Abstract
Background:
Nucleotide repeat expansion disorders constitute a group of clinically and genetically heterogeneous diseases, pathologically characterized by the misfolding, aggregation, and accumulation of proteins. The expanded GGC repeats in the 5′ untranslated region (5’UTR) of the NOTCH2NLC gene translate into uN2CpolyG, a toxic polyglycine protein that leads to neuronal intranuclear inclusion disease (NIID). However, the precise composition of uN2CpolyG and its pathogenic mechanisms remain fully unclear.
Objectives:
We aimed to investigate the proteomic profile of uN2CpolyG and explore the novel neuropathology in NIID patients, which may underlie the disease pathogenesis.
Methods:
Mass spectrometry analysis was performed on purified intranuclear inclusions to investigate the proteomic profile. Three patients with genetically confirmed NIID were enrolled; two participants underwent skin biopsy, and one underwent brain autopsy. Skin and brain tissues derived from these patients were used to examine NOTCH2NLC-related pathological changes.
Results:
A group of enriched proteins interacting with uN2CpolyG were identified, characterized by significant intrinsically disordered regions (IDRs). Among these, we detected the co-localization of uN2CpolyG with PML and FUS in notably distinct patterns, causing significant DNA damage and impaired stress response. Furthermore, FUS- and PML-positive inclusions were confirmed in NIID patients’ tissues.
Conclusion:
Our findings provide novel insights into the proteomic profile and neuropathology of NIID, potentially enlightening the pathogenesis and therapeutic strategies for protein aggregation-related neurodegenerative diseases.
1 Introduction
Nucleotide repeat expansion disorders constitute a group of neurodegenerative diseases characterized by clinical and genetic heterogeneity, with the pathological hallmarks of protein misfolding, aggregation, and accumulation. Examples include polyglutamine (polyQ) tracts in Huntington’s disease and spinocerebellar ataxias (), and dipeptide repeat proteins in familial amyotrophic lateral sclerosis and frontotemporal dementia (). The precise mechanisms underlying the formation and toxicity of these inclusions remain incompletely understood, posing a significant challenge for clinical therapeutics. Neuronal intranuclear inclusion disease (NIID), a recently identified and rare neurodegenerative disorder, is characterized by the widespread distribution of eosinophilic, hyaline intranuclear inclusions in both the nervous system and various visceral organs (Sone et al., 2019). Clinically, the vast majority of cases are adult-onset and exhibit a highly variable phenotype, encompassing diverse combinations of cognitive impairment, cerebellar ataxia, parkinsonism, peripheral neuropathy, autonomic dysfunction, and episodic encephalopathy (; Okubo et al., 2019; Yu et al., 2021). Following the application of skin biopsy and the identification of a GGC repeat expansion in the 5′-untranslated region (5’-UTR) of the NOTCH2NLC gene, the associated phenotypic spectrum has broadened to include a range of neurological disorders (; Tai et al., 2023; Tian et al., 2022). Pathologically, the GGC repeat expansions, located within a small upstream open reading frame in the 5′ UTR (uN2C), are translated into a polyglycine-containing protein (uN2CpolyG), which serves as the primary component of the intranuclear inclusions (IIs) (). These IIs are not exclusive to NIID but are also present in other neurological diseases, such as fragile X-associated tremor/ataxia syndrome () and distal oculopharyngodistal myopathy (Yu et al., 2022). These disorders share considerable clinical overlap, similar pathological outcomes, and even some genetic commonalities.
To date, the detailed composition of IIs remains unclear. Electron microscopy reveals that these inclusions are round-shaped, filamentous materials lacking surrounding membranes. Located near nucleoli, they display typical p62/ubiquitin staining and do not contain other pathological proteins such as α-synuclein, Tau, β-amyloid, or TDP-43 (Mao et al., 2022; McFadden et al., 2005), suggesting that dysfunction of the protein quality control system is involved in NIID pathogenesis. In addition to p62 and ubiquitin, various non-specific proteins—such as promyelocytic leukemia (PML/TRIM19) protein (Nakano et al., 2017), SUMO molecule (Takahashi-Fujigasaki et al., 2006), and heat shock proteins (Pountney et al., 2008), ave. been found in IIs in diseases with unknown genetic etiologies. The retention of these proteins is closely related to their functions, providing potential clues to the pathogenic mechanism. To further investigate the detailed composition of uN2CpolyG, we developed constructs expressing expanded NOTCH2NLC with 104 GGC repeats. Mass spectrometry revealed that the proteins enriched with uN2CpolyG possess significant intrinsically disordered regions. Among these enriched proteins, FUS (Wang et al., 2024), HNRNPM (Liu et al., 2022), and PML (Takahashi et al., 2003) have been previously reported in various neurodegenerative diseases featuring cytoplasmic or nuclear inclusions. Furthermore, we detected distinct co-localization patterns of uN2CpolyG with PML and FUS, which were associated with significant DNA damage and impaired stress response. PML- and FUS-positive nuclear inclusions were subsequently confirmed in skin and brain tissues derived from NIID patients. Our findings provide a novel proteomic profile and neuropathological characteristics of NIID, shedding new light on the contribution of IIs to pathogenesis and highlighting potential avenues for therapeutic development.
