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Elucidation of MBNL1 function in the nervous system of myotonic dystrophy type 1

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Elucidation of MBNL1 function in the nervous system of myotonic dystrophy type 1

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Content Elucidation of MBNL1 function in the nervous system of myotonic dystrophy type 1 By Yuntong Liu A Thesis Presented to the FACULTY OF THE USC KECK SCHOOL OF MEDICINE UNIVERSITY OF SOUTHERN CALIFORNIA In partial Fulfilment of the Requirement for the Degree MASTER OF SCIENCE (BIOCHEMISTRY AND MOLECULAR MEDICINE) August 2022 Copyright 2022 Yuntong Liu i ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to my mentor, Dr. Lucio Comai. This thesis would not be completed without his patience, continued guidance, and invaluable feedback throughout the project. I would also like to extend my sincere thanks to my thesis committee members, Dr. Sita Reddy and Dr. Judd Rice for their insightful comments and suggestions. Thanks to Dr. Parvin Valiulahi for her patience in training me in the experimental techniques and in discussing the details of the protocols. And thanks to Yilin Liu for a cherished time spent together in the lab. Lastly, I thank my parents and my friend, Shuyu Fang, for giving me all the emotional support. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ..................................................................................................................... i LIST OF FIGURES ............................................................................................................................... iii ABSTRACT ........................................................................................................................................... iv Introduction ............................................................................................................................................. 1 Methods and Materials ............................................................................................................................ 5 Results ................................................................................................................................................... 10 Discussion ............................................................................................................................................. 18 References ............................................................................................................................................. 22 iii LIST OF FIGURES Fig. 1 Immunoprecipitation of MBNL1 interacting partners from mouse brain. ................................. 11 Fig. 2 Function of MBNL1 interacting partners. .................................................................................. 11 Fig. 3 Cytoplasmic and nuclear extraction from HT22 cell. ................................................................. 13 Fig. 4 Cytoplasmic and nuclear extraction from HT22 cell using commercial kit. .............................. 13 Fig. 5 Vector construct containing 3xFLAG-MBNL1Δ5. .................................................................... 15 Fig. 6 FLAG-MBNL1Δ5 expression in HT22 cell. .............................................................................. 15 Fig. 7 FLAG-MBNL1Δ5 expression in cytoplasmic and nuclear fractions of HT22 cell. ................... 15 Fig. 8 Neurite outgrowth of mouse neuronal cell HT22. ...................................................................... 17 iv ABSTRACT Myotonic dystrophy type 1 (DM1) is an autosomal dominant, multi-system disorder. DM1 patients develop muscular dystrophy as well as neurological symptoms. The disease is primarily caused by the expansion of trinucleotide CTG repeats in the 3’ untranslated region (UTR) of the DMPK gene which sequesters the RNA-binding muscleblind-like protein 1 (MBNL1) in the mutant transcript. This MBNL1 has confirmed function in regulating alternative splicing. Other suggested roles of MBNL1 in the cytoplasm are RNA transport, translation, and miRNA processing. However, how the loss of MBNL1 function results in neurodegeneration is poorly understood. In this study, we set up multiple systems to study the role of MBNL1 in the nervous system. We identified MBNL1 interacting partners in the mouse brain using immunoprecipitation (IP) to elucidate its functional mechanism. The interacting protein identified in the experiment have roles in splicing, translation, and transportation. Since the cytoplasmic MBNL1 isoform (MBNL1Δ5) plays a role in neurite outgrowth, we optimized the protocol to separate the nuclear and cytoplasmic proteins to specifically study the function of this isoform. Alternatively, the FLAG-tagged MBNL1Δ5 was transduced into mouse hippocampal neuronal cells HT22. The transduced cells showed more rapid neurite outgrowth than control cells upon differentiation. This cell line thus offers a cell model to gain novel information on the role that this MBNL1 isoform plays in neuron development. Altogether, this project contributes to understanding the mechanisms of neuronal dysfunction in DM1. 