NOP56 is essential for mammalian generation and maintenance of multiple central nervous systems, associated with SCA36 pathology

Abstract

NOP56, a core nucleolar component involved in small nucleolar ribonucleoprotein assembly, has been genetically implicated in spinocerebellar ataxia type 36. However, the role of NOP56 in mammalian neurodevelopment and disease remains poorly defined. We investigated NOP56 pathobiology using both in vitro induced pluripotent stem cell-derived neurons and in vivo NOP56 knockout mouse models. NOP56 expression significantly decreased both in the spinocerebellar ataxia type 36 patients induced pluripotent cells and induced pluripotent cell-derived neurons, which suggests the possibility that the NOP56 loss of function is involved in the spinocerebellar ataxia type 36 phenotype. Therefore, we generated and validated the NOP56 knockout mouse phenotype. Homozygous NOP56 deletion resulted in total embryonic lethality; no NOP56^−/− progeny was viable at birth. Heterozygous knockouts showed clasping at 8 months of age and had a larger body size with aging, although there was no significant difference in survival between heterozygous and wild type. Heterozygous knockout mice showed deterioration in rotarod performance and a decrease in exploration behavior. Immunohistochemical analysis of the heterozygous knockouts revealed widespread, significant central nervous system abnormalities, particularly cerebellar degeneration, accompanied by motor cortex and spinal cord disturbances. Widespread ubiquitin-positive inclusions were detected in the cerebellum, motor cortex, and anterior spinal cord of the heterozygous knockout mice at the 12-month age, and it was positive from the 6-month age in the cerebellum. Colocalizations of TDP-43 and ubiquitin were observed in the motor cortex, spinal cord, and cerebellum. Along with findings from previous reports showing early downregulation of NOP56 in SOD1 G93A transgenic mice, this finding indicates that NOP56 might be involved in a wide range of motor neuron diseases. The pathological characteristics of the NOP56 heterozygous knockouts are like those of a patient with spinocerebellar ataxia type 36. Results reveal that NOP56 is indispensable for mammalian embryogenesis and central nervous system maintenance, and that its reduction contributes to molecular pathology in spinocerebellar ataxia type 36. These findings uncover a convergent neurodegenerative mechanism and identify NOP56 as a potential therapeutic target.

Clinical trial registration

This study was registered with the Japan Clinical Trials Registry (http//umin.ac.jp/ctr/index/htm), under the number UMIN000047097.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s40478-026-02379-8.

Introduction

NOP56 is a nucleolar protein with a molecular mass of 66 kDa that is involved in RNA metabolism and is highly conserved from yeast to humans [9, 39]. Small nucleolar RNAs (snoRNAs) are abundant noncoding RNAs that are enriched in the nucleoli of eukaryotic cells. These snoRNAs are classified into two groups, box C/D and box H/ACA, according to the presence of conserved sequence elements. NOP1, NOP56, NOP58, and SNU13 bind to box C/D snoRNAs, forming snoRNA-protein (snoRNP) complexes that methylate specific target RNAs [8, 16]. NOP56 is associated with the final maturation and activation of catalytically active box C/D snoRNPs, and NOP56 pre-mRNA levels are tightly regulated [3, 16]. The snoRNA-protein complex plays a vital role in the early to middle steps of pre-ribosomal RNA processing, including 2′-O-methylation. Therefore, NOP56 has roles in ribosomal RNA processing and ribosome biogenesis.

Regarding the relationship between NOP56 and diseases, a hexanucleotide GGCCTG repeat expansion in intron 1 of NOP56 causes spinocerebellar ataxia type 36 (SCA36) [13]. In addition, dysregulation of NOP56 is closely related to the occurrence and development of various malignant tumors [11, 17]. Recently, structural insights into an antiparallel chair-type G-quadruplex were revealed, and the relationship between a reduction in NOP56 mRNA and an oncogene has been demonstrated [38].

Considering these facts, NOP56 may play an important role in mammals, including humans. A zebrafish NOP56 loss-of-function model exhibited a severe neurodegenerative phenotype [34]. In this fish model, NOP56 homozygous knockout resulted in the absence of a cerebellum, a reduced number of spinal cord neurons, high levels of apoptosis in the central nervous system (CNS), and impaired movement, resulting in death before 7 days post-fertilization. However, the effect of NOP56 knockout in mammals remains unclear.

Addressing the influence of NOP56 dysfunction is crucial for understanding the pathology of neurological diseases. SCA36 not only disrupts the cerebellum but also causes various CNS abnormalities, affecting areas such as motor nerves [12] and the frontal cortex [1]. Recently, several pathologies associated with SCA36 have been identified, notably the production of aggregating dipeptide repeat proteins through repeat-associated non-AUG translation [20]. This pathology is common in both SCA36 and amyotrophic lateral sclerosis (ALS) and frontotemporal dementia associated with C9orf72 GGGGCC repeat expansion. However, it remains unclear why SCA36 results in multiple CNS abnormalities or the effects of NOP56 knockout in this context.

To clarify these important points, we performed this study (Fig. 1). First, we compared human SCA36 and patient-derived induced pluripotent stem cell (iPSC) & derived neurons with those from healthy controls. Secondly, we established NOP56 knockout mice and analyzed their pathology.

Fig. 1.

Study design. a Analysis of spinocerebellar ataxia type 36 (SCA36) patient induced pluripotent stem cells (iPSCs) and iPS-derived neurons (in vitro part). b Analysis of NOP56 knockout mouse model (in vivo part)

Materials and methods

Ethics and research governance

This study was approved by the Ethics Committees of Jichi Medical University and Omuta National Hospital, and it obtained approval from the Institutional Review Board (approval no.: Rin-Dai 22–165). Written informed consent was obtained from all participants. All analyses were performed in accordance with the Declaration of Helsinki and with the relevant guidelines and regulations.

The animal protocol was approved by the Institutional Animal Care and Use Committee of Jichi Medical University and complied with the Guide for the Care and Use of Laboratory Animals (approval no.: 23014-03). Mice were kept on a 12-h light/dark cycle with food and water available ad libitum.

iPSC generation and genotyping

Human healthy control [21] and SCA36 [18] iPSCs were provided by RIKEN BioResource Center through the National BioResource Project of MEXT/AMED, Japan [27]. They were cultured by the feeder-free and xenofree culture system with StemFit (Ajinomoto, Tokyo, Japan) with penicillin/streptomycin [24].

The GGCCTG hexanucleotide repeat expansion in intron 1 of NOP56 was examined by repeat-primed PCR as described previously [13]. Three primers were used in each disease: fluorescent dye-conjugated forward primer located upstream of the repeat region, first reverse primer consisting of units of the GGCCTG repeat, and second reverse primer as an anchor. PCR was performed with TaKaRa LA Taq with GC buffer (Takara Bio, Kusatsu, Japan). Fragment analysis of PCR products were conducted with SeqStudio Genetic Analyzer (Thermo Fisher Scientific). Detailed information about the primers is provided in Table S5.