2 Materials and methods
2.1 Plasmid constructs
To better recapitulate the pathological characteristics observed in NIID patients, we modified the EGFP-tagged uN2CpolyG construct (containing 104 GGC repeats) originally obtained from the Wang lab (). Briefly, a nuclear localization signal (SV40-NLS) was inserted at the N-terminus of the uN2C sequence, followed by fusion to EGFP, mRuby3, or an HA tag at the C-terminus. To maintain the stability of the expanded GGC repeats, all plasmids were transformed into E. coli (Shanghai Tsingke Biotechnology) and propagated at 37 °C. The coding sequence of FUS was amplified from a HeLa cDNA library, while the fluorescent protein sequences were PCR-amplified from pmEmerald-C1 plasmids. All final constructs were verified by Sanger sequencing.
2.2 Cell culture and transfection
HEK-293 cells were cultured in 6-well or 12-well plates using DMEM (high glucose) supplemented with 10% FBS, under a humidified atmosphere of 5% CO2 at 37 °C. For transient transfection, cells were seeded and cultured until they reached approximately 80% confluence. Plasmid DNA was then delivered into the cells using PEIpro (Polyplus) according to the manufacturer’s protocol. The culture medium was replaced 8–12 h post-transfection. Cells were harvested 36 h or 48 h after transfection for subsequent analyses.
2.3 Purification of intranuclear inclusions
Purification of IIs was performed as previously reported. Briefly, cells were plated in 6 cm dishes for 24 h and then transfected with SV40-NLS uN2CpolyG (GGC104)-EGFP plasmids using PEIpro. Cells were harvested 48 h post-transfection and resuspended in 10 mM Tris–HCl, pH 7.4, containing 2 mM MgCl2, 1% NP-40, 2 mM dithiothreitol (DTT), 1 mM PMSF, and complete protease inhibitor EDTA-free (MCE). Cells were ultrasonic lysed with 35% power lasting 10 s each time, with an interval of 10 s, repeated 3 times, and nuclei were collected by centrifugation at 300 g for 6 min at 4 °C, followed by resuspended in Washing buffer (10 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 0.25 M sucrose, 2 mM DTT, 1 mM PMSF, and MCE) with 1% NP-40, 500 U/mL DNaseI (Beyotime), and 15 U/mL RNase A (Yeasen), mixed for 60 min at 37 °C, and centrifuged at 1500 g for 5 min. The precipitates were resuspended in Washing buffer with 2 M NaCl, mixed for 60 min at 4 °C, and centrifuged at 1,500 g for 5 min. The precipitates then were resuspended in Washing buffer with 4% sarcosyl (Sangon), mixed for 60 min at 4 °C, and centrifuged at 18,600 g for 30 min. Again, the precipitates were washed with Washing buffer with 4% sarcosyl, centrifuged at 18,600 g for 30 min, and collected as the purified inclusions component. The protein was dissolved in 1x SDS loading buffer (50 mM Tris pH 6.8, 10% glycerol, 2% SDS, 0.0012% bromophenol blue) for further mass spectrometry analysis or SDS-PAGE electrophoresis. Due to there were no intranuclear aggregates in the control group, no comparison between the uN2CpolyG and control was performed. To ensure the stability of the experiment, two independent purification-based proteomics analysis have been conducted.
2.4 Immunofluorescence and immunohistochemistry
For immunofluorescence of cultured cell, the post-transfected 48 h cells were fixed with 4% paraformaldehyde (PFA) for 30 min, followed by washing with 1 × PBS for 5 min, 3 times. Cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min and blocked with 2% bovine serum albumin (BSA) for 1 h at room temperature. Cells then were incubated with primary antibodies diluted in 2% BSA for 1 h at room temperature, followed by PBS washing, and incubated with secondary antibodies for 1 h. After washing with PBS, cells were incubated with DAPI (2 μg/mL) for 5 min and the slides were mounted by Antifade Mounting Medium (Vector Lab).
For immunofluorescence of human skin tissues, the samples were plated on adherent slides, rewarmed to room temperature and washed with 1 × PBS three times. The tissues were fixed with 4% PFA for 30 min and permeabilized with 0.2% Triton X-100 in PBS for 20 min, then blocked with 2% BSA for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C, and the tissues were washed with PBS for 10 min, 3 times, then incubated with secondary antibodies for 1 h at room temperature, followed by PBS washing. Sections were incubated with DAPI (2 μg/mL) for 5 min and mounted with Antifade Mounting Medium. All images were taken Dragonfly microscope (Andor).
For immunohisochemistry of human skin or brain tissues, sections were fixed in 4% PFA, paraffin-embedded, cut into 8 μm thickness, and deparaffinized in xylene and rehydrated in ethanol (100–75%). The sections were performed antigen retrieval, followed by blocking with 2% BSA for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C in blocking buffer, and the sections were washed with 1 × PBS for 10 min, 3 times, incubated with secondary antibodies for 1 h at room temperature, followed by PBS washing. Then sections were incubated for 5 min in diaminobenzidine and rinsed with running water for 1 min, followed by staining cell nucleus with Hematoxylin for 3 min, and differentiating tissues with 1% hydrochloric acid alcohol solution for 10 s, and returning to blue with 1% ammonia water for 30 s. Sections were finally rehydrated in ethanol (95–100%) and permeabilized in xylene, and sealed with neutral gum after drying. Images were captured by a Zeiss AxioImager M2 microscope (Zeiss, Jena, Germany) equipped with a CCD digital camera (Vistron Systems, Tuchheim, Germany).