1 Introduction Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder. With prevalence from 1/8,000 in Caucasians to 1/20,000 in Asian, Japanese, and African populations, it is the most common muscular dystrophy in adults (Foff & Mahadevan, 2011). The genetic basis of DM1 is related to the expansion of the CTG trinucleotide repeats in the 3’ untranslated region (3’ UTR) of dystrophia myotonia protein kinase gene (DMPK) on chromosome 19 (Brook et al., 1992). Normally, people have 3 to 37 of the CTG repeats in this gene. In contrast, DM1 patients have 50 to several thousand of the repeats (Brook et al., 1992). The severity of the disease increases with an increase in repeat size and as the repeat is inherited, the disease severity increases from one generation to the next (Harley et al., 1993). The toxicity of this mutation is not from the decrease in DMPK protein level and function, but from the production of the mutant transcript. The mRNA with expanded CTG repeats forms aggregates in the nuclei which sequester RNA-binding proteins causing their loss of functions in the downstream events (Ranum & Day, 2004). Especially, a class of protein called muscleblind- like protein (MBNL) is found to colocalize with the expanded CUG repeats (Mankodi et al., 2001; Miller et al., 2000). Several studies using Mbnl knockout mouse models have reproduced characteristic DM1 pathologies in muscle, eye, heart, and central nervous system (CNS) (Charizanis et al., 2012; J. Choi et al., 2016; Dixon et al., 2015; Du et al., 2010; Kanadia et al., 2003), supporting the MBNL loss-of-function mechanism in DM1 pathology. The RNA-binding family of muscleblind-like proteins (MBNLs) is evolutionarily conserved and consists of three paralogs, MBNL1, MBNL2, and MBNL3, which are encoded by three different genes. Each of these proteins possesses four zinc-finger motifs that recognize the consensuses sequence in their RNA targets. They are expressed across many tissues and are known in mediating alternative splicing in the nucleus (Kanadia et al., 2003; Pascual, Vicente, Monferrer, & Artero, 2006). Since MBNLs are present in both the nucleus and the 2 cytoplasm of cells, additional functions in regulating precursor and mature RNA metabolism including RNA transport and miRNA processing are suggested (Batra et al., 2014; Rau et al., 2011). Previous studies from our lab and others have suggested that MBNL1 as the primary determinant of DM1 focus formation in DM1 cells and has the highest mobility to expanded CUG RNA (Dansithong, Paul, Comai, & Reddy, 2005; Sznajder et al., 2016). It forms a ring- like structure which binds to the hairpin structure in double-stranded CUG and mediates homotypic interactions with its C-terminal region to stabilize intra- and/or inter-ring interactions (Yuan et al., 2007). Its importance in DM1 pathology is further supported by experiments showing that rescue of splice defects, a key molecular feature of DM1 patients, can be achieved by releasing MBNL1 from the expanded repeats to restore normal functional MBNL1 levels in DM1 mouse models (Warf, Nakamori, Matthys, Thornton, & Berglund, 2009; Wheeler et al., 2009). Significantly, a splicing microarray study demonstrated that of the more than 200 mis-spliced targets in CUG-expressing DM1 mouse models, over 80% of these events are attributed to loss of MBNL1 function (Du et al., 2010). DM1 is a multi-system disease that affects various organs in addition to skeletal muscle. Typical symptoms include myotonia, muscle weakness and wasting, cardiac conduction defects, cataracts, endocrine abnormalities, etc. There are also extensive data demonstrating impairment of the CNS in DM1, as patients show various levels of mental retardation, excessive daytime sleepiness, cognitive decline, intelligence and verbal impairments, depression, apathy, and avoidant personality (Abe et al., 1994; de Leon & Cisneros, 2008; Meola et al., 2003). Magnetic resonance imaging (MRI) of DM1 patients’ brain has shown abnormal structural integrity with generalized brain atrophy and an increase in ventricular space (Hashimoto et al., 1995; Okkersen et al., 2017). Both widespread gray and white matter reductions are also found in DM1 patients’ brains (Di Costanzo, Di Salle, Santoro, Bonavita, & Tedeschi, 2002; Minnerop, Gliem, & Kornblum, 2018; Okkersen et al., 2017). In contrast to 3 the substantial understanding of muscular pathology, the molecular events leading to alterations in the nervous system of DM1 patients still need to be investigated. Several studies have identified MBNL1 as a key regulator of neuron development in DM1. A behavioral study in mouse demonstrated that mice with decreased MBNL1 levels exhibit cognitive and behavioral abnormalities in open field and maze tasks (Matynia et al., 2010). In agreement with these findings, profound structural alterations have been observed in mice with depleted levels of MBNL1 and MBNL2 (Maria et al., 2021). Another study has observed cortical neuron distributional defects, dendritic complexity reduction, and postsynaptic density alterations in Mbnl1/2 knockout mice (Lee, Chang, Seah, & Lee, 2019). Moreover, synapse formation was dysregulated that distal neuromuscular junction (NMJ) was lost in motor neurons was shown in C. elegans lacking neuronal MBNL homolog (Spilker, Wang, Tugizova, & Shen, 2012). Cell-based studies demonstrated reduced axon