lum (ER) as in the setting of increased protein synthesis, leads to 5-wk-old brain areas of Uch-l1d/d mice (Fig. 4E). At 3 wk of age, G76V a condition referred to as ER stress. To counteract the accu- UB -GFP protein levels were comparable (cerebrum and hip- mulation of misfolded proteins, cells activate the /threo- pocampus) or decreased (cerebellum and brainstem) in comparison nine IRE1α to up-regulate molecular chaperones such as +/+ with Uch-l1 littermates, possibly reflecting an enhanced removal Grp78 via activation of the factor X-box binding of UBG76V-GFP through the elevated proteasomal activity. Taken protein (26). Grp78 is situated in the ER lumen (27) where it is together, these results indicate that proteasomal removal of target involved in the folding of newly synthesized proteins (28) and in proteins is impaired in aged UCH-L1–deficient mice, following a the retrograde transport of aberrant proteins across the ER phase of enhanced removal in juvenile mice. membrane for degradation by the proteasome (29). Western blot analyses exhibited a significant activation of IRE1α in brain ly- + + Protein Synthesis Is Enhanced in UCH-L1–Deficient Neurons. Protein sates (Fig. 6A) of 3- to 5-wk-old Uch-l1d/d mice versus Uch-l1 / , homeostasis regulates the concentration, localization, and in- whereby immunofluorescence analyses confirmed enhanced pIRE1α

7966 | www.pnas.org/cgi/doi/10.1073/pnas.1812413116 Reinicke et al. Downloaded by guest on October 1, 2021 Fig. 4. Proteasomal activity is enhanced in young UCH-L1–deficient mice followed by an age-dependent decline. (A) WB for the 20S core proteasomal subunit β5, which carries the chymotrypsin-like activity of the proteasome, of the regulatory 19S cap protein RPT1 and of the PA28α regulatory cap protein exhibits a postnatal and persistent up-regulation of proteasomal capacity in brain lysates of postnatal day 8 (P8), week 3 (W3), and week 20 (W20) Uch-l1d/d + + and Uch-l1 / . Right graphs exhibit densitometric quantifications; 3 exp. n = 5–9; §§P < 0.01, §§§P < 0.001, §§§§P < 0.0001 (effect of genotype by two-way + + ANOVA). (B) Chymotrypsin-like activity of the proteasome; n = 5; *P > 0.05 to Uch-l1 / .10μM MG132 was added to the brain lysate as an assay control. To assess proteasome function, UCH-L1–deficient mice were crossed to UBG78V-UB transgenic mice. (C) WB for UCH-L1, β5, and UBG76V-GFP in primary neurons from Uch-l1+/+, Uch-l1+/d, and Uch-l1d/d. Uch-l1d/d UBG76V-GFP neurons exhibit GFP accumulation despite increased proteasome levels; 3 exp. n = 7–10; *P < 0.05 to Uch-l1+/+.(D) Confocal images of UBG78V-GFP (green; DNA = blue) in the cerebellum of 20-wk-old Uch-l1d/d and Uch-l1+/+ UBG76V-GFP transgenic MEDICAL SCIENCES littermates exhibit GFP accumulation in neurons of Uch-l1d/d mice. (E) WB for UBG78V-GFP accumulation in four distinct brain areas of 3-wk- and 20-wk-old + + Uch-l1d/d versus Uch-l1 / UBG76V-GFP littermates, graph demonstrates densitometric quantification; 2 exp. n = 8–10; *P < 0.05; **P < 0.01; ***P < + + 0.001 to Uch-l1 / .

expression in neurons, as shown exemplarily for the cerebellum sufficiency, mTORC1 phosphorylates S6K, S6, and 4E-BP to co- (Fig. 6B). In line with these observations, Western blot analyses ordinately up-regulate protein translation and biosynthesis (25). In + + exhibited an early and persistent induction of Grp78 in brains and comparison with Uch-l1 / littermates, Uch-l1d/d brains exhibited cultured neurons of Uch-l1d/d mice, compared with wild-type lit- enhanced phosphorylation and thereby activation of mTOR ex- termates (Fig. 6 A and C). Together, these findings support the clusively during the postnatal period (SI Appendix,Fig.S13A). occurrence of ER stress and the involvement of UCH-L1 in reg- Furthermore, levels of raptor but not rictor were enhanced in ulation of protein synthesis in neurons at the age of 3 wk, shortly postnatal day 5 and 3- to 5-wk-old Uch-l1d/d brains (SI Appendix, before the first symptoms are observed in 5-wk-old UCH-L1– Fig. S13B). This result suggests an enhanced activity of mTOR deficient mice. Since processes such as protein synthesis and through mTORC1 rather than mTORC2 and corroborates our proteasomal degradation highly depend on sufficient ATP levels, finding of enhanced phosphorylation of mTORC1 downstream we searched for further signs of neuronal stress due to energy targets S6K, S6, and 4E-BP phosphorylation demonstrated in Fig. depletion or reactive oxidative species. Whereas the mitochon- 5A. Given the findings of enhanced mTORC1 activity in the drial membrane ATP synthase subunit beta (ATPB) is involved in postnatal period, we hypothesized that inhibition of mTORC1 ATP production from ADP, the manganese superoxide dismutase