2.5 Microirradiation experiment
Twenty-four hours after transfection with Emerald-FUS with uN2cploy-mRuby3 or mRuby3, HEK-293 cells were plated onto a glass-bottom 3.5 cm dish. Then a region of interest was selected and photo-bleached with a 405 nm laser line at 100% intensity with three cycles. Adjusted the photography time and recorded all fluorescence images within 10 min after laser irradiation. The intensity of FUS at bleached sites was collected. The microarray analysis was performed on FV3000 (Olympus). Data were analyzed with Fiji/Image J.
2.6 Co-immunoprecipitation
Cells seeded in 6-well plates were transfected with EGFP and NLS uN2CpolyG (GGC104)-EGFP. 48 h post transfection, cells were treated with or without heat shock (HS, 43 °C) for 1 h. Then, cells were lysed in 500 μL Lysis buffer (Sangon) for 30 min followed by 3 × 5 s sonication. After centrifuge at 13,000 ×g for 10 min, one aliquot of the supernatants was saved for input, and the rest were incubated with 1.5 μL anti-PML stock solution (Proteintech) overnight at 4 °C. Then the cell lysates were incubated with protein A/G magnetic beads (Beyotime) at 4 °C overnight. After incubation, beads were washed 3 × 5 min with lysis buffer and suspended in 1 x SDS loading buffer (50 mM Tris pH 6.8, 10% glycerol, 2% SDS, 0.0012% bromophenol blue), boiled for 10 min at 100 °C, and analyzed by western blotting.
2.7 Immunoblotting
The protein samples were lysed in 1 x SDS loading buffer and boiled at 100 °C for 10 min. The samples were resolved by SDS-PAGE, and the gel were transferred to 0.45 mm PVDF membranes, followed by blocking with 5% BSA. The primary antibodies were diluted at the recommended ratio and incubated at 4 °C overnight. Blots were then incubated with HRP-conjugated secondary antibodies (1:10000, Beyotime). Bands were scanned by enhanced chemiluminescence using Western Blot Enhancer reagents (Thermo Fisher Scientific). For the immunoblot analysis of purified inclusions, the cytoplasmic protein GAPDH served as the internal loading control for the input samples. In the FUS rescue experiments, HEK293T cells were co-transfected to express uN2CpolyG-EGFP and FUS-HA at the indicated ratios. Following treatment with etoposide (ETO; 10 μM for 1 h) and a subsequent 2-h recovery period, cell lysates were analyzed by immunoblotting. The expression of phosphorylated ATM (pATM) was assessed, with the HA tag serving as a transfection control.
2.8 Human participants and tissues
Two patients diagnosed with NIID were recruited from the Department of Neurology at Shanghai Sixth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. The diagnosis was established based on a combination of characteristic clinical manifestations and the identification of abnormal GGC repeat expansions (>60 repeats) within the NOTCH2NLC gene. Systematic clinical evaluations and brain magnetic resonance imaging (MRI) assessments were performed and confirmed by at least two senior neurologists to ensure diagnostic accuracy. Informed consent was obtained from all participants for the collection of skin tissue biopsies, which were harvested from the distal lateral calf (approximately 10 cm above the lateral malleolus). To serve as a control for investigating NOTCH2NLC-related pathological changes, a patient diagnosed with diabetic peripheral neuropathy (without GGC repeat expansions) was included. Genetic testing, including repeat-primed PCR and fluorescence amplicon length PCR, was performed to determine the GGC repeats within the NOTCH2NLC gene as previously described (Sone et al., 2019). The brain autopsy tissue of a patient with genetically confirmed NIID was obtained from Department of neurology, Peking University First Hospital (Wang et al., 2024). The study was approved by the ethics committee of Shanghai Sixth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (Approval No: 2021–219). All the participants or their guardians provided written informed consent.
2.9 Statistical analysis
Representative data for immunoblotting and microscopy imaging were obtained from at least two independent experiments. SPSS Statistics Version 26.0 (IBM Corp, Armonk, New York, USA) and GraphPad Prism Version 9 (GraphPad Software, San Diego, California, USA) were used to analyze the data. The difference between two independent groups were analyzed by non-parametric testing. For p < 0.05, differences were considered statistically significant (*p < 0.05; **p < 0.01).
3 Results
3.1 Design of constructs expressing intranuclear inclusions
The original plasmid expressing uN2CpolyG was A gift from Wang’s lab from Department of Neurology, Peking University First Hospital. During our initial experiments involving transient transfection, we observed that the majority of aggregates formed cytoplasmic inclusions. This observation was inconsistent with the established pathology of NIID patients, particularly considering the significant differences in signal transduction and protein quality control mechanisms between the cytoplasm and nucleus. To more accurately recapitulate the pathological hallmark of NIID, specifically, the formation of intranuclear inclusions—we engineered the plasmid by inserting a nuclear localization signal (SV40-NLS) at the N-terminus of the uN2C sequence. This modification significantly increased the proportion of intranuclear inclusions (Figure 1A). Furthermore, we validated that these intranuclear uN2CpolyG aggregates exhibited strong co-localization with established landmark proteins, including P62 and SUMO2/3 (Figure 1B).