and dendrite outgrowth in cultured primary neurons with reduced MBNL1 levels that are similar to those shown in neuronal cells expressing expanded CUG RNA (P. Y. Wang, Chang, Lin, Kuo, & Wang, 2018). Interestingly, this study also suggested that deubiquitination of MBNL1 by expanded CUG RNA may be involved in pathogenesis. However, the precise role of MBNL1 in neuron development is not fully understood. To investigate how loss of MBNL1 results in neurodegeneration, I pursued the identification of MBNL1 interacting partners in mice brains and neuronal cells. The idea is that these interacting proteins will provide clues on the neuronal pathways in which MBNL1 functions. For this purpose, proteins bound to MBNL1 were immunoprecipitated from extracts prepared from mice brains and subsequently identified by mass spectrometry. As an alternative approach, I attempted to isolate nuclear and cytoplasmic fractions from either mice brains or neuronal cells in culture. The rationale for this approach is because MBNL1 is reported to have 9 different isoforms generated from alternative splicing (Fardaei et al., 2002). The inclusion or 4 exclusion of exon 5 in MBNL1 is suggested to determine whether MBNL1 localized in the nucleus or cytoplasm, respectively (Dhaenens et al., 2008; Terenzi & Ladd, 2010). Since the functions of MBNL1 in the nucleus and cytoplasm are likely distinct, the identification of proteins that specifically interact with the cytoplasmic isoform (MBNL1Δ5) is predicted to provide novel information on the function of this protein in non-nuclear processes. Lastly, I generated a hippocampal cell line that expressed an epitope tagged MBNL1Δ5 to study how MBNL1 influences neuronal differentiation. Collectively, my experiments provide a preliminary understanding of the role of MBNL1 in neurodegeneration and establish a cell culture system that mimics neurite outgrowth, which will be used to further examine the function of MBNL1 in the cytoplasm. 5 Methods and Materials Cell culture. The HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin solution (P/S). Mouse hippocampal neuronal HT22 cells were cultured in DMEM supplied with 10% FBS, 1% P/S, and 1% L-glutamine. All cells were incubated at 37℃ with 5% CO2. Plasmid preparation. The N-terminus 3xFLAG-tagged MBNL1Δ5 vector with CBh promotor was designed in the lab and procured through VectorBuilder. The 2 nd generation lentiviral packaging plasmid, envelope plasmid, and transfer plasmid were separately transformed into DH5α competent E. coli by heat shock method for mass production. These plasmids were then extracted and prepared using Plasmid Midi Kits from Qiagen. Restriction enzymes EcoRV-HF (New England Bio-labs, R3195) and HincII (New England Bio-labs, R0103) were used to test the FLAG-MBNL1Δ5 construct. Both enzymes required incubation at 37℃ for 1h and deactivation at 65℃ for 20min in rCutSmart™ Buffer (New England Bio-labs, B6004), as indicated in the manufacturer’s protocol. The digested fragments were separated in 1% agarose gel. Lentivirus production and transduction. The lentivirus with 3xFLAG-tagged MBNL1Δ5 or EGFP was produced using 2 nd generation system in HEK293T cells. Before transfection, HEK293T cells were incubated in DMEM with 10% HyClone FBS (Thermo Scientific). The transfection used 1mg/ml polyethyleneimine in DMEM and 1x Tris-EDTA buffer with 7µ g transfer plasmid, 7µ g packaging plasmid, and 7µ g envelope plasmid. After 16h, 10mM sodium butyrate was added to the DMEM containing 10% HyClone FBS to increase the efficiency of transfection and 6 expression(Goding & Russell, 1983). The medium was changed to DMEM with 10% HyClone FBS 8h later and collected after 24h. The collected medium was filtered through a 0.45µ m filter to obtain the lentivirus for transduction. To generate the HT22 cell line expressing 3x FLAG-tagged MBNL1Δ5 or EGFP, HT22 cells were incubated with the lentivirus-containing medium for 1h at 37℃. 48h post- transduction, the HT22 cells were selected with 1µ g/ml puromycin. The cells were kept in selection medium for 9 days. The EGFP expression was checked using an all-in-one fluorescence microscope KEYENCE BZ-X710. FLAG-MBNL1Δ5 expression was confirmed by Western blot using an anti-FLAG antibody. Total protein extraction and subcellular fractionation. A freshly dissected mouse brain was flash-freezed and pulverized in liquid nitrogen. Total proteins extract was prepared by incubating the tissue powder in lysis buffer containing modified RIPA buffer (50mM Tris-HCl pH7.4, 150mM NaCl, 0.25% deoxycholic acid, 1% NP-40) with 1mM EDTA pH8.0, 1mM NaF, 1mM Na3VO4 1mM phenylmethylsulfonyl fluoride (PMSF), and 1x protease inhibitor cocktail (PI) (Sigma-Aldrich) for 1h at 4℃ while rotating. Proteins were recovered in the supernatant after centrifugation for 30min at 13,000rpm. Cells were harvested and washed in cold 1x Dulbecco’s phosphate-buffered saline (DPBS) and lysed in modified RIPA buffer with 1mM EDTA, 1mM dithiothreitol (DTT), 1mM PMSF, and 1x PI. The mixture was incubated for 30min at 4℃ on a rotator and centrifuged at 13,000rpm for 30min to obtain the whole-cell protein. For subcellular fractionation, the cytoplasmic and nuclear protein was extracted in low salt and high salt buffers. The low salt buffer had 10mM Tris-HCl