Neuronal differentiation from iPSCs

All iPSC clones were differentiated into neural cells containing motor neurons by serum-free floating culture of embryoid-body-like aggregates using the quick re-aggregation method [18, 19]. Briefly, iPSCs were dissociated to single cells and quickly reaggregated in low-cell-adhesion U-shaped 96-well plates (Lipidule-Coated Plate A-U96; NOF Corporation, Tokyo, Japan). Aggregates were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 (Thermo Fisher Scientific) containing 5% KSR (Thermo Fisher Scientific), minimum essential medium-nonessential amino acids (Thermo Fisher Scientific), L-glutamine (MERCK), 2-mercaptoethanol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 2 mM dorsomorphin (MERCK), 10 mM SB431542 (FUJIFILM Wako Pure Chemical Corporation), 3 mM CHIR99021 (FUJIFILM Wako Pure Chemical Corporation), and 12.5 ng/mL fibroblast growth factor (FUJIFILM Wako Pure Chemical Corporation) in a neural inductive stage for 11 days. On day 4, 100 nM retinoic acid (MERCK) and 500 nM Smoothened ligand (Enzo Life Sciences, Farmingdale, NY, USA) were added. After patterning with neurobasal medium (Thermo Fisher Scientific) supplemented with B27 (Thermo Fisher Scientific), 100 nM retinoic acid, 500 nM Smoothened ligand, and 10 mM DAPT (FUJIFILM Wako Pure Chemical Corporation), the aggregates were separated by Accumax (Stem Cell Technologies), dissociated into single cells, and adhered to Matrigel-coated (MERCK) dishes on day 16. Adhesive cells were cultured in neurobasal medium with 10 ng/mL brain-derived neurotrophic factor (R&D Systems, Minneapolis, MN, USA), 10 ng/mL glial cell line-derived neurotrophic factor (R&D Systems), and 10 ng/mL neurotrophin-3 (R&D Systems) for 8 days.

Mouse breeding and genotyping

Wild-type pregnant mice were maintained on a C57BL/6JJcl background. C57BL/6JJcl wild-type mice were obtained from CLEA Japan, inc. (Tokyo, Japan). The NOP56 exon 1–11 deletion construct (GRCm39 (GCF_000001635.27) Chr 2 130112515:130120644) was electroporated into C57BL/6JJcl zygote by the CRISPR-Cas9 system. The NOP56^+/− and wild-type NOP56^+/+ mice used in these experiments were obtained through either intercrossing NOP56^+/− mice (NOP56^+/− x NOP56^+/−) or crossing NOP56^+/− mice with NOP56^+/+ mice (NOP56^+/− x NOP56^+/+), or a combination of both methods. As a result, mutant mice were backcrossed multiple generations into the C57BL6JJcl background.

To validate embryonic lethality, in addition to mating, we conducted in vitro fertilization (IVF) and embryo transfer using three parental combinations; NOP56^+/– × NOP56^+/+, NOP56^+/+ × NOP56^+/–, and NOP56^+/– × NOP56^+/–. We checked all the offspring genotyping at Day 12.5 embryo stage, at birth, and at weaning for all mating combinations. Super-ovulated female mice (strain, age) were mated with males of the desired genotype. Oocytes were collected and subjected to IVF using Human Tubal Fluid medium (ARK Resource. Co., Ltd., Kumamoto, Japan), according to standard protocols. Successfully fertilized zygotes were cultured at 37 °C in 5% CO₂ to the 2-cell stage in modified Whitten’s medium (ARK Resource. Co., Ltd.). Two-cell embryos were transferred into the oviducts of pseudopregnant recipient females.

For mouse genotyping, genomic DNA was extracted from tail tips, and PCR was performed using three primers: a forward primer in common with the two genotypes (5′F, 5′-ACCTATGGGGGAGGTCCATT-3′), one reverse primer for the detection of the wild-type NOP56 allele (5′R, 5′-GGCTGGACATTGATGTTGGA-3′), and one reverse primer for the detection of the mutant NOP56 allele (3′R, 5′-TCAAGGTCACTGGCCAACTC-3′). The following PCR cycling conditions were used: 95 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and one cycle of 72 °C for 10 min. The PCR products were 526 bp for the wild-type allele and 637 bp for the mutant allele.

Histochemistry

NOP56^+/+ and NOP56^+/− mice were sacrificed at 6, 8 and 12 months. Each animal was anesthetized by intraperitoneal injection of 0.75 mg/kg medetomidine, 4 mg/kg midazolam, and 5 mg/kg butorphanol, then perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer. After post-fixation overnight, 50-µm-thick sections were cut with a vibrating blade microtome (LinearSlicer PRO10; DOSAKA EM CO., LTD., Kyoto, Japan). We targeted the CA1 region of the hippocampus, and in Lobule V and Lobule VI of the cerebellum for immunocytochemistry. For adipose tissue histological analysis, we obtained inguinal white adipose tissue (iWAT) from 8 months of age mice. Adipose tissues gradually permeabilized with 10%, 20%, and 30% sucrose buffer after post-fixation. After permeabilization, 10-µm-thick sections were cut with Leica CM3050 S Cryostat (Leica Biosystems, Nussloch, Germany).

Immunocytochemistry

For immunostaining of iPSCs and iPSC-derived neurons, the cells were fixed in 4% paraformaldehyde (pH 7.4) for 30 min at room temperature and rinsed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by rinsing with PBS. After blocking with 5% bovine serum albumin for 60 min at room temperature, the cells were incubated with the following primary antibodies overnight at 4 °C: anti-Nanog (1:500; REPROCELL, Yokohama, Japan, RCAB004P-F), anti-SSEA4 (1:500; GeneTex, Irvine, CA, USA, MC-813-70), anti-neurofilament-H (1:500; SMI-32, BioLegend, San Diego, CA, USA, SMI-32P), anti-ßΙΙΙ-tubulin (1:2,000; D71G9, Cell Signaling Technology, Danvers, MA, USA, 5568), anti-MNX (1:250; HB9, MERCK, ABN174), and anti-Islet1 (1:200; 39.4D5, Developmental Studies Hybridoma Bank, Iowa City, IA, USA, AB-2314683). After three rinses with PBS, the cells were incubated with appropriate Alexa Fluor-conjugated secondary antibodies for 1 h at room temperature, and DAPI (Thermo Fisher Scientific) was used to label nuclei. Cell images were acquired with an Operetta CLS (Revvity, Inc., Waltham, MA, USA). To evaluate the positive count ratio of immunocytochemistry, the cells were imaged using automated microscopy by an Operetta CLS and then the immunostained structures were quantified automatically with an Operetta CLS High Content Analysis System (Revity, Inc.).