Figure 1
3.2 Proteome associated with uN2CpolyG possess intrinsically disordered regions
To further characterize the interactome of intranuclear inclusions, mass spectrometry (MS) was performed on purified uN2CpolyG aggregates. In contrast to previous studies utilizing affinity tags for immunoprecipitation, this direct analysis of purified inclusions identified over 450 unidentified proteins that were sequestered within the aggregates (; Liu et al., 2022). Gene Ontology biological process enrichment analysis found the enriched proteins were mainly involved in various RNA metabolism and SUMOylation modification. From the subcellular localization, these proteins were closely associated with multiple cytoplasmic organelles and nuclear bodies (Figure 1C). Due to these nuclear bodies are membraneless organelles with liquid-like properties formed by liquid–liquid phase separation, which can be driven by proteins or nucleic acids with special intrinsically disordered domains (; Uversky, 2017). Indeed, structural characterization analysis further confirmed that the majority of these enriched proteins contained intrinsically disordered regions (Figure 1D). Among them, we noted that FUS (97.4%), HNRNPM (61.4%), and PML (32.6%) had previously been implicated in various neurodegenerative diseases characterized by nuclear inclusions. Co-immunoprecipitation assays demonstrated a direct interaction between uN2CpolyG and FUS or PML (Figure 1E). In our cellular model, we observed distinct co-localization patterns between uN2CpolyG aggregates and PML or FUS. Specifically, PML formed a ring-like structure surrounding the uN2CpolyG aggregates (Figure 1F), whereas FUS was entirely sequestered within the inclusions (Figure 1G).
3.3 uN2CpolyG cause significant DNA damage and impaired stress response
Previous studies have shown that both PML and FUS are involved in DNA damage repair, especially the FUS dependent liquid–liquid phase separation is necessary for initiating DNA damage responses (; ). To investigate the cellular response to DNA damage, we utilized microirradiation to induce DNA double-strand breaks. We observed that FUS was rapidly recruited to the damage sites in a time-dependent manner (Figure 2A). However, this recruitment was significantly delayed in cells expressing uN2CpolyG compared to controls (Figure 2B). Consequently, due to this impaired recruitment, we further confirmed a significant increase in DNA damage markers—including pATM, γH2AX, and 53BP1 foci—in uN2CpolyG-expressing cells, indicating the accumulation of unresolved DNA damage (Figure 2C). Additionally, PML nuclear bodies were found to be involved in the cellular stress response under these conditions (). To investigate the functional impact of PML under stress conditions, we subjected cells to heat shock (HS, 43 °C, 1 h). In control cells, the number of PML nuclear bodies significantly increased following HS. However, cells expressing uN2CpolyG aggregates failed to mount this stress response, exhibiting abnormal PML distribution (Figure 2D). Furthermore, compared to controls, the interaction between PML and SUMO2/3 was dramatically disrupted in cells containing uN2CpolyG inclusions post-HS (Figure 2E), suggesting that intranuclear uN2CpolyG sequesters and incapacitates PML bodies, thereby impairing their ability to respond to cellular stress. We further found that FUS overexpression significantly reduced DNA damage (pATM foci) in a dose-dependent manner in uN2CpolyG-expressing cells (Figure 2F), indicating that the sequestration of FUS by uN2CpolyG directly contributes to its functional impairment.
Figure 2
3.4 Clinical and pathological characteristics of NIID patients
To further validate the interaction between uN2CpolyG and FUS or PML in vivo, we enrolled three patients with genetically confirmed NIID. Patient 1 (NIID case #1) was a 67-year-old male presenting with leg weakness as the initial symptom, which gradually progressed to drowsiness, memory impairment, and urinary/defecatory dysfunction. Patient 2 (NIID case #2) was a 68-year-old female with a history of recurrent stroke-like episodes accompanied by urinary difficulty and constipation. Brain MRI for both patients revealed abnormalities, primarily including high-intensity signals along the corticomedullary junction on DWI, diffuse T2WI/FLAIR white matter lesions, and cerebral atrophy (Figure 3A). Notably, no microbleeds were observed on SWI imaging. Skin pathological examinations further identified p62-positive intranuclear inclusions within sweat gland cells (Figures 3B,C). Intranuclear inclusions were captured as round-shaped filament materials without separating membranes under electron microscope (Figures 3D,E). Intranuclear inclusions were captured as round-shaped filament materials without separating membranes under electron microscope (Figures 3D,E). The third patient (NIID case#3) is a 61-year-old female with 8-year history of paroxysmal headache, and slowing progressive personality changes, abnormal behavior, cognitive decline and repeated syncope. She gradually developed to stay in bed, hypersomnia, urinary and fecal incontinence, recurrent high fever in the later stage. The patient ultimately died of respiratory failure and underwent brain autopsy. The detailed clinical, pathological, and genetic characteristics of all NIID patients are shown in Supplementary Table S1.