pH7.5, 1mM MgCl2, 1mM EDTA, 1mM DTT, 1mM PMSF, and 1x PI. The high salt buffer added 420mM NaCl and 10% 7 glycerol to the low salt buffer. The cell pellet was incubated in a low salt buffer first to obtain cytoplasmic fraction. The pellet remaining after centrifugation was incubated with high salt buffer and centrifuged to extract nuclear protein. Another protocol used ice-cold 1x DPBS as lysis buffer and extracted cytoplasmic proteins by passed cell through a 25-gauge needle. In this protocol, the nuclear fraction was obtained by incubating the remaining cell pellet in RIPA buffer. The Boster’s cytoplasmic and nuclear protein extraction kit (AR0106) was also used, and the manufacturer’s protocol was followed. Protein concentrations were determined using the Pierce BCA protein assay kit (Thermo Scientific). Immunoprecipitation assay (IP). To reduce non-specific binding in immunoprecipitation, control mouse IgG (Santa Cruz sc-2025) was added to the protein extract, together with suspended (25%v/v) protein A-agarose (Invitrogen 10-1141). This mixture was incubated at 4℃ for 30min and centrifuged at 3,000rpm for 30s at 4℃. 5% supernatant was saved as input for SDS-PAGE. The rest of the supernatant was incubated with ribonuclease A (Sigma) and anti-MBNL1(3A4) conjugated agarose (Santa Cruz sc-47740) at 4℃ overnight with rotating. Other antibody conjugated beads used for IP were anti-FLAG M2 affinity gel (Sigma A2220) and anti-FLAG M2 in TH/0.1 + NaN3, in PBS. The immunoprecipitants were collected by centrifugation at 3,000rpm for 30s at 4℃ and supernatant was saved. The pellet was washed 5 times using modified RIPA buffer with 1mM DTT and 1mM PMSF for 10min at 4℃ with rotating. The bound proteins were eluted from the MBNL1-agarose beads using BCO buffer containing 20mM Tris-HCl pH 8.0, 0.5mM EDTA pH8.0, 20% glycerol, 1M KCl, 1% sodium deoxycholate, 1mM DTT, and 0.5mM PMSF. The beads were removed by centrifugation at 3,000rpm for 30min at 4℃. Proteins were precipitated by 100% trichloroacetic acid and 0.4% deoxycholic acid. They were washed with 100% acetone, air-dried, and suspended in sample 8 buffer. The eluted proteins were subjected to SDS-PAGE and Western blot analyses. For protein identification, the gel was sent to Dr. James Wohlschlegel’s lab at the University of California, Los Angeles (UCLA, Los Angeles, CA) to perform liquid chromatography tandem mass spectrometry (LC-MS/MS). The identified proteins were analyzed by QIAGEN ingenuity pathway analysis (IPA). Western blot analysis (WB). Proteins were resolved by 10% SDS-PAGE and transferred to Whatman TM Protran BA 85 nitrocellulose membranes (GE Healthcare Life Sciences) using semi-dry transfer. The membranes were blocked with 5% skim milk in 1x TBST (10mM Tris-HCl pH 7.5, 100mM NaCl, 0.05% Tween-20) for 1h at room temperature. The primary antibodies used were mouse MBNL1 (MB1a) monoclonal antibody (1:100, gift from Ian Holt), mouse GAPDH monoclonal antibody (100µ g/ml, 1:2000, Santa Cruz), rabbit LAMIN A/C polyclonal antibody (200µ g/ml, 1:2000, Santa Cruz), mouse DYKDDDK (FLAG) tag monoclonal antibody (500µ g/ml, 1:3000, Proteintech). They were incubated with the membranes overnight at 4℃. The membranes were then washed with 1x TBST and incubated with corresponding secondary antibodies, anti- mouse IgG HRP conjugate (1mg/ml, 1:2500, Promega) or anti-rabbit IgG HRP conjugate (1mg/ml, 1:2500, Promega), for 1h at room temperature, followed by washing in 1x TBST. Signal was detected on Chemi TM Doc MP imaging system (Bio-Rad) with Pierce TM ECL plus Western blotting substrate (Thermo Scientific). Signal intensities were analyzed using Fiji (Schindelin et al., 2012). Cell differentiation. For the differentiation of HT22 cells, cells were maintained in NeuroBasal medium (Gibco) with 1% L-glutamine and 100mM cyclic AMP (cAMP). Cells were cultured in the differentiation medium for 2 days. Images of cells were taken under phase-contrast microscopy 9 using KEYENCE BZ-X710 with the same settings. Cell differentiation data are presented as mean ± standard error and analyzed by unpaired two-tailed Student’s t-test assuming equal variance. The statistically significant difference between groups was considered as p<0.05. 10 Results Immunoprecipitation of MBNL1 interacting partners from mouse brain. In order to elucidate the function of MBNL1, proteins that interact with MBNL1 in the brain were pulled down using IP. The experimental conditions for the detection and immunoprecipitation of MBNL1 from mouse brain were optimized in a small scale/pilot experiment (Fig. 1A). Samples of protein from pre-cleared extract (input) and supernatant after IP (sup) were loaded to the gel as control. In this experiment, a small amount of MBNL1 was not precipitated as shown in the supernatant lane on the Western blot membrane at 42kD. The two intense bands at 50kD and 25kD are heavy chain and light chain of the dissociated antibody. In collaboration with Dr. Parvin Valiulahi, a lab member, the large-scale isolation of MBNL1- interacting proteins from wildtype (WT) mouse whole brain was performed using 50mg of protein extracts and 25µ g MBNL1-conjugated agarose. The protein extract was treated with RNase to exclude proteins that were indirectly bound to MBNL1. As a control, the same