For immunostaining mouse tissue, the sections were incubated with the following primary antibodies overnight at 4 °C after blocking with 5% bovine serum albumin for 60 min at room temperature: anti-choline acetyltransferase (ChAT) (1:500; CL3169, Thermo Fisher Scientific, MA5-31382), anti-neuronal-nuclei (NeuN) (1:100; 14H6L24, Thermo Fisher Scientific, 702022), anti-calbindin (1:200; C26D12, Cell Signaling Technology, 2173), anti-glial fibrillary acidic protein (GFAP) (1:500; 4B2E10, Thermo Fisher Scientific, 60190-1-IG), anti-ionized calcium-binding adaptor molecule 1 (Iba1) (1:500; GT10312, Thermo Fisher Scientific, MA5-27726), anti-Poly GP antibody (1:500; MERCK, ABN1358), anti-Poly PR antibody (1:500; MERCK, ABN1354), anti-agouti-related protein (AGRP) antibody (1:200; Proteintech Group, Rosemont, IL, USA, 21249-1-AP), anti-cocaine and amphetamine regulated transcript prepropeptide (CARTPT) antibody (1:100; MERCK, SAB4301768), anti-Neuropeptide Y antibody (1:200; D7Y5A, Cell Signaling Technology, #11976), anti-pro-opiomelanocortin (POMC) antibody (1:500; EPR22534-165, Abcam, ab254257), anti-uncoupling protein 1 (UCP1) antibody (1:2,000, RM1066, Abcam, ab314239), and anti-perilipin-1 antibody (1:400, Proteintech Group, 27716-1-AP). After three rinses with PBS, the cells were incubated with appropriate Alexa Fluor-conjugated secondary antibodies for 1 h at room temperature, and DAPI (Thermo Fisher Scientific) was used to label nuclei. Tissue images were acquired with Dragonfly 200 (Oxford Instruments, Abingdon-on-Thames, UK).

According to the ubiquitin staining, sections were incubated in 0.3% hydrogen peroxide for 30 min to bleach endogenous peroxidase activity. Then, they were incubated at 4 °C overnight with anti-ubiquitin antibody (1:100; EPR8830, Abcam, ab134953). Positive staining was visualized using the avidin-biotin-peroxidase complex method with diaminobenzidine as the chromogen by VECTASTAIN Elite ABC Kit (Vector Laboratories Inc, Newark, CA, USA), and the images were acquired with BZ-X700 All-in-one Microscope (KEYENCE, Osaka, Japan). According to the double immunofluorescence staining to validate the colocalization with ubiquitin, we used the following primary antibody sets: anti-mouse ubiquitin antibody (1:2,000, P4D1, Abcam, ab303664) and anti-rabbit TDP43 antibody (1:100, D9R3L, Cell Signaling Technology, #89789), or anti-rabbit Poly GP antibody, or anti-rabbit Poly PR antibody. Another set as anti-rabbit ubiquitin antibody and anti-mouse NOP56 antibody (1:500, MERCK, AMAB91013). Tissues were incubated with appropriate species corresponding Alexa Fluor-conjugated secondary antibodies pair, and DAPI was used to label nuclei. Tissue images were acquired with Dragonfly 200, and Z-stack integrating and 3D images were obtained for the additional three-dimensional analysis.

Fluorescence in situ hybridization

All procedures according to RNA fluorescence in situ hybridization were performed as the previous established methods [18]. Images acquired with Dragonfly 200.

qRT-PCR

Mouse tissue samples were infiltrated with RNAlater™ Stabilization Solution (Thermo Fisher Scientific) and homogenized using a MagNA Lyser Instrument (F. Hoffmann-La Roche, Ltd., Basel, Switzerland). All iPSCs, iPSC-derived neurons, and mice sample total RNA were isolated using an RNeasy Plus Mini Kit (QIAGEN), and RNA was reverse transcribed to cDNA using a ReverTra Ace-α Kit (TOYOBO, Osaka, Japan). Real-time quantitative reverse transcription PCR (qRT-PCR) was performed with SYBR Green Master Mix (Thermo Fisher Scientific) using the ∆∆CT method. qRT-PCR of NOP56 and GAPDH was performed using primer sets and detailed primer information is described in Table S5.

Western blotting

iPSCs and iPSC-derived neurons were lysed with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate, pH 7.5). Mouse tissue samples were homogenized to a final concentration of 10% in Dulbecco’s PBS (Thermo Fisher Scientific) by a MagNA Lyser Instrument. Halt™ protease inhibitor cocktail (Thermo Fisher Scientific) was added to the sample buffer. Protein concentration was determined by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) using bovine serum albumin as a standard. Total proteins were separated by 12% SDS-PAGE with a NuPAGE™ MOPS SDS Running Buffer (Thermo Fisher Scientific) at 100 V for 90 min. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane using a wet transfer apparatus with transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) at 35 V for 120 min. Membranes were blocked in 5% skimmed milk in PBS-0.05% Tween 20 for 60 min and then probed with a primary antibody overnight at 4 °C. The following primary antibodies were used: rabbit anti-NOP56 (1:500; Thermo Fisher Scientific, 18181-1-AP) and rabbit anti-β-actin (1:1,000; Cell Signaling Technology, 4967). β-Actin was used as a loading control. Anti-rabbit IgG (whole molecule)–alkaline phosphatase antibody (1:30,000; MERCK, A3687) was used as a secondary antibody for 60 min at room temperature. The bands were visualized using Novex™ AP chemiluminescent substrate (CDP-Star; Thermo Fisher Scientific) and analyzed with an Amersham ImageQuant 800 (Danaher Corporation, Washington, D.C., USA).

Mouse motor function testing

Every 3 weeks, the mice performed five trials on an accelerating rotarod (Ugo Basile, Varese, Italy) at 4 rpm with an acceleration of 0.2 rpm/s for 180 s, and the latency to fall was recorded. The mice were habituated by exposing them to a rotating rod and test validation was performed after 3 consecutive days of training. The evaluation was terminated when the mice fell from the rotarod or reached the criterion level. Grip strength of the front paws was measured every 3 months using a grip strength meter (Bioseb, Vitrolles, France). Strength was measured five times for each mouse. Both behavioral tests were performed on 30 mice per genotype. We also used hindlimb clasping test and calculated scores as previously established [10] at several points. All motor function tests were recorded by video camera and deposited.

Mouse locomotor activity and indirect calorimetry

Mouse spontaneous motor activity was recorded using the ACTIMO-100 activity monitoring system (Shin Factory Co., Ltd., Fukuoka, Japan). Average oxygen consumption (VO₂) was measured using an ARCO-2000 (ARCO System, Chiba, Japan) with an airflow of 0.3 L/min. The mice were placed in individual calorimeter chambers. Activity was recorded every 1 min and VO₂ (mL/kg/min) was measured every 5 min for 7 days. The measurements were conducted in male mice with free access to food (9 weeks old and 1 year old). Basic VO₂ values were calculated by averaging the values on the 6th and 7th days after the start, after 5 days of acclimation.