Figure 3
3.5 Detection of PML- and FUS-positive intranuclear inclusions in NIID tissues
We next examined the relationship between intranuclear inclusions and PML or FUS in skin and brain tissues from NIID patients. Immunofluorescence double staining for PML and SUMO2/3 revealed that PML was recruited around SUMO2/3-labeled inclusions in a ring-like structure in patient skin cells, whereas it exhibited a typical dot-like pattern in controls (Figure 4A). This specific distribution pattern was similarly confirmed in brain tissue (Figure 4B). Furthermore, compared to controls, cells containing intranuclear inclusions showed a significant decrease in the number of PML nuclear bodies alongside a marked increase in their volume, indicating that nuclear inclusions profoundly affect the morphology and properties of PML bodies (Figures 4C,E), potentially leading to functional impairment. Additionally, FUS was almost completely sequestered into intranuclear inclusions in NIID skin cells, contrasting with its dispersed nuclear distribution in the control group (Figure 4F). Immunohistochemical staining also identified several circular, FUS-immunopositive aggregates in both NIID skin (Figure 4G) and brain (Figure 4H) tissues. The presence of PML- and FUS-positive intranuclear inclusions suggests that the abnormal sequestration of these proteins plays a crucial role in the pathological mechanisms of NIID.
Figure 4
4 Discussion
NIID, a rare progressive neurodegenerative disorder, is named based on the characteristic pathology with the findings of eosinophilic nuclear inclusions in the nervous system and various visceral organs of patients (; Sung et al., 1980). Since the discovery of the GGC repeat expansion in the NOTCH2NLC gene, extensive efforts have been devoted to elucidating the pathogenesis of NIID; however, the underlying molecular mechanisms remain largely unknown. While intranuclear inclusions (IIs) are defined by the abnormal aggregation of diverse proteins, their specific composition is not yet fully characterized. Moreover, there is currently no definitive explanation for how uN2CpolyG proteins, which lack a canonical nuclear localization signal (NLS), enter the nucleus. The NOTCH2NLC gene expresses two primary transcript variants, giving rise to two distinct proteins: an early-terminated uN2CpolyG and a longer uN2CpolyG-N2C fusion protein. Whether differential expression of these isoforms in patient tissues contributes to their divergent localization patterns requires further investigation. To better model the pathological features of NIID, we modified the plasmid by inserting an SV40-NLS at the N-terminus of the uN2C sequence. MS analysis of purified intranuclear inclusions identified a specific interactome associated with uN2CpolyG. Notably, the most enriched proteins contained intrinsically disordered regions (IDRs), a key molecular feature that drives liquid–liquid phase separation (LLPS) (Mohanty et al., 2022). IDR is typically composed of low complexity domain. Proteins containing IDR are called intrinsic disordered proteins, which lack a stable three-dimensional structure, providing an structural basis for the multivalent weakly adhesive molecules interaction (Trivedi and Nagarajaram, 2022). The essence of LLPS of intracellular biomolecules is the spontaneous dynamic aggregation and separation, thereby efficiently exerting their complex and diverse biological functions (Zhang et al., 2020). Previous studies indicated that specific protein and nucleic acid components are separated by LLPS to form various membraneless organelles in the nucleus [nucleolus, Cajal bodies, nuclear plaques, perinuclear plaques, PML nuclear body (NB) and cytoplasm] (P bodies, stress granules), which participate in complex cellular biological reactions, such as RNA metabolism, transcriptional regulation, genome stability, and stress response (; Zhao and Zhang, 2020). Recently, studies have found that abnormal phase separation can cause protein aggregation, deposition, and denaturation, which is closely related to various neurodegenerative diseases (; Patel et al., 2015; Ray et al., 2020; Solomon et al., 2021). Thus, we propose that aberrant phase transitions driven by repetitive peptides with low-complexity domains play a pivotal role in protein aggregate formation, warranting further investigation to delineate their specific contributions.
Furthermore, we observed abnormal sequestration effects mediated by uN2CpolyG. Specifically, PML encircled the uN2CpolyG aggregates in a ring-like structure, whereas FUS was completely sequestered into the inclusion core, leading to significant DNA damage and an impaired cellular stress response. PML, also known as tripartite motif-containing 19 (TRIM19), serves as a major scaffold protein for PML nuclear bodies (NBs). These membrane-less nuclear organelles play diverse roles in maintaining cellular homeostasis, including functions in apoptosis, DNA repair, gene regulation, and antiviral defense (). Previous study showed PML can promote the degradation of misfolded proteins through SUMO-targeted ubiquitin ligase pathway (). The loss of PML significantly exacerbate the behavior and pathological phenotype of spinocerebellar ataxia type 1 mice (), while the aggregation of toxic polyQ proteins can be inhibited by inducing endogenous PML overexpression (). And FUS is mainly distributed in nucleus, and can continuously shuttle between nucleus and cytoplasm (). As a multifunctional RNA binding protein, FUS mainly participates in the process of RNA metabolism, stress response and DNA damage repair (; Masuda et al., 2016; Naumann et al., 2018). Mutant FUS is involved in ALS and frontotemporal lobar degeneration with the pathological findings of FUS-positive, while tau and TDP-43-negative cytoplasmic aggregates (Neumann et al., 2009). Moreover, PML or FUS-positive inclusions have also previously reported in patients with Huntington’s disease, spinocerebellar ataxia, or neuronal intranuclear hyaline inclusion disease with unknown genetic etiology (Mori et al., 2012; Nakano et al., 2017; Takahashi et al., 2003; Takahashi-Fujigasaki et al., 2006; Woulfe et al., 2010), suggesting the dysfunction of PML and FUS play a common pathogenic role in IIs-related neurodegenerative diseases. Very recently, Deng’s study showed the aberrant interaction between uN2CpolyG and FUS may lead to impaired stress granule formation and dysregulated miRNA biogenesis (Wang et al., 2024). Accumulation of DNA damage and abnormal stress response are believed to induce cell death and/or protein dysfunction, leading to chronic inflammation, which is an important risk factor for promoting disease or aging (Wang et al., 2021). Therefore, our identification of PML- and FUS-positive inclusions in NIID patient tissues further corroborates their critical pathogenic role. Further investigation is required to elucidate the common molecular mechanisms underlying the dysfunction of proteins sequestered by IIs, paving the way for potential therapeutic strategies for these disorders.