immunoprecipitation experiment was conducted with mouse IgG and protein-A-agarose instead of MBNL1-agarose. An elution step was included in this experiment after IP. The elution buffer was optimized in the lab to only elute proteins bound to MBNL1 so that the antibody and most of MBNL1 would remain attached to the agarose beads. Eluted proteins were separated by SDS-PAGE and the Coomassie-stained gel is shown in Fig. 1B. From IgG IP, no intense bands were detected after elution. The α-MBNL1 elution lane contained all the MBNL1 interacting proteins. As compared to small-scale IP results, the antibody bands did not produce interfering signals after elution. All visible bands in the α-MBNL1 elution lane were cut out and sent for LC-MS/MS by Dr. Wohlschlegel’s team at UCLA. The MS identification for proteins from 60kD to 140kD was obtained and analyzed by the number of peptides detected in LC-MS/MS and IPA. MBNL1 was found to interact with proteins both in the nucleus and in the cytoplasm. These proteins function as splicing factors, transcription 11 regulators, translation regulators and transporters. The summarized result was presented in Fig. 2. A B Fig. 1 Immunoprecipitation of MBNL1 interacting partners from mouse brain. Protein extracts were obtained from freshly dissected WT mouse’s whole brain. Protein extracts were pre-cleaned with mouse IgG (input) and incubated with antibody-conjugated beads overnight at 4℃. The protein-bead complexes were collected by centrifugation and the supernatant was saved (sup). The immunoprecipitants were washed before processing. A. Small-scale immunoprecipitation to test protocol and antibody. 1mg of WT mouse brain protein was used. The MBNL1 interacting partners attached to the beads (IP) were separated by SDS-PAGE and analyzed by Coomassie staining and WB. Left: Coomassie-stained gel; Right: WB. B. Optimized large-scale immunoprecipitation with elution for protein identification. 50mg of protein extracts were used and treated with RNase after pre-cleared by IgG. MBNL1 interacting proteins were eluted after IP and separated by SDS-PAGE followed by Coomassie staining. In all IP experiments, 5% of input and supernatant were used for SDS-PAGE. Fig. 2 Function of MBNL1 interacting partners. Interacting partners of MBNL1 were immunoprecipitated and identified by LC-MS/MS. Based on the number of peptides of the protein detected in MS and IPA, the important interacting proteins were selected and grouped by their functions and locations. Representation proteins are included in this chart. 12 Subcellular fractionation of HT22 cell. The in vitro approach was intended to study the function of the cytoplasmic isoform of MBNL1. As the first step, distinct protocols were tested to separate nuclear and cytoplasmic protein from mouse hippocampal neuronal HT22 cells. These protocols varied in incubation time, centrifugation condition, and lysis buffer content. The cell fractions were analyzed by Western blot that LAMIN A/C and GAPDH were used as nuclear and cytoplasmic markers, respectively. Selected results are presented in Fig. 3. Fig. 3A and 3B were results of the same low salt and high salt extraction. Fig. 3B was a modification with which the incubation time and centrifugation speed were decreased. Fig. 3C was the result of extraction using 1x DPBS. This protocol had enrichment of cytoplasmic protein in the cytoplasmic fraction. In all experiments, both the nuclear and the cytoplasmic fractions had LAMIN A/C and GAPDH presence. None of the protocols showed apparent improvement in separating the nuclear and cytoplasmic fractions. The result of nuclear and cytoplasmic protein separation using the Boster’s kit is shown in Fig. 4. LAMIN A/C still existed in both fractions, but GAPDH only presented in the cytoplasmic fraction. 13 HT22 cell line expressing FLAG-tagged MBNL1Δ5 As an alternative approach to studying the MBNL1 cytoplasmic isoform, FLAG-tagged MBNL1Δ5 was introduced into the WT HT22 cell. The map of the vector containing FLAG- MBNL1Δ5 is shown in Fig. 5A. The recombinant protein expression is driven by the CBh promoter. To validate the construct, restriction digestion was performed using EcoRV and HincII. Each enzyme has only one restriction site on the plasmid at 2828bp and 3617bp (within Fig. 3 Cytoplasmic and nuclear extraction from HT22 cell. HT22 cells were washed and harvested in A-B. 1x DPBS, C. 1x DPBS with 1mM PMSF. The cell pellet was obtained by centrifugation at A-B. 3,000rpm for 5min, at 4℃. Cells were not pelleted in C. Pellet from A was washed in ice-cold 1x DPBS with 5mM MgCl 2 and centrifuged at 3,000rpm for 5min at 4℃. A-B. To extract cytoplasmic proteins, the pellet was resuspended in 5x pellet volume of low salt buffer and incubated at 4 ℃ for 10min. C. The cell was passed through a 25-gauge needle 10 times and left on ice for 20min. The cytoplasmic extract was collected in the supernatant after centrifugation at 4℃ for A. 3,500rpm for 10min, B. 3,000rpm for 5min, C. 13,000rpm for 10min. A washing step was included for A-B using 3x pellet volume of low salt buffer and centrifuged at 4℃ for A. 4,000rpm for 7min, B. 3,000rpm for 7min. The nuclear proteins were extracted by incubating the remaining pellet at 4℃ in A-B. 3x pellet volume of high slat buffer for 30min, C. modified RIPA buffer with 0.1%SDS and 1mM PMSF. After centrifuging for 13,000rpm at 4℃ for A-B. 30min, C. 2min, the nuclear fraction was obtained as the supernatant. The same amount of protein was loaded to every lane for each blot. WB: anti-LAMIN A/C (69/62kD) for top panels, anti-GAPDH (37kD) for bottom panels. A C B Fig. 4 Cytoplasmic and nuclear extraction from HT22 cell using commercial kit. The Boster’s cytoplasmic and nuclear protein extraction kit was used in this experiment. The same amount of protein was loaded into each lane. WB: α-LAMIN A/C (69/62kD) for top panels, α-GAPDH (37kD) for bottom panels. 