activity could be an effective measure to dampen the postnatally (MnSOD) scavenges reactive oxygen species (ROS) in stress- enhanced protein synthesis and resultant neuronal strain and induced diseases. Time course analyses exhibited elevated levels neurodegeneration in Uch-l1d/d brains.First,rapamycin,amTORC1 of both ATPB and of MnSOD, which were most pronounced in inhibitor which acutely inhibits mTORC1 signaling activity (30), postnatal days 5–9 and 3- to 5-wk-old Uch-l1d/d brains (Fig. 6D). d/d was used to treat primary cultured neurons. Indeed, as in vivo, This finding was corroborated in primary culture Uch-l1 neu- + pS6/S6 levels were significantly elevated both in Uch-l1 /d and rons, in which MnSOD levels were significantly enhanced and d/d ATPB levels slightly up-regulated in comparison with primary Uch-l1 primary culture neurons with a concomitant increase in +/+ protein synthesis as shown by L-AHA incorporation (Fig. 7A). culture Uch-l1 neurons (Fig. 6E). Immunofluorescent analyses + Rapamycin treatment reduced levels of pS6 in Uch-l1 /d and Uch- exhibited an enhanced signal for ATPB in neurons, as demon- d/d strated in neurons of the forebrain (Fig. 6F). Taken together these l1 primary culture neurons with a concomitant reduction in protein synthesis (Fig. 7A). Notably, rapamycin treatment in- results indicate enhanced neuronal strain in the form of ATP +/+ +/d depletion and ER stress in UCH-L1–deficient mice secondary to creased UCH-L1 protein in Uch-l1 and Uch-l1 neurons (Fig. accelerated protein synthesis and degradation. 7B), suggesting that mTORC1 inhibition by rapamycin can also induce protein synthesis of particular proteins such as UCH-L1 in mTORC1 Inhibition Restricts Protein Synthesis and Ubiquitin Accumulation the regulation of protein synthesis. Analysis of ubiquitin staining d/d in UCH-L1–Deficient Neuronal Cultures. To address how UCH-L1 on neurons showed strong ubiquitin accumulation in Uch-l1 deficiency might affect protein synthesis, and whether suppression neurons, which was reduced following rapamycin treatment. of enhanced protein synthesis could ameliorate the phenotype of Also, neurofilament staining was ameliorated (Fig. 7C). To- UCH-L1–deficient mice, we analyzed activity levels of mammalian gether these findings indicate that reduction of unbalanced target of rapamycin (mTOR). mTOR complexes with raptor to protein synthesis in UCH-L1 deficiency restores protein ho- form the mTOR complex 1 (mTORC1) or with rictor to form the meostasis and prevents accumulation of polyubiquitinated mTOR complex 2 (mTORC2). During growth factor and nutrient proteins in primary culture neurons.

Reinicke et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 7967 Downloaded by guest on October 1, 2021 Fig. 5. Protein synthesis is enhanced in UCH-L1–deficient neurons. (A) Heat map of significantly changed eIF ( initiation) subunit protein expression determined by proteomic analysis. Proteins of the eIF complex quantified with fold change of log2(d/d/control) or log2(d/+/control) larger 1 and FDR < 0.2, means per genotype; n = 3. (B and D) WB for the activation [phosphorylation (p)] of kinases involved in transcriptional activation such as the p70 ribosomal S6 protein kinases (S6K), ribosomal protein S6 (S6), the eukaryotic initiation factor 4E-binding proteins (4E-BPs), and for the large ribosomal subunit RPL7 in (B) postnatal day 5 (P5), 3-wk-old (W3), and 20-wk-old (W20) brain lysates of Uch-l1d/d and Uch-l1+/+ and in (D) cultured neurons. Right graphs demonstrate densitometric quantification in B 3 exp. n = 5–9; §P < 0.05, §§§P < 0.001, §§§§P < 0.0001 (effect of genotype by two-way ANOVA). In D 3 exp. n = 5–9; *P < 0.05 (Bonferroni test after one-way ANOVA). (C) Confocal images for ribosomal RPL7 (a and b), which localizes to NeuN positive neurons + + + + + (a′ and b′) in frontal cortical neurons of 3-wk-old Uch-l1d/d and Uch-l1 / .(E) Overlay of polysome profiles from neocortices of Uch-l1 / , Uch-l1 /d,andUch-l1d/d + + + postnatal day 5. Graph: normalized polysome-to-monosome (p/m) ratio for Uch-l1 /d and Uch-l1d/d vs. Uch-l1 / neocortices; 3 exp. n = 3–6; *P < 0.05. (F)Bin + + + + + analysis of changes in polysome fractions; 3 exp. n = 3–6; #P < 0.05 Uch-l1 / versus Uch-l1 /d and *P < 0.05 Uch-l1 / versus Uch-l1d/d at the indicated fraction, §P < 0.05 (effect of genotype x fraction, mixed two-way ANOVA). Note the significant shift of ribosomes from lighter to heavier polysomes in Uch-l1+/d and Uch-l1d/d. (G) Measurement of new protein synthesis rate in primary neurons by incorporation of the methionine analog L-AHA HCl during a 1-h pulse. Densitometric +/+ quantification of L-AHA incorporation; 3 exp. n = 8–14; **P < 0.01; ***P < 0.01 to Uch-l1 .