In summary, our study demonstrates that proteins enriched in uN2CpolyG inclusions contain prominent IDRs, and that abnormal uN2CpolyG sequestration induces significant DNA damage and impairs the stress response. Furthermore, we provide novel neuropathological insights into NIID. These findings may illuminate new avenues for understanding the pathogenesis and developing potential therapies for protein aggregation-related neurodegenerative diseases.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.
Ethics statement
The studies involving humans were approved by the ethics committee of Shanghai Sixth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (approval no: 2021–219). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Author contributions
FZ: Data curation, Investigation, Methodology, Resources, Writing – original draft. JZ: Data curation, Investigation, Methodology, Resources, Writing – original draft. YW: Data curation, Formal analysis, Resources, Writing – original draft. YC: Investigation, Writing – original draft. XS: Investigation, Writing – original draft. WL: Investigation, Writing – original draft. JW: Investigation, Writing – original draft. XH: Resources, Writing – original draft. SH: Formal analysis, Resources, Writing – review & editing. XL: Investigation, Methodology, Resources, Supervision, Writing – review & editing. LC: Funding acquisition, Project administration, Resources, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was financed by the National Natural Science Foundation of China (82371255), Program for Shanghai Outstanding Academic Leaders (23XD1402500), Shanghai Science and Technology Innovation Action Plan (23DZ2291500), Program for Outstanding Medical Academic Leader of Shanghai (2022LJ011).
Acknowledgments
We gratefully acknowledge the patients and their families who provided the tissues for this study. We also sincerely appreciate Prof. Z. X. Wang from the Department of Neurology, Peking University First Hospital, Beijing, China, for kindly providing the human brain samples and the original uN2CpolyG104 × plasmid.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi.2026.1827292/full#supplementary-material
References
1
AhmadA.UverskyV. N.KhanR. H. (2022). Aberrant liquid-liquid phase separation and amyloid aggregation of proteins related to neurodegenerative diseases. Int. J. Biol. Macromol.220, 703–720. doi: 10.1016/j.ijbiomac.2022.08.132,
2
AnderssonM. K.StahlbergA.ArvidssonY.OlofssonA.SembH.StenmanG.et al. (2008). The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response. BMC Cell Biol.9:37. doi: 10.1186/1471-2121-9-37,
3
BoivinM.DengJ.PfisterV.GrandgirardE.Oulad-AbdelghaniM.MorletB.et al. (2021). Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders: the polyG diseases. Neuron109, 1825–1835.e5. doi: 10.1016/j.neuron.2021.03.038,
4
DeJesus-HernandezM.MackenzieI. R.BoeveB. F.BoxerA. L.BakerM.RutherfordN. J.et al. (2011). Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron72, 245–256. doi: 10.1016/j.neuron.2011.09.011,
5
DellaireG.Bazett-JonesD. P. (2004). PML nuclear bodies: dynamic sensors of DNA damage and cellular stress. BioEssays26, 963–977. doi: 10.1002/bies.20089,
6
FanY.XuY.ShiC. (2022). NOTCH2NLC-related disorders: the widening spectrum and genotype-phenotype correlation. J. Med. Genet.59, 1–9. doi: 10.1136/jmedgenet-2021-107883,
7
FoninA. V.AntifeevaI. A.KuznetsovaI. M.TuroverovK. K.ZaslavskyB. Y.KulkarniP.et al. (2022). Biological soft matter: intrinsically disordered proteins in liquid-liquid phase separation and biomolecular condensates. Essays Biochem.66, 831–847. doi: 10.1042/EBC20220052,
8
GaoC.GuJ.ZhangH.JiangK.TangL.LiuR.et al. (2022). Hyperosmotic-stress-induced liquid-liquid phase separation of ALS-related proteins in the nucleus. Cell Rep.40:111086. doi: 10.1016/j.celrep.2022.111086,
9
GuoL.GiassonB. I.Glavis-BloomA.BrewerM. D.ShorterJ.GitlerA. D.et al. (2014). A cellular system that degrades misfolded proteins and protects against neurodegeneration. Mol. Cell55, 15–30. doi: 10.1016/j.molcel.2014.04.030,
10
HagermanR. J.HagermanP. (2016). Fragile X-associated tremor/ataxia syndrome - features, mechanisms and management. Nat. Rev. Neurol.12, 403–412. doi: 10.1038/nrneurol.2016.82,