14 the insert), respectively. The digestion product was separated by agarose gel electrophoresis (Fig. 5B). Two bands around 8500bp and 800bp were visible on the gel, matching the expected fragment sizes (8543bp and 789bp) of the double digested plasmid. The HT22 cell line expressing FLAG-MBNL1Δ5 plasmid was then generated by lentivirus transduction. Puromycin-selected cells were tested for expression with an anti-FLAG antibody using Western blot (Fig. 6A). FLAG was detected from protein extract of these cells whereas no signal appeared from that of the WT HT22 cells. To quantify the MBNL1 level, total MBNL1 expression was analyzed by WB using an anti-MBNL1 antibody. The expression level was normalized to the GAPDH level and compared between transduced cell and WT cell (Fig. 6B-C). Since MBNL1 has several isoforms, multiple bands were detected by an anti- MBNL1 antibody with several bands from 40-43kD. From transduced HT22 cells, the top band became prominent. However, it was insufficient to discriminate between the MBNL1 isoforms. In general, the anti-MBNL1 WB result showed higher signal intensity from the transduced cell sample compared to the WT cell sample. Accordingly, the FLAG-MBNL1Δ5 cell sample showed a higher normalized MBNL1 level. The FLAG expression was also analyzed in the cytoplasmic and nuclear fractions of the FLAG-MBNL1Δ5 expressing HT22 cells (Fig. 7). Using the commercial subcellular fractionation kit, the nuclear fraction was free of cytoplasmic protein, but the cytoplasmic fraction had nuclear protein. Although FLAG was detected in both fractions, the cytoplasmic extract had a more intense FLAG-MBNL1Δ5 band. 15 A B Fig. 5 Vector construct containing 3xFLAG-MBNL1Δ5. A. Map of the vector with 3xFLAG-MBNL1Δ5 (green). The picture was created with SnapGene. B. Restriction digestion of the plasmid by EcoRV and HincII. Restriction sites were at 2828bp and 3617bp. Expected fragment sizes were 8543bp and 789bp. The fragments were loaded to the digested lane and indicated by the black arrow. Fig. 6 FLAG-MBNL1 Δ5 expression in HT22 cell. An equal amount of whole cell extract was loaded to each lane for SDS-PAGE. Western blot was performed to detect the proteins. A. The recombinant protein was expressed in the HT22 cell line. WB: anti-FLAG. B. Comparison of MBNL1 expression level between HT22 WT cells and FLAG-MBNL1Δ5 cells. Top panel: anti- MBNL1. Bottom panel: anti-GAPDH. C. Total MBNL1 level was normalized with respect to GAPDH level for comparison. B A C α-FLAG Fig. 7 FLAG-MBNL1Δ5 expression in cytoplasmic and nuclear fractions of HT22 cell. HT22 cytoplasmic and nuclear fractions were prepared using the Boster’s kit. An equal amount of protein was loaded to each lane. WB: anti-LAMIN A/C for the top panel, anti-GAPDH for the middle panel, anti-FLAG for the bottom panel. 16 HT22 differentiation With the new HT22 cell line, the effect of MBNL1Δ5 overexpression on neuron growth was studied. Cultured HT22 cells reached 40% confluency in normal medium before the differentiation process. After the medium was changed to differentiation medium, neurite outgrowth was monitored for 2 days. Images of cells were taken before differentiation (D0), at 24h after differentiation (D1), and at 48h after differentiation (D2). The length of the longest neurite from each cell was recorded for further analysis. From the images, it was observed that the FLAG-MBNL1Δ5 cells survived better than the other two cell lines during differentiation (Fig. 8A). The cells with EGFP had longer neurites before differentiation. The measurement data agreed with this observation (D0 average neurite length: EGFP: 45.26µ m, WT: 35.32µ m, FLAG-MBNL1Δ5: 37.51µ m). For comparison, the neurite length was normalized to the average neurite length at D0 and presented as a relative length in Fig. 8B. At D1, the relative neurite length of FLAG-MBNL1Δ5 cells was significantly different than that of the other two cell lines. At D2, the relative length of FLAG-MBNL1Δ5 cells showed no significant difference with WT cells but was significantly different from that in EGFP cells (p=0.0756, p=1.81*10 -4 ). The relative neurite length of WT HT22 cells and HT22 cells with EGFP were not significantly different at D1 or D2. The other quantification method noted the percentage of cells that had neurite longer than the D0 mean in each cell line (Fig. 6C). No significant difference existed between cell lines at D0 or D2. At D1, the difference in the percentage of cells was observed between FLAG-MBNL1Δ5 cells and the other two cell lines. 