Postnatal Inhibition of mTORC1 Ameliorates the Neurodegenerative (Fig. 8D). Morphologically, postnatal rapamycin treatment signifi- Phenotype of UCH-L1 Deficiency. Finally, we addressed whether cantly ameliorated the extent of ubiquitin aggregates in the white dampening protein synthesis by rapamycin treatment could also matter of the cerebellum by immunofluorescence when analyzed in + block UCH-L1–dependent motor degeneration in vivo. Postnatal 16-wk-old Uch-l1 /d and Uch-l1d/d mice (Fig. 8 E and F). Taken days 1–8 treatment of litters from heterozygous breeding pairs together, these data show that postnatal dampening of mTOR ac- with rapamycin reduced pS6/S6 levels of all three genotypes tivity ameliorates the neurological phenotype of UCH-L1 deficiency. + + below untreated Uch-l1 / levels (Fig. 8A), demonstrating suc- cessful mTORC1 inhibition. As in cultured neurons, rapamycin Discussion treatment significantly elevated UCH-L1 protein levels in Uch- + + + UCH-L1 is one of the most abundant and enigmatic enzymes of l1 /d mice to almost Uch-l1 / protein levels (Fig. 8A). Postnatal + the CNS. As a synopsis of findings based on existing spontaneous reflexes were normalized in Uch-l1 /d and Uch-l1d/d mice following UCH-L1 knockout models, UCH-L1 is thought to be required treatment with rapamycin (SI Appendix,Fig.S14). We next eval- for the maintenance of axonal integrity, but not for neuronal uated whether rapamycin treatment ameliorated the molecular + development, despite its high expression in neurons (6). We phenotype of Uch-l1 /d and Uch-l1d/d mice. Chymotrypsin-like generated a new transgenic mouse model to investigate the activity of the proteasome was significantly reduced in brains of mechanisms underlying neurodegeneration due to UCH-L1 + Uch-l1 /d and Uch-l1d/d mice at the termination of rapamycin deficiency. Based on our data we propose that UCH-L1 has a treatment on postnatal day 9 (Fig. 8B), suggesting that rapamycin previously unrecognized role in neuronal development. By per- treatment reduced the need for enhanced proteasomal activity forming biochemical and behavioral analyses at pre- and post- caused by UCH-L1 deficiency, as the burden of protein synthesis symptomatic time points, we can show that UCH-L1 deficiency was reduced. Finally, we analyzed the effects of postnatal rapa- results in enhanced neuronal protein synthesis during the first mycin treatment on motor function in adult mice using the open postnatal weeks, leading to ER stress, energy depletion, protea- field and accelerated rotarod tests in 12- and 16-wk-old mice, somal impairment with accumulation of nondegraded ubiquiti- respectively. Rapamycin treatment rescued motor deficits of Uch- nated protein, and neurodegeneration starting around the fifth l1d/d mice in the open field (Fig. 8C) and ameliorated the perfor- postnatal week. Strikingly, we found that treatment of mice + mance of Uch-l1d/d and Uch-l1 /d mice on the accelerated rotarod lacking UCH-L1 with the protein synthesis modulator rapamycin

7968 | www.pnas.org/cgi/doi/10.1073/pnas.1812413116 Reinicke et al. Downloaded by guest on October 1, 2021 Fig. 6. UCH-L1 defiency results in neuronal strain. Uch-l1d/d brains were analyzed for the occurrence of ER stress and for signs of energy depletion. (A)WBfor the activation [phosphorylation (p)] of the sensor of unfolded proteins in the lumen of the ER, IRE1α, and of the molecular Grp78 in brain lysates of + + postnatal day 8 (P8), 3-wk-old (W3), and 20-wk-old (W20) Uch-l1d/d in comparison with Uch-l1 / . Right graphs exhibit densitometric quantifications; 3 exp. n = 5–9; §§P < 0.01, §§§P < 0.001 (effect of genotype by two-way ANOVA). (B) Confocal images of pIRE1α (green), glial fibrillary acidic protein (GFAP, red), and DNA (blue) in cerebella of Uch-l1d/d in comparison with Uch-l1+/+ mice. Arrows: Purkinje cells. Note the enhanced signal for pIRE1α in the stratum granulosum in 3- and 20-wk-old Uch-l1d/d cerebella. (C) WB for Grp78 levels in primary neurons of Uch-l1d/d in comparison with Uch-l1+/+. Lower graph exhibits densitometric = – < +/+ quantifications; 3 exp. n 5 9; *P 0.05 to Uch-l1 . WB for the mitochondrial membrane ATP synthase (ATPB) and the detoxifying manganese MEDICAL SCIENCES superoxide dismutase (MnSOD) in (D) brain lysates of postnatal day 8 (P8), 3-wk-old (W3), and 20-wk-old (W20) Uch-l1d/d; 3 exp. n = 5–15; and (E) in primary + + + + neurons of Uch-l1d/d in comparison with Uch-l1 / . Graphs (D and E) exhibit densitometric quantifications; 3 exp. n = 8; §P < 0.05; *P < 0.05 to Uch-l1 / , n.s., + + not significant. (F) Confocal images for ATBP in forebrain neurons of 3-wk-old Uch-l1d/d mice in comparison with Uch-l1 / mice. Arrows: Purkinje cells, note the enhanced signal for ATBP in Uch-l1d/d neurons.