11
HuangX. R.TangB. S.JinP.GuoJ. F. (2022). The phenotypes and mechanisms of NOTCH2NLC-related GGC repeat expansion disorders: a comprehensive review. Mol. Neurobiol.59, 523–534. doi: 10.1007/s12035-021-02616-2,
12
JanerA.MartinE.MurielM. P.LatoucheM.FujigasakiH.RubergM.et al. (2006). PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins. J. Cell Biol.174, 65–76. doi: 10.1083/jcb.200511045,
13
Keiten-SchmitzJ.WagnerK.PillerT.KaulichM.AlbertiS.MullerS. (2020). The nuclear SUMO-targeted ubiquitin quality control network regulates the dynamics of cytoplasmic stress granules. Mol. Cell79, 54–67.e7. doi: 10.1016/j.molcel.2020.05.017,
14
LevoneB. R.LenzkenS. C.AntonaciM.MaiserA.RappA.ConteF.et al. (2021). FUS-dependent liquid-liquid phase separation is important for DNA repair initiation. J. Cell Biol.220:8030. doi: 10.1083/jcb.202008030,
15
LiebermanA. P.ShakkottaiV. G.AlbinR. L. (2019). Polyglutamine repeats in neurodegenerative diseases. Annu. Rev. Pathol.14, 1–27. doi: 10.1146/annurev-pathmechdis-012418-012857,
16
LindenbergR.RubinsteinL. J.HermanM. M.HaydonG. B. (1968). A light and electron microscopy study of an unusual widespread nuclear inclusion body disease. A possible residuum of an old herpesvirus infection. Acta Neuropathol.10, 54–73. doi: 10.1007/bf00690510,
17
LiuQ.ZhangK.KangY.LiY.DengP.LiY.et al. (2022). Expression of expanded GGC repeats within NOTCH2NLC causes behavioral deficits and neurodegeneration in a mouse model of neuronal intranuclear inclusion disease. Sci. Adv.8:eadd6391. doi: 10.1126/sciadv.add6391,
18
MaoC.ZhouL.LiJ.PangJ.ChuS.JinW.et al. (2022). Clinical-neuroimaging-pathological relationship analysis of adult onset neuronal Intranuclear inclusion disease (NIID). BMC Neurol.22:486. doi: 10.1186/s12883-022-03025-1,
19
MasudaA.TakedaJ.OhnoK. (2016). FUS-mediated regulation of alternative RNA processing in neurons: insights from global transcriptome analysis. Wiley Interdiscip. Rev.: RNA7, 330–340. doi: 10.1002/wrna.1338,
20
McFaddenK.HamiltonR. L.InsalacoS. J.LavineL.Al-MateenM.WangG.et al. (2005). Neuronal intranuclear inclusion disease without polyglutamine inclusions in a child. J. Neuropathol. Exp. Neurol.64, 545–552. doi: 10.1093/jnen/64.6.545,
21
MohantyP.KapoorU.Sundaravadivelu DevarajanD.PhanT. M.RizuanA.MittalJ. (2022). Principles governing the phase separation of multidomain proteins. Biochemistry61, 2443–2455. doi: 10.1021/acs.biochem.2c00210,
22
MoriF.TanjiK.KonT.OdagiriS.HattoriM.HoshikawaY.et al. (2012). FUS immunoreactivity of neuronal and glial intranuclear inclusions in intranuclear inclusion body disease. Neuropathol. Appl. Neurobiol.38, 322–328. doi: 10.1111/j.1365-2990.2011.01217.x,
23
NakanoY.Takahashi-FujigasakiJ.SengokuR.KanemaruK.AraiT.KandaT.et al. (2017). PML nuclear bodies are altered in adult-onset neuronal Intranuclear hyaline inclusion disease. J. Neuropathol. Exp. Neurol.76, 585–594. doi: 10.1093/jnen/nlx039,
24
NaumannM.PalA.GoswamiA.LojewskiX.JaptokJ.VehlowA.et al. (2018). Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation. Nat. Commun.9:335. doi: 10.1038/s41467-017-02299-1,
25
NeumannM.RademakersR.RoeberS.BakerM.KretzschmarH. A.MackenzieI. R. (2009). A new subtype of frontotemporal lobar degeneration with FUS pathology. Brain132, 2922–2931. doi: 10.1093/brain/awp214,
26
OkuboM.DoiH.FukaiR.FujitaA.MitsuhashiS.HashiguchiS.et al. (2019). GGC repeat expansion of NOTCH2NLC in adult patients with leukoencephalopathy. Ann. Neurol.86, 962–968. doi: 10.1002/ana.25586,
27
PatelA.LeeH. O.JawerthL.MaharanaS.JahnelM.HeinM. Y.et al. (2015). A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell162, 1066–1077. doi: 10.1016/j.cell.2015.07.047,
28
PountneyD. L.RafteryM. J.CheginiF.BlumbergsP. C.GaiW. P. (2008). NSF, Unc-18-1, dynamin-1 and HSP90 are inclusion body components in neuronal intranuclear inclusion disease identified by anti-SUMO-1-immunocapture. Acta Neuropathol.116, 603–614. doi: 10.1007/s00401-008-0437-4,
29
RayS.SinghN.KumarR.PatelK.PandeyS.DattaD.et al. (2020). Α-Synuclein aggregation nucleates through liquid–liquid phase separation. Nat. Chem.12, 705–716. doi: 10.1038/s41557-020-0465-9,
30