17 A Fig. 8 Neurite outgrowth of mouse neuronal cell HT22. A. Representative pictures of transduced and WT HT22 cells taken before differentiation, at 24h, and at 48h. Same settings were applied in phase-contrast microscopy for all photos. The enlarged picture showed example of neurite length measurement in red. B. Relative average length of neurite in each cell line. The longest neurite of each cell was recorded in the experiment. The length was normalized to D0 mean of each cell line for comparison. Cells were randomly selected and photographed for each cell line. C. Percent of cells that had neurite length longer than the average neurite length at D0. *p<0.05, **p<0.01. B C 18 Discussion In this study, I explored the role of MBNL1 in the nervous system by pursuing the identification of the binding partners of MBNL1 in the brain. Since the protein extracts were treated with RNase, these proteins are thought to interact directly with MBNL1. The IP results show that MBNL1 binds to proteins known to play roles in a variety of cellular processes (Fig. 2). Notably, some of these MBNL1 interacting proteins have been linked to neurodegeneration and/or neurological symptoms (Lim, James, Huang, & Lee, 2020; Zatsepina, Evgen'ev, & Garbuz, 2021). As anticipated, MBNL1 also interacts with many splicing factors in the nucleus. In the cytoplasm, a large amount of MBNL1 interacting proteins are parts of translation machinery, hinting at a potential new role of MBNL1 in translation. These results suggest that loss of MBNL1 function could affect several nuclear and cytoplasmic processes in the nervous system and consequently be directly implicated in neurodegeneration and brain structural abnormality in DM1. Using the same approach, another project in the lab identified the interacting protein partners of MBNL2 from mouse brains (unpublished). We compared the results between these two data sets and noted that the majority of the proteins that interact with MBNL1 also interact with MBNL2. Since MBNL1 and MBNL2 are members of the same protein family and are thought to have overlapping roles in the cell (Lee et al., 2013; Thomas et al., 2017; E. T. Wang et al., 2012), the finding that the almost identical set of interacting proteins are identified in MBNL1 and MBNL2 immunoprecipitation, gives us confidence on the biological relevance of these interactions. Nonetheless, the interactions between these proteins and MBNL1 (and MBNL2) need to be confirmed by co-IP assays. In the in vitro experiment, the separation of nuclear and cytoplasmic fractions from HT22 cells was unsuccessful using the low salt/high salt buffer or the DPBS protocols (Fig. 3). A possible explanation for these results is that the nuclei of HT22 cells are very fragile and need to be handled with extra care. I attempted to use a commercial fractionation kit and was 19 able to obtain a nuclear fraction free of any cytoplasmic contamination. However, the cytoplasmic fraction was contaminated with nuclear proteins (Fig. 4). Others have successfully separated the nuclear and cytoplasmic fractions using SH-SY5Y cells, LNCAP cells, and CRC cells (Bai et al., 2021; Chen et al., 2021; J. N. Wang et al., 2020). Referring to the manufacturer’s troubleshooting guide, a lower incubation time and longer vortex time may yield better separation. It is also possible that some nuclei did not pellet in the centrifugation and were collected with the cytoplasmic supernatant during separation. As discussed above, the aim of nuclear and cytoplasmic proteins separation is to study the function of the cytoplasmic isoform of MBNL1 in neuron development. Even though the separation remains to be optimized, it is still useful doing MBNL1 IP from the whole cell extract of this hippocampal neuronal cell line because any (or most) identified protein will have a known localization in the cell. It would also be interesting to compare the profiles of proteins co- immunoprecipitating with MBNL1 before and after neuron differentiation to gain new insights on its function in this process. In the alternative approach to study the cytoplasmic isoform of MBNL1, I generated an HT22 cell line expressing FLAG-MBNL1Δ5. HT22 cells are a mouse hippocampal neuronal cell line subcloned from the HT4 cell line (Davis & Maher, 1994). HT22 cells possess essential properties similar to those of immature hippocampal neuronal precursor cells and mature hippocampal neurons and have been used as a model system to study the neurodegenerative Alzheimer's disease (A. Y. Choi et al., 2010; He, Liu, Cheng, Xing, & Suo, 2013; Liu, Li, & Suo, 2009; Zhao et al., 2012). Before lentiviral transduction, it was confirmed that the FLAG- MBNL1Δ5 vector had the correct insert (Fig. 5B). After transduction and selection, the FLAG- MBNL1Δ5 HT22 cell line showed a mild increase in overall MBNL1 expression (Fig. 6C). By western blot analysis using anti-FLAG antibody, I detected FLAG-MBNL1Δ5 in the cytoplasmic and nuclear extracts from this cell line had, but the signal was much stronger in 20 the cytoplasmic fraction (Fig. 7). Because the cytoplasmic fraction I prepared using the protocol described above contains nuclear proteins, I will have to perform additional experiments to confirm that the majority of MBNL1Δ5 localizes into the cytoplasm. On the other hand, the nuclear extract was not contaminated by cytoplasmic protein, and we could infer that the