directly after birth could rescue neurological phenotypes later lation of tubulovesicular structures and loss of synaptic vesicles in life. in UCH-L1 deficiency could result from UCH-L1–dependent Our Uch-l1d/d mice show a phenotype of sensory and motor dysregulation of protein synthesis that may involve both up- ataxia consistent with that described in gad and nm3419 spon- and down-regulation of protein synthesis of particular proteins. taneous UCH-L1 mutants (11, 12). In contrast to the proposal Intriguingly, we observed enhanced postnatal reflexes in UCH- that neurodegeneration is caused by reduced levels of mUB (10, L1–deficient animals reflecting an UCH-L1–dependent accel- 12), we found that mUB levels are grossly normal in postnatal, eration of neonatal development of sensory and motor capabil- and 3- to 5-wk-old Uch-l1d/d mice and in primary neurons, sug- ities. Currently we do not know how UCH-L1 mechanistically gesting that UCH-L1 is not primarily required to maintain the modulates neuronal protein synthesis. The enhanced postnatal mUB pool in young neurons. UCH-L1’s proposed role in activity of mTORC1 in UCH-L1–deficient mice suggests that maintaining a pool of ubiquitin in the is based on its UCH-L1 modulates protein synthesis through the mTOR path- hydrolytic activity to cleave short from the C terminus way. mTORC1 activation can increase protein synthesis of some, of ubiquitin in vitro (6), which was minimally reduced in Uch- yet repress translation of other specific proteins. Thereby devi- l1d/d mice. Loss of UCH-L1 did not result in a compensatory up- ations to an optimal range of protein synthesis that adapts to regulation of other known DUBs. Rather, levels of other DUBs demand results in the behavioral impairments that develop with were found to be down-regulated by proteomic analyses. Thus, age (34–37). Notably, our data showing that rapamycin treat- + + + UCH-L1 is so unique from its structural (and thereby potentially ment increases UCH-L1 protein in Uch-l1 / and Uch-l1 /d functional) characteristics (31) (quoted as “the most complicated neurons demonstrates that mTORC1 inhibition can also induce eukaryotic discovered to date”) (6), that a protein synthesis of particular homeostatic proteins, such as compensation is not possible by other DUBs. Since reduced UCH-L1, important in the regulation of protein synthesis. Pre- levels of mUB could not explain the early behavioral phenotype viously it was shown that UCH-L1 regulates mTORC1 stability of the Uch-l1d/d mice, we focused on UCH-L1’s role in other but not levels (38). In our mice, however, amounts of phos- processes of the UPS system. phorylated mTOR and raptor were postnatally elevated, sug- Protein turnover was increased in total brains of postnatal and gesting a different level of mTORC1 regulation. UCH-L1 was 3-wk-old UCH-L1–deficient mice and more specifically in neu- further ascribed to affect protein synthesis in B cell malignancy rons. Support for UCH-L1’s function in controlling protein by bypassing mTORC1 through association with the translation synthesis comes from recent work examining the role of UCH- initiation complex eIF4F, the downstream target of 4E-BP1 (39). L1 in synaptic transmission at neuromuscular junctions (13). In In our mice, however, postnatal amounts of phosphorylated UCH-L1–deficient mice, synaptic activity is impaired, with loss 4E-BP1 were significantly enhanced, and dampening of protein of synaptic vesicles and accumulation of branched tubulove- synthesis by postnatal rapamycin treatment attenuated the de- sicular structures at presynaptic nerve terminals (13). Indeed, velopment of neurodegeneration, pointing toward a regulatory presynaptic protein synthesis is required for synapse formation effect of UCH-L1 upstream of mTOR. and components of the mTOR pathway have been shown to The GFP degron mouse reporter was designed to give in- control synaptic bouton size, active zone number, and synaptic formation about the functional status of the ubiquitin/protea- function (32, 33). Therefore, it is conceivable that the accumu- some system as a whole (21). Using this reporter system, we

Reinicke et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 7969 Downloaded by guest on October 1, 2021 Fig. 7. mTORC1 inhibition by rapamycin reduces protein synthesis and ubiquitin accumulation in Uch-l1d/d cultured neurons. Primary neurons were treated for 1 h with rapamycin (rapa) or vehicle (veh). (A)Mea- surement of new protein synthesis rate by in- corporation of the methionine analog L-AHA during a 1-h pulse and WB for biotin; pS6 to S6 ratio to control for mTORC1 inhibition. Right graph exhibits densito- metric quantification of L-AHA incorporation; 4 exp. + + n = 4–22; *P < 0.05; **P < 0.01; ***P < 0.001 to Uch-l1 / + vehicle, §P < 0.05 to Uch-l1 /d or to Uch-l1d/d vehicle. (B) WB for UCH-L1. Densitometric analyses exhibit the change of UCH-L1 protein levels with rapamycin treatment in individual (paired) neuronal cultures + + + from Uch-l1 / or Uch-l1 /d littermate embryos; 2 exp. n = 4–9; *P < 0.05; **P < 0.01. (C) Confocal images for ubiquitin (green) and neurofilament (white) in primary neurons of Uch-l1d/d and Uch-l1+/+ with or without treatment with rapamycin or vehicle for 24 h. Arrows point toward ubiquitin aggregates in neurofilament positive processes. Graph exhibits quantification of mean intensity of fluorescence (MIF) of ubiquitin to + + neurofilament; 2 exp. n = 6; *P < 0.05 to Uch-l1 / vehicle; §P < 0.05 to Uch-l1d/d rapamycin.