SolomonD. A.SmikleR.ReidM. J.MizielinskaS. (2021). Altered phase separation and cellular impact in C9orf72-linked ALS/FTD. Front. Cell. Neurosci.15:664151. doi: 10.3389/fncel.2021.664151,
31
SoneJ.MitsuhashiS.FujitaA.MizuguchiT.HamanakaK.MoriK.et al. (2019). Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease. Nat. Genet.51, 1215–1221. doi: 10.1038/s41588-019-0459-y,
32
SungJ. H.Ramirez-LassepasM.MastriA. R.LarkinS. M. (1980). An unusual degenerative disorder of neurons associated with a novel intranuclear hyaline inclusion (neuronal intranuclear hyaline inclusion disease). A clinicopathological study of a case. J. Neuropathol. Exp. Neurol.39, 107–130. doi: 10.1097/00005072-198003000-00001,
33
TaiH.WangA.ZhangY.LiuS.PanY.LiK.et al. (2023). Clinical features and classification of neuronal Intranuclear inclusion disease. Neurol Genet.9:e200057. doi: 10.1212/NXG.0000000000200057,
34
TakahashiJ.FujigasakiH.IwabuchiK.BruniA. C.UchiharaT.El HachimiK. H.et al. (2003). PML nuclear bodies and neuronal intranuclear inclusion in polyglutamine diseases. Neurobiol. Dis.13, 230–237. doi: 10.1016/S0969-9961(03)00080-9,
35
Takahashi-FujigasakiJ.AraiK.FunataN.FujigasakiH. (2006). SUMOylation substrates in neuronal intranuclear inclusion disease. Neuropathol. Appl. Neurobiol.32, 92–100. doi: 10.1111/j.1365-2990.2005.00705.x,
36
TianY.ZhouL.GaoJ.JiaoB.ZhangS.XiaoQ.et al. (2022). Clinical features of NOTCH2NLC-related neuronal intranuclear inclusion disease. J. Neurol. Neurosurg. Psychiatry93, 1289–1298. doi: 10.1136/jnnp-2022-329772,
37
TrivediR.NagarajaramH. A. (2022). Intrinsically disordered proteins: an overview. Int. J. Mol. Sci.23:4050. doi: 10.3390/ijms232214050,
38
UverskyV. N. (2017). Protein intrinsic disorder-based liquid-liquid phase transitions in biological systems: complex coacervates and membrane-less organelles. Adv. Colloid Interf. Sci.239, 97–114. doi: 10.1016/j.cis.2016.05.012,
39
WangH.LautrupS.CaponioD.ZhangJ.FangE. F. (2021). DNA damage-induced neurodegeneration in accelerated ageing and Alzheimer's disease. Int. J. Mol. Sci.22:6748. doi: 10.3390/ijms22136748,
40
WangH.ZhengY.YuJ.MengL.ZhangW.HongD.et al. (2024). Pathologic changes in neuronal intranuclear inclusion disease are linked to aberrant FUS interaction under hyperosmotic stress. Neurobiol. Dis.190:106391. doi: 10.1016/j.nbd.2023.106391,
41
WoulfeJ.GrayD. A.MackenzieI. R. (2010). FUS-immunoreactive intranuclear inclusions in neurodegenerative disease. Brain Pathol.20, 589–597. doi: 10.1111/j.1750-3639.2009.00337.x,
42
YuJ.LuanX. H.YuM.ZhangW.LvH.CaoL.et al. (2021). GGC repeat expansions in NOTCH2NLC causing a phenotype of distal motor neuropathy and myopathy. Ann. Clin. Transl. Neurol.8, 1330–1342. doi: 10.1002/acn3.51371,
43
YuJ.ShanJ.YuM.DiL.XieZ.ZhangW.et al. (2022). The CGG repeat expansion in RILPL1 is associated with oculopharyngodistal myopathy type 4. Am. J. Hum. Genet.109, 533–541. doi: 10.1016/j.ajhg.2022.01.012,
44
ZhangH.JiX.LiP.LiuC.LouJ.WangZ.et al. (2020). Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases. Sci. China Life Sci.63, 953–985. doi: 10.1007/s11427-020-1702-x,
45
ZhaoY. G.ZhangH. (2020). Phase separation in membrane biology: the interplay between membrane-bound organelles and Membraneless condensates. Dev. Cell55, 30–44. doi: 10.1016/j.devcel.2020.06.033,
Summary
Keywords
neuronal intranuclear inclusion disease, neuropathology, NOTCH2NLC gene, protein aggregation, proteomic
Citation
Zhan F, Zhu J, Wang Y, Cao Y, Shen X, Lv W, Wei J, Huang X, Hou SX, Luan X and Cao L (2026) Novel proteomics and neuropathology of NOTCH2NLC-related neuronal intranuclear inclusion disease. Front. Aging Neurosci. 18:1827292. doi: 10.3389/fnagi.2026.1827292
Received
10 March 2026
Revised
27 April 2026
Accepted
19 June 2026
Published
03 July 2026
Volume
18 - 2026
Edited by
Luciana Mosca, Sapienza University of Rome, Italy
Reviewed by
Li Zeng, National Neuroscience Institute (NNI), Singapore
Yangguang Lu, Wenzhou Medical University, China
Updates
Copyright
© 2026 Zhan, Zhu, Wang, Cao, Shen, Lv, Wei, Huang, Hou, Luan and Cao.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xinghua Luan, green_lxh@sina.com; Li Cao, caoli2000@yeah.net
† These authors have contributed equally to this work
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