Clinical SCA36 patient analysis

We analyzed the clinical characteristics of patients diagnosed with SCA36 at the Division of Neurology, Department of Medicine, Jichi Medical University, who agreed to participate in the Jichi Neurological Disease Registry Study. This registry study focuses on recording and analyzing data from patients with neurodegenerative diseases. For comparison, we established a disease control group consisting of patients with SCA6. Both SCA6 and SCA36 diagnoses were confirmed using the SeqStudio Genetic Analyzer. Since most SCA36 symptoms typically develop after the age of 50 [12], we exclude patients who were diagnosed before this age or asymptomatic carriers. We recorded and validated body weight (kg), Body Mass Index (BMI), and cognitive function scores [Mini-Mental State Examination (MMSE), Revised Hasegawa’s Dementia Scale (HDS-R), and Frontal Assessment Battery (FAB)] for both SCA subtypes.

Statistical analysis

Values are expressed as the mean ± standard error. All in vitro experiments were performed using each iPSC and iPSC-derived neuron clone plated in three wells, and significant differences between groups were determined by Student’s t test. In vivo mouse experiment included equal number male and female, and number in each experimental is described in legends. Immunofluorescence was performed on 6 sections for each animal, and mouse tissue image analysis was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA). ChAT, NeuN, and calbindin were evaluated with number calculated from positive intensity, and GFAP and Iba1 were evaluated with positive area. AGRP, CARTPT, Neuropeptide Y, and POMC were evaluated both with number calculated from positive intensity and with positive area. UCP1 and perilipin-1 were evaluated with positive cell area (µm²) and signal intensity calculated by DAPI positive number. Mean time and power results of five trials were analyzed for the mouse motor function test. Repeated measurements analysis of variance and the Wilcoxon signed rank test were used for comparison of the wild-type and heterozygous-knockout groups. Chi-square test was used for comparison of genotype birth rate. Survival analysis was carried out using the Kaplan-Meier method. Differences were considered significant when p < 0.05 or p < 0.01 (three group chi-square test), and statistical analyses were performed with Prism (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Generation of iPSC clones and neural differentiation

iPSC clones were generated from three healthy controls and three SCA36 patient (Table S1) [14, 18, 26, 28, 35]. One iPSC clone was generated for each subject. As a result, a total of six iPSC clones were analyzed. All six clones expressed the pluripotency markers NANOG and SSEA4 in immunostaining (Fig. 2a). All iPSC clones were examined by repeat-primed PCR, which confirmed that SCA36 patient-derived iPSC clones retained the GGCCTG repeat expansion (Fig. 2b). The neural differentiated cells expressed the motor neuron subtype markers MNX1 and Islet1 (Fig. 2c). There was no obvious difference in neuronal differentiation propensity between healthy control and SCA36 iPSC clones (Fig. 2d).

Fig. 2.

Analysis of NOP56 expression of SCA36 patient iPSCs and iPSC-derived neurons. a Induced pluripotent stem cell (iPSCs) from healthy controls and spinocerebellar ataxia type 36 (SCA36) patients showing embryonic stem cell-like morphology (phase) and expressing pluripotent stem cell markers such as NANOG (green) and SSEA4 (red). Scale bars, 200 μm. b Sequence traces of repeat-primed PCR products are shown. Atypical saw-tooth pattern by repeat expansion is observed in SCA36 iPSCs, but not in healthy control iPSCs. c Differentiation of neural cells containing motor neurons from iPSC clones. Generated neurons expressed the neuron markers neurofilament-H and βⅠⅠⅠ-tubulin, and motor neuron markers MTX1 and Islet1. There was no obvious difference in appearance between them. Scale bars, 50 μm. d There were no significant differences in the differentiation propensity of neurons or motor neurons between healthy control and SCA36 iPSC clones. Values are expressed as the mean ± standard error from n = 3 clones. n.s., not significant. e Graph showing NOP56 mRNA expression levels in healthy control and SCA36 patient iPSCs. The mean NOP56 mRNA expression level of control iPSCs was standardized to be 1.0. NOP56 mRNA expression level was significantly lower in SCA36 patient iPSCs than in healthy controls in both primer-1 and primer-2 sets. Primer-2 sets are same ones using in Ref. 13. f Western blot analysis of healthy control and SCA36 patient iPSCs lysates. Anti-NOP56 antibody shows 65–70 kDa bands in three different control clones, which were reduced in SCA36. g Quantification of NOP56 protein in control and SCA36 iPSCs relative to β-actin. h Graph showing NOP56 mRNA expression levels in healthy control and SCA36 patient iPSC-derived neurons. i Western blot analysis of healthy control and SCA36 patient iPSC-derived neurons lysates. NOP56 protein levels were reduced in SCA36. j Quantification of NOP56 protein in control and SCA36 patient iPSC-derived neurons relative to β-actin. *p < 0.05. Data are presented as the mean ± standard error from n = 3 clones

Analysis of the NOP56 expression level of iPSCs and iPSC-derived neurons

NOP56 mRNA expression was analyzed between healthy control and SCA36 iPSCs and iPSC-derived neurons with qRT-PCR using two different primer sets, which showed a significant decrease in SCA36 compared to healthy controls (Fig. 2e–h). Western blot analysis also showed significantly decreased NOP56 protein levels in SCA36 compared to healthy controls (Fig. 2f–g and i–j).

Genetic ablation of NOP56 in mice

NOP56 expression levels in SCA36 iPSCs and iPSC-derived neurons were significantly decreased compared to healthy controls. However, the effect of this decrease in NOP56 expression to mammalians remained uncertain. Therefore, we examined its influence on mammals by generating NOP56 knockout mice. NOP56 gene-ablated mice (F0) from exon 1 to exon 11 were generated by CRISPR-Cas9 electroporation with guide RNA (Fig. 3a) [25, 36]. Initially, we intended to examine NOP56 homozygous knockout (NOP56^−/−) mice; however, all progeny from NOP56^+/− and NOP56^+/− mating were wild-type (NOP56^+/+) or NOP56^+/−, and no NOP56^−/− mice were born (Fig. 3b–d and Table S2). Eliminating NOP56^+/− effect to mating, we performed IVF and embryo transfer (Fig. 3e and Table S3). From 144 embryo from NOP56^+/− X NOP56^+/−, no NOP56^−/− mice were born. The birth rate from NOP56^+/− X NOP56^+/− IVF was 22.2%, which is significantly lower compared to NOP56^+/+ X NOP56^+/−. The birth rate between NOP56^+/+ and NOP56^+/− was nearing 1:2. We also terminated the pregnancy and checked the genotyping at Day 12.5 embryo stage from NOP56^+/− X NOP56^+/− IVF, which showed no NOP56^−/− embryo present (Fig. 3f and Table S4). All these results correspond to the embryonic lethality [30]. Since no NOP56^−/− mice were born, NOP56^+/+ and NOP56^+/− mice were compared for this research.