observed increased protein degradation through the proteasome and gad mice showed increased vulnerability to perox- in 3- to 5-wk-old UCH-L1–deficient animals followed by impaired idation (53, 54). Thus, altered protein synthesis caused by UCH- degradation in week 20 mice. However, it needs to be considered L1 deficiency at very early developmental time points has acute that the observed changes in GFP could be due to changes in importance for long-term neuronal health, as suggested by the synthesis, degradation, or both. Nonetheless, chymotrypsin-like finding of early onset progressive neurodegeneration in humans activity measurements of the proteasome supported the finding with missense mutations in the UCH-L1 gene (16). With age, we that GFP levels measured in the degron mouse reflected protea- saw a significant reduction of proteasome activity with concom- somal activity. Interestingly, in gad or nm3419 spontaneous UCH- itant accumulation of polyubiquitinated proteins, suggesting that L1 mutants proteasomal activity was not obviously impaired (11, prolonged dysregulated protein synthesis with accompanying 12). It has however been proposed that UCH-L1 is crucial for , ER stress, and energy depletion are toxic for neu- maintaining proper proteasomal function (10, 40) and is involved rons. In line with this hypothesis, motor neurons could be par- in the determination of proteasomal subtype abundance (41), the ticularly susceptible to UCH-L1 loss, as the high energy and underlying mechanism(s) however being completely unknown. protein turnover burdens required to maintain extensive axonal Nonetheless, enhanced proteasomal abundance and activity in our projections imply that motor neurons operate very close to their Uch-l1d/d mice is in accordance with the function of the UPS maximum capacity rendering them vulnerable to defects that system to maintain homeostasis in the face of stress by synthe- other neuronal types might withstand longer. This idea is sup- sizing and degrading proteins in a synchronized manner (42–44). ported by our finding that reducing protein synthesis by rapa- mycin treatment during the first postnatal week ameliorates the Of note, we found UCH-L1–deficient mice to increase their + neurological phenotype and ubiquitin accumulations in Uch-l1 /d proteasomal capacity by up-regulation of the 26S standard pro- d/d teasome and of the PA28 proteasome. This is of interest, as the and Uch-l1 mice up to 16 wk of age. 26S proteasome results in an ATP- and ubiquitin-dependent In conclusion, our study describes the temporal sequence of ef- proteolysis, whereas the PA28 proteasome enables cells to per- fects caused by UCH-L1 deficiency yielding insights into the mech- form ATP- and ubiquitin-independent proteolysis (45), a process anisms underlying neurodegeneration caused by UCH-L1 loss of shown to be of great importance for cellular stress compensation function. Moreover, we show that a 50% reduction in UCH-L1 can (46). Of note, also the differential distribution of the proteasome have significant effects at the neuronal and behavioral levels, estab- forms in specific brain areas is thought to predispose brain areas lishing the importance of UCH-L1 levels in neuronal development to stress susceptibility as a predisposition to neurodegeneration and neurodegeneration. Finally, our study reveals that UCH-L1 (47, 48). Therefore, the initial up-regulation of proteasomal ca- deficiency affects the ability to regulate translational activity during pacity could reflect the need of UCH-L1–deficient neurons to early synapse development and that this can be pharmacologically addressed, a finding of major importance for patients with juvenile remove flawed newly synthesized proteins and to deal with cellular onset neurodegeneration due to mutations in UCH-L1 (16). This stress through energy depletion in face of high ATP-consuming result has potential implications beyond UCH-L1 deficiency: timing cellular processes. prophylactic interventions to specific windows may be generally Consistent with the observation of ER stress and oxidative useful for the prevention of neurodegenerative processes. stress in juvenile mutant brains and cultured primary neurons of d/d our Uch-l1 mice, Tan et al. (49) demonstrated in an in vitro Materials and Methods cell culture model that pharmacological UCH-L1 inhibition in- duces ER stress and cell death. Further, nm3419 mice were Detailed materials and methods are within SI Appendix. described to have ER stress at the presymptomatic stages in corticospinal motor neurons, correlating with disintegration of Generation of UCH-L1 Knockout Mice. UCH-L1 floxed mice were generated by Genoway (SI Appendix,Fig.S1A), bred to constitutive Cre deleter mice, and the apical dendrite and spine loss (50). Interestingly, UCH-L1 is backcrossed for 12 generations to the C57BL/6 background. Littermates proposed to have an antioxidant function in neurons (17, 51), born from heterozygous parents were used. For rapamycin treatment, pups which could explain the up-regulation of the ROS-detoxifying were treated daily with rapamycin (0.1 mg/kg) or NaCl/ethanol on post- enzyme MnSOD in neurons of our mice lacking UCH-L1 natal days 1–8 between 9 AM and 10 AM. Animal experiments were carried expression. Consistent with this, down-regulation of UCH-L1 out in accordance with the European Community Council Directive (86/609/ in embryonic carcinoma cells resulted in an increased suscepti- EEC), and the procedures used were approved by the State of Hamburg bility to oxygen and glucose deprivation-induced cell death (52) (approval 75/14).

7970 | www.pnas.org/cgi/doi/10.1073/pnas.1812413116 Reinicke et al. Downloaded by guest on October 1, 2021 Fig. 8. Postnatal rapamycin treatment ameliorates neurodegenerative phenotype of UCH-L1–deficient mice. Litters from Uch-l1+/d females crossed with Uch-l1+/d males were treated on a daily basis with rapamycin (0.1 mg/kg) or vehicle (NaCl/ethanol) from postnatal day 1–8. Thereafter, treatment was stopped. (A)WB for the phosphorylation (p) status of mTORC1 substrate S6 ribosomal protein (S6) in brains from one litter. Graph demonstrates densitometric quantification; + + + 4 litters n = 6–22; **P < 0.01, ***P < 0.001, ****P < 0.0001 to Uch-l1 / ; §P < 0.05, §§§§P < 0.0001 to Uch-l1 /d vehicle. (B) Chymotrypsin-like activity of the proteasome in brain lysates postnatal day 9 (P9), 24 h after termination of short-term treatment with rapamycin or vehicle; 3 litters n = 3–11; *P < 0.05 and ***P < 0.001 to Uch-l1+/+ vehicle; §§§P < 0.001 genotype comparison. (C) Measurement of the distance moved in the open field test and the maximal velocity performed at 12 wk of age, demonstrating partially rescued phenotype in Uch-l1d/d mice; 5 litters n = 5–17; *P < 0.05, ***P < 0.001 to Uch-l1+/+ vehicle; §P < + 0.05; §§P < 0.01 genotype comparison. (D) Accelerated rotarod tests performed at 16 wk of age, demonstrating rescued neurological phenotype in Uch-l1 /d + + + mice, 5 litters n = 2–15; ***P < 0.001 to Uch-l1 / vehicle, §§P < 0.01 to Uch-l1 /d vehicle. (E and F) Ubiquitin aggregates within the cerebellar white matter MEDICAL SCIENCES were quantified (E) and visualized (F) by immunofluorescent staining for ubiquitin (green), glial fibrillary acidic protein (GFAP, red), and DNA (blue). Graph + + exhibits quantification of five high power fields (hpf) of n = 3 mice per genotype and treatment; *P < 0.05 to vehicle Uch-l1 / ; §P < 0.05 to respective vehicle- treated genotype.