MBNL1Δ5 isoform is not solely, but mostly expressed in the cytoplasm. A previous study that utilized immunofluorescence to determine the localization of MBNL1Δ5 found that this protein is primarily cytoplasmic (Kino et al., 2015; P. Y. Wang et al., 2018). Once it is confirmed that FLAG-MBNL1Δ5 is primarily cytoplasmic in the HT22 cell line, immunoprecipitations using anti-FLAG antibody can be used to identify cytoplasmic protein partners of MBNL1 in undifferentiated and differentiated HT22 cells. Changes in the identity of MBNL1Δ5 interacting proteins before and after differentiation could provide clues on the pathways that are involved in this process and elucidate the specific function of this isoform in neuron development. In the neurite outgrowth experiment, the overexpression of MBNL1Δ5 promoted neurite outgrowth of differentiated HT22 cells as shown in D1 (Fig. 8B). A better understanding of why ectopic expression of MBNL1Δ5 leads to enhanced outgrowth and cell survival could be gained by the identification of MBNL1 interacting partners. Our preliminary data suggest that MBNL1 interacts with translation regulators and transporters in the cytoplasm. The increase in the levels of cytoplasmic MBNL1 could affect translation and transport processes favoring differentiation and the outgrowth of the neurite. Overexpression of MBNL1Δ5 was found to rescue the defects in axon outgrowth and dendrite development in DMPK-CUG 960 neurons in a dose-dependent manner (P. Y. Wang et al., 2018). The neurite length at D2 was not significantly different between FLAG-MBNL1Δ5 HT22 cell line and WT HT22 cell lines. Contrary to this result, the cultured primary neuron cell with MBNL1Δ5 in another study shows significantly longer dendrite and axon when differentiated (P. Y. Wang et 21 al., 2018). It is reported that the inclusion of exon 5 in MBNL1 is higher in most brain areas in DM1 patients (Nishi et al., 2020), supporting an effect of MBNL1Δ5 in neuron development. The neurite outgrowth in our experiment could be limited by the plate size and cell density that the cells reached maximum growth capacity after 2 days. Conversely, another study conducted in our lab silenced endogenous MBNL1Δ5 in human neuroblastoma SH-SY5Y cells by shRNA. A decrease in neurite outgrowth rate was not observed (unpublished). The effect of MBNL1Δ5 on neuron differentiation could be further evaluated by comparing the number of differentiated and undifferentiated cells in each cell line with differentiation markers. Moreover, it is observed that the cells overexpressing MBNL1Δ5 had reduced levels of cell death. The common cause of neuronal cell death in many neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, is due to glutamate-induced cytotoxicity or oxidative stress (Ambrosi, Cerri, & Blandini, 2014; Arundine & Tymianski, 2004; Doria et al., 2013; Lau & Tymianski, 2010; Olney & de Gubareff, 1978). MBNL1Δ5 may be involved in the pathway of glutamate regulation. 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Abstract (if available)

Abstract Myotonic dystrophy type 1 (DM1) is an autosomal dominant, multi-system disorder. DM1 patients develop muscular dystrophy as well as neurological symptoms. The disease is primarily caused by the expansion of trinucleotide CTG repeats in the 3’ untranslated region (UTR) of the DMPK gene which sequesters the RNA-binding muscleblind-like protein 1 (MBNL1) in the mutant transcript. This MBNL1 has confirmed function in regulating alternative splicing. Other suggested roles of MBNL1 in the cytoplasm are RNA transport, translation, and miRNA processing. However, how the loss of MBNL1 function results in neurodegeneration is poorly understood. In this study, we set up multiple systems to study the role of MBNL1 in the nervous system. We identified MBNL1 interacting partners in the mouse brain using immunoprecipitation (IP) to elucidate its functional mechanism. The interacting protein identified in the experiment have roles in splicing, translation, and transportation. Since the cytoplasmic MBNL1 isoform (MBNL1Δ5) plays a role in neurite outgrowth, we optimized the protocol to separate the nuclear and cytoplasmic proteins to specifically study the function of this isoform. Alternatively, the FLAG-tagged MBNL1Δ5 was transduced into mouse hippocampal neuronal cells HT22. The transduced cells showed more rapid neurite outgrowth than control cells upon differentiation. This cell line thus offers a cell model to gain novel information on the role that this MBNL1 isoform plays in neuron development. Altogether, this project contributes to understanding the mechanisms of neuronal dysfunction in DM1.

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Creator Liu, Yuntong (author)

Core Title Elucidation of MBNL1 function in the nervous system of myotonic dystrophy type 1

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2022-08

Publication Date 07/25/2022

Defense Date 05/27/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cytoplasmic isoform,MBNL1,neuron development,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Comai, Lucio (committee chair), Reddy, Sita (committee member), Rice, Judd (committee member)

Creator Email lytong98@163.com,yuntongl@usc.edu

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