Fig. 3.

NOP56 knockout mouse generation. a Schematic overview of the NOP56 knockout mouse design. The region from pre-exon 1 to intron 11, which spans approximately 8 kilobases, of the NOP56 gene was deleted. b Genotyping results of NOP56 heterozygous knockout (NOP56^+/−) and NOP56^+/− mating. While NOP56 wild-type (NOP56^+/+) mice have a single 526-bp PCR product, hetero-knockout (NOP56^+/−) mice have double 526-bp and 637-bp PCR products. c Results of NOP56^+/+ × NOP56^+/– mating and genotype rates of neonates. d Results of NOP56^+/– × NOP56^+/– mating and genotype rates of neonates. e Results of NOP56^+/– × NOP56^+/– in vitro fertilization (IVF) and embryo transfer, and genotype rates of neonates. From 144 embryos, the number of neonates remained 32, and no NOP56^–/–. f Termination was performed to NOP56^+/– × NOP56^+/– IVF and embryo transfer at Day 12.5, and genotype rates of embryos. g Appearance of 1-year-old (48-weeks-old) NOP56^+/+ and NOP56^+/− mice. NOP56^+/− mice appear slightly larger. The body size was calculated as the length between the nose and the root of tail. Time course data of body size at 12, 24, 36, and 48-weeks-old are showing

h Clasping test. While NOP56^+/+ mice showed the normal response, extending hindlimbs away from the abdomen, NOP56^+/− mice at 8-month-old showed an abnormal response of retracting and clasping. In this photo, clasping was positive in his right hindlimb, and the score was 1. i Graph showing NOP56 mRNA expression levels in NOP56^+/+ and NOP56^+/− mouse brains. The mean NOP56 mRNA expression level was standardized to be 1.0. NOP56 mRNA expression levels were significantly lower in NOP56^+/− mice than in NOP56^+/+ mice. j Western blot analysis of NOP56^+/+ and NOP56^+/− mouse brain homogenates. k Quantification of NOP56 protein in NOP56^+/+ and NOP56^+/− mouse brains relative to β-actin. NOP56 protein levels were significantly lower in NOP56^+/− mice. l NOP56 mRNA expression levels in the cerebellum. Significantly lower in NOP56^+/− mice. m Western blot analysis of cerebellum samples. n Quantification of NOP56 protein in NOP56^+/+ and NOP56^+/− mouse cerebellums relative to β-actin. Significantly lower in NOP56^+/− mice. *p < 0.05. Data are presented as the mean ± standard error from n = 5

There was no noticeable difference in appearance between NOP56^+/+ and NOP56^+/− mice after birth. However, NOP56^+/− mice had a larger body size than NOP56^+/+ mice with aging (Fig. 3g). In addition, clasping was observed in NOP56+/- mice starting at 8 months old, and it became more apparent over time. (Fig. 3h). NOP56 mRNA levels in the brain and cerebellum were significantly lower in NOP56^+/− mice than in NOP56^+/+ mice (Fig. 3i and l). NOP56 protein levels in the whole brain and cerebellum were also significantly lower in NOP56^+/− mice than in NOP56^+/+ mice (Fig. 3j–k and m–n), in agreement with the qRT-PCR results. There was a significant difference of NOP56 mRNA levels among CNS tissues of NOP56^+/+ mice (Fig. S1), with the highest levels in the cerebellum, intermediate levels in the brain cortex, and the lowest in the spinal cord.

Histochemical analysis of NOP56^+/− mice

To examine the influence of decreased NOP56 expression on the CNS, the number of motor neurons in the motor cortex and anterior horn of the spinal cord, number of hippocampal neurons, number of cerebellar Purkinje cells, and thickness of the cerebellar molecular layer were assessed by immunohistochemistry in 6- and 12-month-old NOP56^+/− mice and littermate NOP56^+/+ controls (Fig. 4). Cerebellar molecular thickness was calculated because it consists of Purkinje cell dendrites and parallel fibers and reflects cerebellar function. The number of ChAT-positive neurons in the motor cortex was significantly lower in NOP56^+/− mice than in NOP56^+/+ mice at 6 and 12 months of age (Fig. 4a–b); however, there was no significant difference in the spinal cord (Fig. 4c–d). Additionally, there was no significant difference in the number of NeuN-positive neurons in the hippocampus (Fig. 4e–f). In contrast, the number of Purkinje cells in the cerebellum was significantly lower in NOP56^+/− mice than in NOP56^+/+ mice at 6 and 12 months of age (Fig. 4g–h). The cerebellar molecular layer was also significantly thinner in NOP56^+/− mice than in NOP56^+/+ mice (Fig. 4g–h). None of RNA foci, Poly-GP, or Poly-PR dipeptide repeats aggregates were present in the NOP56^+/− mice brain, spinal cord, or cerebellum.

Fig. 4.

Neuronal loss of NOP56 heterozygous-knockout (NOP56^+/−) mice. Immunofluorescent staining of a 6-month-old, and b 12-month-old motor cortex using an anti-choline acetyltransferase (ChAT) antibody. The number of positive cells was significantly lower in NOP56^+/− mice than in NOP56^+/+ mice. Immunofluorescent staining of c 6-month-old, and d 12-month-old spinal cord using an anti-ChAT antibody. The number of positive cells was significantly lower in NOP56^+/− mice than in NOP56^+/+ mice. Immunofluorescent staining of e 6-month-old, and f 12-month-old hippocampus using an anti-neuronal-nuclei (NeuN) antibody. There was no significance. Immunofluorescent staining of g 6-month-old, and h 12-month-old cerebellar cortex using an anti-calbindin antibody. Both the number of Purkinje cells and thickness of the molecular layer were significantly lower in NOP56^+/− mice than in NOP56^+/+ mice. Scale bars, 50 μm. *p < 0.05. Values are expressed as the mean ± standard error from n = 6. n.s., not significant

To examine the presence of neurodegeneration, GFAP- and Iba1-positive areas were assessed by immunohistochemistry in the motor cortex, spinal cord, hippocampus, and cerebellum in 12-month-old NOP56^+/− mice and littermate NOP56^+/+ controls (Fig. 5). Consequently, the GFAP-positive areas were significantly larger in NOP56^+/− mice than in NOP56^+/+ mice in all regions examined, except for the hippocampus (Fig. 5a). Similarly, the Iba1-positive areas were also significantly larger in NOP56^+/− mice than in NOP56^+/+ mice in all regions examined, except for the hippocampus (Fig. 5b). Ubiquitin strongly positive inclusion bodies were present in the motor cortex, anterior spinal cord, and cerebellum of 12-month-old NOP56^+/− mice but not in hippocampus or NOP56^+/+ mice (Fig. 5c). In the cerebellum of NOP56^+/− mice, ubiquitin strongly positive inclusion bodies were already present from the 6-month-old (Fig. 5d).