Behavioral Studies. Offspring were examined for postnatal reflexes, and in processed mass spectrometry data of this study have been be uploaded to adolescence and adulthood for motor coordination (open field and the PRIDE/ProteomExchange database (55, 56) with the dataset identifier accelerated rotarod test) and strength of the forelimbs (grip strength test). All PXD010449. tests were performed during the dark cycle of the mice under red light. Polysome Profiling and Analysis. Neocortices were lysed and centrifuged, the Primary Neuronal Culture. Brains from E15.5–E16.5 embryos, arising from resulting supernatants were supplemented with Nonidet P-40 and Triton heterozygous breeding pairs were isolated and the cerebellum, brainstem, X-100 (both to 1%), and incubated on ice for 5 min. After centrifugation, and meninges were removed. The remaining cortex was digested and sucrose gradients were generated using the Gradient Master 108 pro- mechanically dissected. A total of 750,000 cells were plated to grow and grammable gradient pourer. Gradients were fractionated and measured for differentiate for 7 d. For mTORC1 inhibition, neurons were treated for 1 h RNA content using a Piston Gradient Fractionator attached to a UV monitor.

with 20 nM rapamycin or dH2O as vehicle. Measurement of Protein Synthesis by Click-iT SPAAC Reaction. L-AHA HCl Ubiquitin-Derived Activity-Based DUB Activity Assay. The ubiquitin-derived (2 mM) was added for 1 h in methionine-free culture DMEM before har- activity-based ubiquitin-vinylmethylester probe (HA-UB-VME), which is an vesting. The conjugation reaction was performed by adding DBCO-PEG12- active site-directed probe with broad reactivity toward DUBs, with the ex- biotin (200 μM final) to the cell extract (10 μL) and incubated for 1 h at 37 °C ception of few metalloproteases, binds covalently to the active-site (Cys) of and analyzed by SDS/PAGE. most DUBs, which is only accessible in active enzymes. The N-terminal tag hemagglutinin (HA tag) allows detecting the modified DUBs after the Immunohistochemistry and Immunofluorescence. Antigen retrieval was per- separation of a protein mixture by SDS/PAGE, followed by immunoblotting. formed by microwave boiling (10 mM citrate buffer, pH 6.1) or by protease digestion [protease XXIV (Sigma) 5 μg/mL]. Unspecific binding was blocked Chymotrypsin-Like Activity Assay. A total of 10 μg total protein was diluted in [5% horse serum (Vector), 30 min at room temperature (RT)]. Antibody in- incubation buffer. After preincubation, the substrate for chymotrypsin-like cubations of primary antibodies were followed by incubation with bio- activity Suc-LLVY-7-amino-4-methylcoumarin (AMC) (Calbiochem, EMD Chem- tinylated or AF488 or Cy3-coupled secondary antibodies (1:400, 30 min, RT). icals, Inc, division of Merck KGaA) was added to the samples at a final con- Color development was performed with the ZytoChem-Plus AP Kit centration of 60 μmol/L. Proteasomal activity was measured at 355 and 460 nm (Zytomed) according to the manufacturer’s instructions with neufuchsin. For after incubation at 37 °C for 1 h in the dark. MG132 at a concentration of immunofluorescence, cultured neurons were fixed with 4% PFA for 8 min at 10 μmol was added to brain lysate as an assay control. RT. Unspecific binding was blocked and 1° antibody incubations (blocking buffer overnight, 4 °C) were followed by incubation with biotinylated or UCH Activity Assay. Briefly, 10 μg of total brain extract was diluted into UCH AF488 or Cy3-coupled 2° antibodies. reaction buffer. To start the reaction, ubiquitin-AMC was added to a final concentration of 400 nM. Fluorescence intensity was measured at 380 nm Electron Microscopy. Mice were transcardially perfused (4% PFA, 1% glu- and 460 nm at 37 °C and recorded at 1-min intervals for 30 min. taraldehyde in 0.1 M PB buffer, pH 7.4). Brains were dissected and postfixed. Infiltration of the embedding medium was performed by immersing the Proteomics. Snap-frozen, unperfused neocortex from postnatal day 5 Uch-l1+/+, pieces in a 1:1 mixture of propylene oxide and epon and finally in neat epon Uch-l1d/+,andUch-l1d/d mice was homogenized in urea buffer followed and hardened at 60 °C. Ultrathin sections were examined in an EM902. by tryptic digestion. Peptides were cleaned up by Stagetips, separated by nLC MS-MS on a 50-cm self-packed C18 fused silica column, and analyzed qPCR and RT-PCR Analysis. Total mRNA was extracted from whole brain using using a Quadrupole-Orbitrap hybrid mass spectrometer. The raw data and TRIzol and 1 μg was reverse transcribed with random hexamer primer and

Reinicke et al. PNAS | April 16, 2019 | vol. 116 | no. 16 | 7971 Downloaded by guest on October 1, 2021 Moloney Murine Leukemia . mRNA expression was Statistical Analysis. Values are expressed as mean ± SEM. Effects of genotype quantified with an AbiPrism NN8860 using SYBR green. For RT-PCR, ampli- were analyzed with t test or one-way ANOVA followed by Bonferroni tests fication was performed in a Biometra thermocycler. when appropriate. If data did not reach the criteria for parametric sta- tistics, nonparametric statistics (Mann–Whitney’s u test or Kruskal–Wallis’s Immunoblot. Samples were lysed in T-PER (containing 1 mM sodium fluoride, test followed by Dunn’s tests) were used. Within groups, designs were 1 mM sodium vanadate, 100 nM calyculin A, complete) and denatured with analyzed with mixed multifactorial ANOVA with Bonferroni post hoc tests 4× lithium dodecyl sulfate. A total of 10 μg total brain or brain area lysates when appropriate. Tests were two tailed and significance was set at or 3- to 5-μg neuronal cell lysate were separated on a 4–12% or 16% Bis-Tris P < 0.05. NuPage gel. Protein transfer was performed onto PVDF membranes which were blocked before incubation with primary antibodies. Binding was de- ACKNOWLEDGMENTS. A.T.R. and J.R. are supported by the Else Kröner Fre- tected by incubation with HRP-coupled secondary antibodies (1:10,000, 3% senius Stiftung; W.S., M. Sachs, and C.M.-S. by the DFG (Grant SFB1192); and nonfat milk) and visualized with ECL SuperSignal with Amersham Imager 600. M.M.R. by the DFG [Grants RI2811/1 and RI2811/2 (FOR2743)].

1. Komander D, Clague MJ, Urbé S (2009) Breaking the chains: Structure and function of 31. Sułkowska JI, Rawdon EJ, Millett KC, Onuchic JN, Stasiak A (2012) Conservation of the deubiquitinases. Nat Rev Mol Cell Biol 10:550–563. complex knotting and slipknotting patterns in proteins. Proc Natl Acad Sci USA 109: 2. Day IN, Thompson RJ (2010) UCHL1 (PGP 9.5): Neuronal biomarker and ubiquitin E1715–E1723. system protein. Prog Neurobiol 90:327–362. 32. Cheng L, Locke C, Davis GW (2011) S6 kinase localizes to the presynaptic active zone 3. Wilkinson KD, et al. (1989) The -specific protein PGP 9.5 is a ubiquitin carboxyl- and functions with PDK1 to control synapse development. J Cell Biol 194:921–935. terminal hydrolase. Science 246:670–673. 33. Speese SD, Trotta N, Rodesch CK, Aravamudan B, Broadie K (2003) The ubiquitin 4. Meyer-Schwesinger C, et al. (2009) A new role for the neuronal ubiquitin C-terminal proteasome system acutely regulates presynaptic protein turnover and synaptic ef- hydrolase-L1 (UCH-L1) in process formation and podocyte injury in human ficacy. Curr Biol 13:899–910. glomerulopathies. J Pathol 217:452–464. 34. Auerbach BD, Osterweil EK, Bear MF (2011) Mutations causing syndromic autism 5. Miyoshi Y, et al. (2006) High expression of ubiquitin carboxy-terminal hydrolase- define an axis of synaptic pathophysiology. Nature 480:63–68. L1 and -L3 mRNA predicts early recurrence in patients with invasive breast cancer. 35. Niere F, et al. (2016) Analysis of proteins that rapidly change upon mechanistic/ Cancer Sci 97:523–529. mammalian target of rapamycin complex 1 (mTORC1) repression identifies Parkinson 6. Bishop P, Rocca D, Henley JM (2016) Ubiquitin C-terminal hydrolase L1 (UCH-L1): protein 7 (PARK7) as a novel protein aberrantly expressed in tuberous sclerosis Structure, distribution and roles in brain function and dysfunction. Biochem J 473: complex (TSC). Mol Cell Proteomics 15:426–444. 2453–2462. 36. Raab-Graham KF, Haddick PC, Jan YN, Jan LY (2006) Activity- and mTOR-dependent 7. Larsen CN, Krantz BA, Wilkinson KD (1998) Substrate specificity of deubiquitinating suppression of Kv1.1 channel mRNA translation in dendrites. Science 314:144–148. enzymes: Ubiquitin C-terminal hydrolases. Biochemistry 37:3358–3368. 37. Niere F, Raab-Graham KF (2017) mTORC1 is a local, postsynaptic voltage sensor reg- 8. Kurihara LJ, Semenova E, Levorse JM, Tilghman SM (2000) Expression and functional ulated by positive and negative feedback pathways. Front Cell Neurosci 11:152. analysis of Uch-L3 during mouse development. Mol Cell Biol 20:2498–2504. 38. Hussain S, et al. (2013) Ubiquitin hydrolase UCH-L1 destabilizes mTOR complex 1 9. Das C, et al. (2006) Structural basis for conformational plasticity of the Parkinson’s by antagonizing DDB1-CUL4-mediated ubiquitination of raptor. Mol Cell Biol 33: disease-associated ubiquitin hydrolase UCH-L1. Proc Natl Acad Sci USA 103: 1188–1197. 4675–4680. 39. Hussain S, et al. (2018) UCH-L1 bypasses mTOR to promote protein biosynthesis and is 10. Osaka H, et al. (2003) Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes required for MYC-driven lymphomagenesis in mice. Blood 132:2564–2574. monoubiquitin in neuron. Hum Mol Genet 12:1945–1958. 40. Moss A, et al. (2002) A role of the ubiquitin-proteasome system in neuropathic pain. 11. Saigoh K, et al. (1999) Intragenic deletion in the gene ubiquitin carboxy- J Neurosci 22:1363–1372. terminal hydrolase in gad mice. Nat Genet 23:47–51. 41. Radón V, et al. (2018) Ubiquitin C-terminal hydrolase L1 is required for regulated 12. Walters BJ, et al. (2008) Differential effects of Usp14 and Uch-L1 on the ubiquitin protein degradation through the ubiquitin proteasome system in kidney. Kidney Int proteasome system and synaptic activity. Mol Cell Neurosci 39:539–548. 93:110–127. 13. Chen F, Sugiura Y, Myers KG, Liu Y, Lin W (2010) Ubiquitin carboxyl-terminal hy- 42. Schubert U, et al. (2000) Rapid degradation of a large fraction of newly synthesized drolase L1 is required for maintaining the structure and function of the neuromus- proteins by . Nature 404:770–774. cular junction. Proc Natl Acad Sci USA 107:1636–1641. 43. Ding Q, Cecarini V, Keller JN (2007) Interplay between protein synthesis and degra- 14. Setsuie R, Wada K (2007) The functions of UCH-L1 and its relation to neurodegen- dation in the CNS: Physiological and pathological implications. Trends Neurosci 30: erative diseases. Neurochem Int 51:105–111. 31–36. 