Fig. 5.

Neurodegeneration of NOP56 heterozygous-knockout (NOP56^+/−) mice. a Immunofluorescent staining of 12-month-old motor cortex, spinal cord, hippocampus, and cerebellum using an anti-glial fibrillary acidic protein (GFAP) antibody. All GFAP-positive areas, except for the hippocampus, were significantly larger in NOP56^+/- mice than in NOP56^+/+ mice. b Immunofluorescent staining of 12-month-old motor cortex, spinal cord, hippocampus, and cerebellum using an anti-ionized calcium binding adaptor molecule 1 (Iba1) antibody. Scale bars, 50 μm. *p < 0.05. Values are expressed as the mean ± standard error from n = 6. n.s., not significant. c Immunofluorescent staining of 12-month-old NOP56^+/- mice motor cortex, spinal cord, hippocampus, and cerebellum, and of d 6-month-old NOP56^+/- mice cerebellum using ubiquitin antibody. Arrows were ubiquitin strongly positive inclusions. Left panels showing low magnification and right panels showing high magnification. Scale bars, 20 μm

We also analyzed the proteins colocalized with the ubiquitin accumulation (Fig. 6). None of the NOP56, Poly-GP, and Poly-PR dipeptide repeats showed colocalization with TDP-43 (Fig. 6a, c and d), whereas TDP-43 was colocalized with ubiquitin (Fig. 6b). Colocalizations of TDP-43 and ubiquitin were observed in the motor cortex, spinal cord, and cerebellum. Notably, these colocalizations were most prominent in the cerebellum (Fig. 6e). In the three-dimensional image (Fig. 6f), the colocalizations were located in the cytoplasm outside the nucleus, suggesting a potential dislocation of TDP-43 to the cytoplasm in NOP56^+/− mice.

Fig. 6.

Colocalization protein to ubiquitin inclusion of NOP56 heterozygous-knockout (NOP56+/-) mice. Immunofluorescent staining of 12-month-old motor cortex, spinal cord, and cerebellum using double antibodies sets. NOP56, TDP-43, and two different dipeptide repeats (Poly GP and Poly PR) were examined to validate colocalization to ubiquitin inclusion. a NOP56. NOP56 (red) showed no merge to ubiquitin (green). b TDP-43. While TDP-43 (red) strongly expressed in nucleus, intemediate high signal distributed in cytoplasm. This TDP-43 signal merges with ubiquitin accumulation (yellow) in all motor cortex, spinal cord, and cerebellum. c Poly GP. d Poly PR. Neither merges with ubiquitin and only non-specifin signals. e Z-stack integrating and f three-dimentinal images of TDP-43 and ubiquitin. Scale bars, 10 μm

Behavioral analysis of NOP56^+/− mice

A significant increase in body weight was observed in NOP56^+/− mice after they reached 15 weeks of age (Figs. 3g and 7a–c). Rotarod analysis, which assesses motor ability including muscle function and cerebellar performance, revealed that NOP56^+/− mice performed significantly worse compared to NOP56^+/+ mice, although this difference became non-significant after 39 weeks of age (Fig. 7d). In contrast, there was no significant difference in survival between NOP56^+/− and NOP56^+/+ mice (Fig. 7e). A grip strength test showed no significant difference in muscle strength between NOP56^+/− and NOP56^+/+ mice at 12, 36, and 48 weeks of age; however, grip strength was stronger in NOP56^+/− mice than in NOP56^+/+ mice at 24 weeks of age (Fig. 7f).

Fig. 7.

Behavioral characterization of NOP56 heterozygous-knockout (NOP56^+/-) mice. a Total mice body weight. A significant body weight increase occurred in NOP56^+/- mice after 15 weeks of age. b Male mice exhibited a significant increase in body weight at all time points from 9 to 48 weeks of age. c Female mice body weight also showed the significant difference between NOP56^+/+ and NOP56^+/- mice with repeated measurements analysis of variance, but there was no significance in each time point. d Rotarod performance was significantly worse in NOP56^+/- mice compared to NOP56^+/+ mice. e Survival rates as shown by a Kaplan Meier curve. Survival of NOP56^+/- mice were similar to those of NOP56^+/+ control mice. f The grip strength test also showed that muscle strength was not significantly different between NOP56^+/- and NOP56^+/+ mice. *p < 0.05. Data are shown as the mean ± standard error, n = 30 for each genotype. n.s., not significant

Focused on determining the reason for the difference in body weight between both groups, energy consumption with circadian rhythms were analyzed (Fig. 8). Briefly, exploratory behavior of a novel environment was significantly decreased at 9 weeks and 1 year of age in NOP56^+/− mice compared to NOP56^+/+ mice (Fig. 8a and e). The VO₂ in the dark phase was significantly lower in 9-week-old NOP56^+/− mice (Fig. 8d). In contrast, there was no significant difference in food intake between NOP56^+/− and NOP56^+/+ mice (Fig. 8i and j). These findings indicate that the increased body weight observed in NOP56^+/− mice is not likely due to increased food intake, and suggest that reduced exploratory behavior and lower energy expenditure may contribute to this phenotype, although the involvement of other physiological mechanisms cannot be ruled out.

Fig. 8.

Changes in energy consumption of NOP56 heterozygous-knockout (NOP56^+/-) mice. a Motor activity, and b oxygen consumption (VO₂) in a novel environment during measurement of 9-week-old mice. Each left column shows motor activity and VO₂ every hour for 6 h after measurement initiation, and the right column shows total motor activity and average VO₂ for 6 h after measurement initiation. c Basal spontaneous motor activity, and d average basal VO₂ on days 6 and 7 after acclimation to the measurement environment in 9-week-old mice. Each left column shows spontaneous motor activity and VO₂ every hour for 24 h, and the right column shows total spontaneous motor activity and average VO₂ during the light and dark periods. e Motor activity, and f VO₂ in 1-year-old mice. g Basal spontaneous motor activity, and h average basal VO₂ in 1-year-old mice. The increase in motor activity under a novel environment was significantly lower in NOP56^+/- mice than in NOP56^+/+ mice at both ages examined. Average VO₂ in the dark phase was significantly lower in NOP56^+/- mice at 9 weeks of age, while there was no difference at 1 year of age. i Food intake in NOP56^+/- mice at the age of 9–13 weeks, and j 12–13 months. There was no significant difference in food intake between NOP56^+/- and NOP56^+/+ mice when there was a difference in body weight. Data are shown as the mean ± standard error. n = 10 for each genotype (9 weeks old); n = 8 for wild-type and n = 9 for NOP56^+/- (1 year old)

Arcuate nucleus hormones and adipose tissue analyses of NOP56^+/− mice

For further analysis focusing on the significant increase in body weight observed in NOP56^+/− mice, we examined arcuate nucleus hormone expressions and adipose tissue histology (Fig. 9). According to the arcuate nucleus hormones, there were no significant differences in in any of the four hormones between NOP56^+/+ and NOP56^+/− mice (Fig. 9a–d). However, immunohistochemical characteristics of adipose tissue histology were significantly different between NOP56^+/+ and NOP56^+/− mice at 8-month-old stage (Fig. 9e, f). The size and signal intensity of UCP1 positive cell areas significantly decreased in NOP56^+/− mice iWAT, with the UCP1/perilipin-1 results being more prominent (Fig. 9f).

Fig. 9.

Arcuate nucleus hormones and adipose tissue analyses of NOP56+/- mice. Immunofluorescent staining of 6-month-old arcuate nucleus using a anti-agouti-related protein (AGRP) antibody, b anti-cocaine and amphetamine regulated transcript prepropeptide (CARTPT) antibody, c anti-Neuropeptide Y antibody, and d anti-pro-opiomelanocortin (POMC) antibody. e Immunofluorescent staining of 8-month-old inguinal white adipose tissue (iWT) using anti-uncoupling protein 1 (UCP1) antibody and anti-perilipin-1 antibody. f Calculation results of iWAT. Scale bars, 50 μm. *p < 0.05. Values are expressed as the mean ± standard error from n = 6. n.s., not significant

Clinical SCA36 patient characteristics

We finally analyzed clinical data from twelve SCA6 and five SCA36 patients who were confirmed to have been diagnosed. The results are shown in Table 1. The BMI data of clinical SCA36 patients were significantly lower than those of SCA6 patients, which was the opposite of the results in NOP56^+/− mice. Only FAB scores in the cognitive function tests were significantly lower in SCA36 patients.

Table 1.

Results of clinical SCA patient data

	SCA6 (n = 12)	SCA36 (n = 5)	p
Age at diagnosis
	71.0 ± 2.5	69.6 ± 2.8	0.82
Sex
Male	7 (58.3%)	4 (80.0%)	0.60
Fmale	5 (41.7%)	1 (20.0%)
Body weight (kg) at diagnosis
	55.7 ± 2.5	51.2 ± 3.2	0.33
BMI at diagnosis
	22.0 ± 0.7	19.3 ± 0.8	< 0.05*
Cognitive function at diagnosis
MMSE	25.4 ± 2.0	21.8 ± 2.5	0.32
HDS-R	24.8 ± 2.0	17.3 ± 3.3	0.06
FAB	15.5 ± 1.3	9.3 ± 1.0	< 0.05*

BMI, Body mass index; FAB, Frontal assessment battery; HDS-R, Revised Hasegawa’s Dementia Scale; MMSE, Mini-Mental State Examination; SCA, Spinocerebellar ataxia.*p < 0.05

Discussion

Using iPSCs and iPSC-derived neurons, we found that NOP56 expression levels were significantly lower in SCA36 than in healthy controls. In the NOP56 knockout mouse model, NOP56 homozygous knockout was lethal during embryogenesis. There was no difference in survival between NOP56^+/− and NOP56^+/+ mice, but NOP56^+/− mice showed clasping, a deterioration of rotarod performance, and a decrease of exploratory behavior. Histochemical analysis showed that broad CNS abnormalities except for hippocampus were present in NOP56^+/− mice, especially in the cerebellum. These NOP56^+/− mice characteristics correspond to those of SCA36 patients. Ubiquitin inclusion bodies are present in the motor cortex, anterior spinal cord, and cerebellum in NOP56^+/− mice, which are colocalized with TDP-43 in cytoplasm.

It is expected that NOP56 expression is decreased in SCA36 because some repeat expansions in non-coding regions are known to cause loss-of-function in a number of genes associated with repeat expansion diseases, e.g., fragile X syndrome (CGG repeat expansion in FMR1) and Friedreich’s ataxia (GAA repeat expansion in FTX) [2, 7]. Additionally, in a recent study revealing structural insights into an antiparallel chair-type G-quadruplex, the G4 ligand pyridostatin dramatically decreased NOP56 mRNA expression levels in multiple cell lines [38], which supports and corresponds to our experimental results. We highlight this recent report showing NOP56 expression was dramatically decreased by pyridostatin, which shows the possibility that SCA36 making G-quadruplex interferes with splicing and suppress NOP56 expression (Fig. 10). Although a previous study reported no significant change in NOP56 expression in SCA36 lymphoblastoid cell lines (LCLs), which does not align with our results, we speculate that the difference between LCLs and iPSC systems may account for this discrepancy. Recent findings showed that standard cell line maintenance induces a loss of donor-specific gene expression signatures in LCLs, but this loss is recovered when using an iPSC system [37]. In addition, the Epstein-Barr virus used for the LCLs establishment interferes with gene expression levels and methylation profiles [6]. NOP56 is a molecule involved in RNA metabolism, particularly in methylation profiles. We speculate that the technology used in LCLs has certain limitations when analyzing NOP56. However, iPSC technology overcomes these limitations, which explains the discrepancy between the results of previous studies and ours. RNA dysregulation is considered a key contributor to ALS pathogenesis [5]. Many ALS causative genes, e.g., SOD1, TARDBP, and FUS are involved in aspects of RNA metabolism such as mRNA transcription, alternative splicing, RNA transport, mRNA stabilization, and miRNA biogenesis [4, 32, 33]. Considering NOP56 expression is selectively decreased in SOD1 G93A transgenic ALS model mice in the early phase [22], NOP56 loss-of-function itself may be involved in motor neuron disease’s pathology.

Fig. 10.