15. Hegde AN, Upadhya SC (2007) The ubiquitin-proteasome pathway in health and 44. Zhang Y, et al. (2014) Coordinated regulation of protein synthesis and degradation by disease of the nervous system. Trends Neurosci 30:587–595. mTORC1. Nature 513:440–443. 16. Bilguvar K, et al. (2013) Recessive loss of function of the neuronal ubiquitin hydrolase 45. Cascio P (2014) PA28αβ: The enigmatic magic ring of the proteasome? 4: UCHL1 leads to early-onset progressive neurodegeneration. Proc Natl Acad Sci USA 566–584. 110:3489–3494. 46. Li J, et al. (2011) Enhancement of proteasomal function protects against cardiac 17. Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat proteinopathy and ischemia/reperfusion injury in mice. J Clin Invest 121:3689–3700. Med 10:S10–S17. 47. McNaught KS, Belizaire R, Isacson O, Jenner P, Olanow CW (2003) Altered protea- 18. Zhang M, et al. (2012) Control of BACE1 degradation and APP processing by ubiquitin somal function in sporadic Parkinson’s disease. Exp Neurol 179:38–46. carboxyl-terminal hydrolase L1. J Neurochem 120:1129–1138. 48. McNaught KS, Jnobaptiste R, Jackson T, Jengelley TA (2010) The pattern of neuronal 19. Cartier AE, et al. (2012) Differential effects of UCHL1 modulation on alpha-synuclein loss and survival may reflect differential expression of proteasome activators in Par- in PD-like models of alpha-synucleinopathy. PLoS One 7:e34713. kinson’s disease. Synapse 64:241–250. 20. Wang YL, et al. (2004) Accumulation of beta- and gamma-synucleins in the ubiquitin 49. Tan YY, Zhou HY, Wang ZQ, Chen SD (2008) Endoplasmic reticulum stress contributes carboxyl-terminal hydrolase L1-deficient gad mouse. Brain Res 1019:1–9. to the cell death induced by UCH-L1 inhibitor. Mol Cell Biochem 318:109–115. 21. Lindsten K, Menéndez-Benito V, Masucci MG, Dantuma NP (2003) A transgenic mouse 50. Jara JH, et al. (2015) Corticospinal motor neurons are susceptible to increased ER model of the ubiquitin/proteasome system. Nat Biotechnol 21:897–902. stress and display profound degeneration in the absence of UCHL1 function. Cereb 22. Sengupta S, Peterson TR, Sabatini DM (2010) Regulation of the mTOR complex Cortex 25:4259–4272. 1 pathway by nutrients, growth factors, and stress. Mol Cell 40:310–322. 51. Spillantini MG, et al. (1997) Familial multiple system tauopathy with presenile de- 23. Hay N, Sonenberg N (2004) Upstream and downstream of mTOR. Dev 18: mentia: A disease with abundant neuronal and glial tau filaments. Proc Natl Acad Sci 1926–1945. USA 94:4113–4118. 24. Morita M, et al. (2012) A novel 4EHP-GIGYF2 translational repressor complex is es- 52. Shen H, Sikorska M, Leblanc J, Walker PR, Liu QY (2006) Oxidative stress regulated sential for mammalian development. Mol Cell Biol 32:3585–3593. expression of ubiquitin carboxyl-terminal hydrolase-L1: Role in cell survival. Apoptosis 25. Ma XM, Blenis J (2009) Molecular mechanisms of mTOR-mediated translational con- 11:1049–1059. trol. Nat Rev Mol Cell Biol 10:307–318. 53. Kikuchi T, Mukoyama M, Yamazaki K, Moriya H (1990) Axonal degeneration of as- 26. Zhang L, Zhang C, Wang A (2016) Divergence and conservation of the major UPR cending sensory neurons in gracile axonal dystrophy mutant mouse. Acta Neuropathol branch IRE1-bZIP signaling pathway across . Sci Rep 6:27362. 80:145–151. 27. Hendershot LM, Valentine VA, Lee AS, Morris SW, Shapiro DN (1994) Localization of 54. Yokota T, et al. (2001) Delayed-onset ataxia in mice lacking alpha -tocopherol transfer the gene encoding human BiP/GRP78, the endoplasmic reticulum cognate of the protein: Model for neuronal degeneration caused by chronic oxidative stress. Proc HSP70 family, to 9q34. Genomics 20:281–284. Natl Acad Sci USA 98:15185–15190. 28. Simons JF, Ferro-Novick S, Rose MD, Helenius A (1995) BiP/Kar2p serves as a molecular 55. Vizcaíno JA, et al. (2016) 2016 Update of the PRIDE database and its related tools. chaperone during carboxypeptidase Y folding in yeast. J Cell Biol 130:41–49. Nucleic Acids Res 44:D447–D456. 29. Stolz A, Wolf DH (2010) Endoplasmic reticulum associated protein degradation: A 56. Deutsch EW, et al. (2017) The ProteomeXchange Consortium in 2017: Supporting the chaperone assisted journey to hell. Biochim Biophys Acta 1803:694–705. cultural change in proteomics public data deposition. Nucleic Acids Res 45:D1100–D1106. 30. Guertin DA, Sabatini DM (2009) The pharmacology of mTOR inhibition. Sci Signal 2: 57. Borodovsky A, et al. (2002) Chemistry-based functional proteomics reveals novel pe24. members of the family. Chem Biol 9:1149–1159.

7972 | www.pnas.org/cgi/doi/10.1073/pnas.1812413116 Reinicke et al. Downloaded by guest on October 1, 2021