Pathology of NOP56 loss-of-function in SCA36. The GGCCTG repeat in NOP56 intron 1 inhibits NOP56 transcription. This decrease in NOP56 induces multiple central nervous system abnormalities, but their severity varies in a location-dependent manner. The cerebellum is the most severely affected, while the spinal cord is relatively spared. Adding NOP56 loss-of-function, previously established pathologies such as dipeptide repeat proteins through repeat-associated non-AUG translation and RNA foci toxicity are related to spinocerebellar ataxia type 36 (SCA36). Our established NOP56^+/- mice corresponded to the loss of NOP56 expression status. They showed several pathological characteristics and also induced multiple central nervous system disturbances. NOP56 expression levels are also different among central nervous system locations, and this difference might link to the severity difference among locations. The SCA36 pathology is complicated and results in multiple central nervous system disturbances

Based on in vitro experiment results, we next clarified the effect of NOP56 loss-of-function by using NOP56 knockout mice, which revealed several important findings. First, NOP56 was essential for the generation of progeny unlike a zebrafish model [34]. We analyzed and concluded this fact from multiple different approaches, mating and IVF, both of which resulted in no NOP56^−/− mice born. Second, the phenotypes observed in NOP56^+/− mice correspond to the characteristics of SCA36 patients [1, 12, 23]. Multiple CNS abnormalities except for hippocampus occurred in the NOP56^+/− mice, and the pathological distributions are the same as those of human SCA36 patients [12]. As the behavior, deterioration in rotarod performance, clasping, and a decrease in exploration behavior appeared in the NOP56^+/− mice. These behaviors showed the abnormalities of the cerebellum and apathy [1]. As the histochemistry, loss of neurons and neurodegeneration were detected in the NOP56^+/− mice. These abnormalities were most severe in the cerebellum, followed by the motor cortex and anterior spinal cord. Adding these abnormalities, ubiquitin strongly positive inclusion bodies were present in the broad CNS of NOP56^+/− mice, mainly in the cerebellum, which is also the same as human SCA36 patient tissue [15]. In addition, colocalizations of TDP-43 and ubiquitin throughout the motor cortex, spinal cord, and cerebellum suggest that the NOP56 loss of function links to the TDP-43 pathology of motor neuron diseases. Considering NOP56’s role in box C/D snoRNP assembly [16], there is a possibility that the abnormality of 2’-O-methylation of rRNA is presented in NOP56^+/− mice, which might induce this result. In contrast, we must acknowledge that the TDP-43 abnormality was not observed in the previous autopsy findings for SCA36 patients. This discrepancy suggests that one possible reason may be the differences between humans and mice. In addition, our modeling mice might enhance the loss of function effect of NOP56 more than in clinical SCA36 patients, due to the deletion designed from Exon 1 to Exon 11 of NOP56 (Fig. 3a). This is the first report of NOP56 knockout mice, and there remains some preliminary. Not only immunofluorescence but also additional detailed analyses are required to clarify the relationship between NOP56 and TDP-43, which is a future issue. Despite having multiple CNS abnormalities, the NOP56^+/− mice had a long survival rate, similar to that of NOP56^+/+ mice. Escaping pathology in the hippocampus and avoiding body weight loss may have contributed to the maintenance of survival in NOP56^+/− mice. Even though they had multiple CNS abnormalities, food intake was maintained in NOP56^+/− mice (Fig. 8i and j). SCA36 patients also show the relative maintenance of swallowing, which is a different characteristic from patients with ALS or spinal bulbar muscular atrophy [23]. The body weight of SCA36 patients has not been focused upon, but there were no reports of body weight loss or malnutrition [12]. Considering the body weight increase observed in NOP56^+/− mice, we considered the possibility that patients with SCA36 might show increase in body weight. However, our clinical survey results showed the opposite; the BMI data for clinical SCA36 patients were significantly lower. Several factors can explain this clinical outcome. SCA36 is characterized as a motor neuron disease, which differentiates it from other SCA subtypes [12]. The muscle atrophy resulting from motor neuron dysfunction can lead to a BMI decrease in SCA36 patients. Additionally, apathy that arises from frontal lobe impairment, as indicated by our FAB results, can also contribute to a reduction in appetite and food intake. According to the discrepancy between NOP56^+/− mice and clinical SCA36 patients, we speculate that the difference in body size increase between humans and mice is related. In suitable environments, mice continue to gain weight as they age, but not in human beings [29, 31]. As results of behavioral and arcuate nucleus hormones analyses, the reason why the body weight increases in NOP56^+/− mice is not the food intake acceleration. Based on tissue immunohistochemical results (Fig. 9e–f), almost all beige adipocytes disappear in NOP56^+/− mice, which suggests the adipocyte characteristics changes. Adding to ubiquitin accumulation, adipocyte metabolic change supports the aging acceleration possibility, which can explain the body weight increase, and further validation is required. This present research focuses on the relationship between the NOP56 loss of function and SCA36 pathology, which is shown in Fig. 10.

The present study has several limitations that should be discussed. First, we examined a limited number of samples in each group in the iPSC study. We analyzed only one clone per patient, resulting in a total of six clones. This limitation affects both the statistical power and the generalizability of the findings. A greater number of clones confirmed the quality of the experiment using iPSC technology and contributed to the statistical findings. Considering the discrepancies between our research results and those reported previously using LCLs, an increase in the number of iPSC clones is desired for future analysis. Second, we did not completely characterize NOP56 loss-of-function pathology. Although NOP56^+/− mice showed that decreased NOP56 expression induces broad CNS abnormalities, additional study using conditional NOP56^−/− mice is required to examine the pathology in more detail. Rescue experiments using Adeno-associated virus vectors will clarify the loss-of-function effect of NOP56 more. Conducting these rescue experiments in our developing transgenic mice is also a future task. To determine more precise timing of NOP56^−/− lethality was difficult because obtaining sufficient DNA samples for genotyping was challenging before Day 12.5 in our institution. Further evidence regarding NOP56 and TDP-43 was not obtained in this study, aside from their colocalization with ubiquitin. To understand NOP56’s role in box C/D snoRNP assembly, it is necessary to evaluate its loss-of-function impact on the regulation of 2’-O-methylation of rRNA.

In conclusion, we found that NOP56 expression is significantly decreased in SCA36. NOP56 is essential for the generation of mouse progeny and NOP56 loss-of-function induces multiple systemic CNS abnormalities, especially in the cerebellum. NOP56^+/− mice phenotypes correspond to the characteristics of SCA36 patients, and the NOP56^+/− mouse is a suitable model for future SCA36 research.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

K.M. conceived the project. K.M., Y.N., H.E., and T.O. designed the experiments. K.M., C.S., Y.T., E.S., H.H., K.S., A.M., and K.S. performed the experiments. K.M., Y.N., Y.T., H.H., and E.S. analyzed the data. K.M., Y.T., and E.S. wrote the manuscript. S.F. supervised the project. All authors read and approved of the final manuscript.

Funding

This work was partly supported by a scholarship grant from the Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care (53-2020) and a Grant-in-Aid for Scientific Research (Early-Career) 18K15373 from the Ministry of Education, Science, Culture, and Sports of Japan. SeqStudio Genetic Analyzer was subsidized by JKA through its promotion funds from KEIRIN RACE (2023 M-182).

Data availability

All experimental raw data included in this study have been deposited and opened in Mendeley Data (https://doi.org/10.17632/2jds5sv823.1). All data according to this study are available from the corresponding authors on request.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committees of Jichi Medical University and Omuta National Hospital, and it obtained approval from the Institutional Review Board (approval no.: Rin-Dai 22–165).

Consent for publication

Not applicable.

Informed consent

Informed consent was obtained from all participants.

Competing interests

The authors declare no competing interests.