{'fraction_non_alphanumeric': 0.03943903835145964, 'fraction_numerical': 0.030223239839725243, 'mean_word_length': 5.081099895579533, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The enhanced association between mutant CHMP2B and spastin is a novel pathological link between frontotemporal dementia and hereditary spastic paraplegias

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-022-01476-8.


Introduction
Frontotemporal dementia (FTD), associated with progressive atrophy of the frontal and/or temporal lobes of the brain, is the second most common form of dementia before 65 years of age [17]. FTD is characterized by progressive deterioration in social behavior, personality and language, and regarded as part of the spectrum disorder with the motor neuron disease amyotrophic lateral sclerosis (ALS). In particular, genetic mutations in a number of genes can cause both FTD and ALS, suggesting common pathogenic molecular mechanisms [6,8]. Among them, mutations in charged multivesicular body protein 2B (CHMP2B) are especially interesting, as they are highly pathogenic in FTD linked to chromosome 3 (FTD-3) [19] and also found in some ALS cases [4,18] and patients with early-onset Alzheimer's disease (AD) [9].
CHMP2B encodes a subunit of the endosomal sorting complex required for transport III (ESCRT-III) complex whose molecular function was first shown to be essential during the formation of multivesicular bodies (MVBs) [2]. ESCRTs also play key roles in other cellular processes such as cytokinesis, virus budding, nuclear membrane repair, and autophagy [16]. In FTD3, a splicing site mutation in CHMP2B results in a C-terminal truncation of the protein missing the Microtubule Interaction Motif (MIM), named CHMP2B Intron5 [19]. A series of cell biology studies indicate that this mutant CHMP2B protein Open Access *Correspondence: fen-biao.gao@umassmed.edu exhibits enhanced association to its binding partner CHMP4B and blockage in ESCRT-III disassembly [10,11], leading to compromised endosomal functions [13,22,23,25] and autophagy defects [10,11,14]. It remains to be identified what other cellular and molecular pathways are affected by CHMP2B Intron5 .
Spastic paraplegia 4 (SPG4), the most common autosomal dominant form of hereditary spastic paraplegias (HSP), is caused by loss of function mutations in the SPAST gene that encodes spastin, a member of microtubule severing protein [5,20,21]. SPG4 patients show symptoms of clinical dementia but the underlying mechanisms remain unclear [26]. In this study, we find that spastin associates with greater affinity to CHMP2B Intron5 than to wildtype CHMP2B, revealing a novel potential pathological link between FTD and HSP.

Mice and genotyping
The tTA:CHMP2B Intron5 and tTA:CHMP2B WT mice used in this study have been described [7]; both males and females were used. All procedures involving mice were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts Chan Medical School.

Drosophila genetics
Flies were maintained on a 12-h light/12-h dark cycle on standard cornmeal-yeast agar medium at 25 °C. UAS-CHMP2B Intron5 flies used were described previously [1]. GMR-Gal4, UAS-RNAi SPAST (#27,570), and UAS-RNAi_SPAST (#53,331) fly lines were from the Bloomington Drosophila Stock Center. For genetic interaction studies, the recombined fly line (GMR-Gal4:UAS-CHMP2B Intron5 ) was crossed with UAS-RNAi_SPAST flies. To quantify the retinal degeneration phenotype, we classified the eye phenotype, with or without SPAST downregulation, into three groups: severe (+ + +), medium (+ +), and weak ( +). This classification was based on the relative abundance of black spots on the eye, ranging from a dozen or so scattered spots ( +) to spots covering approximately 50-70% or more of the eye surface (+ + +).

Proteomic analysis of CHMP2B Intron5 interacting proteins
To identify proteins that interact with CHMP2B Intron5 , proteins in experimental and control IP samples were electrophoresed a short distance into a polyacrylamidesodium dodecyl sulfate gel and stained with the Coomassie Brilliant Blue (Bio-Rad). In-gel digestion and liquid chromatography-tandem mass spectrometry analysis were done by the Mass Spectrometry Facility at the University of Massachusetts Chan Medical School. Protein abundance was estimated with IBAQ quantification, in which summed peptide intensities are normalized to the number of theoretically observable peptides of the protein. pCMV-3/FLAG served as a control to exclude nonspecific interacting proteins. Interacting proteins that were not associated with FLAG proteins but bound more to FLAG-CHMP2B Intron5 than FLAG-CHMP2B WT were selected for further analyses. Total proteins were further ranked by iBAA value from most to least abundant. Mass spectrometry (MS) analysis was done by the UMass Chan Medical School Mass Spec Core with a standard protocol as published before [12].

Western blots
The mouse cortex was dissected, quickly frozen at − 80 °C, homogenized, and sonicated in RIPA buffer with proteinase and phosphatase inhibitors (CST, catalog no. 5872). The cultured cells were lysed in RIPA buffer (Thermo Scientific). The protein extract was centrifuged to remove tissue debris, and boiled for 5 min. Protein (20 μg) from each sample was subjected to SDS-PAGE using 4-20% precast gels (Bio-Rad) and immunobloted with the following primary antibodies: rabbit anti-spastin (Proteintech, catalog no. 22792-1-AP; 1:1000) and mouse anti-β-actin (Sigma-Aldrich, catalog no. A2228; 1:3000), overnight at 4 °C. After incubation, immunoblots were washed and incubated with IRDye fluorescent anti-rabbit and anti-mouse secondary antibodies (LI-COR Biosciences). Images were acquired with a LI-COR CLx Odyssey System.

Subcellular fractionation and solubility analysis
HEK293 cells were collected 48 h after transfection and subjected to subcellular fractionation with a ProteoExtract Subcellular Proteome Extraction Kit (Millipore, catalog no. 539790), according to the manufacturer's protocol for adherent cells. If some cells became nonadherent during the protocol, the cytosolic, membrane, and nuclear fractions were spun at 750 g, 5500 g, and 6800 g, respectively, for 10 min at 4 °C, to remove any contamination from later fractions. Proteins were resolved by SDS-PAGE and immunoblotted with spastin antibody (Proteintech, catalog no. 22792-1-AP; 1:1000). For SPAST solubility analysis, cells were seeded into six-well dishes at 250,000 cells/well; 48 h after transfections, cells were washed with PBS, released with 0.25% trypsin, and resuspended in DMEM pre-warmed to 37 °C. The cells were then spun down, washed with PBS, and resuspended in 20 μl of PBS. The cells were lysed by two cycles of flash freezing on dry ice and rapidly thawing at 42 °C. The lysate was spun at 1000 g, and the resulting supernatant was transferred to a new tube and re-spun to remove any insoluble material. The pellet was rinsed 3 times with PBS and resuspended in the corresponding volume of supernatant and briefly sonicated with a tip sonicator (Sonopuls, catalog no. 2070). Equivalent fractions of total volume for 100 ng of supernatant and resuspended pellet were boiled with SDS loading buffer (50 mm Tris-Cl, pH 6.8, 2% (2 w/v) SDS, 0.1% (w/v) bromophenol blue) and 10 mm dithiothreitol, separated by SDS-PAGE on 10% polyacrylamide-sodium dodecyl sulfate gels and immunoblotted with spastin antibody (Proteintech, catalog no. 22792-1-AP; 1:1000).

Immunostaining of mouse brain sections
Paraffin-embedded tissue sections were deparaffinized and hydrated in a series of graded alcohols. After antigen retrieval with citrate buffer (Sigma, C9999), the sections were washed once with water, treated with BLOXALL Endogenous Blocking Solution (Vector Lab, SP-6000-100) for 10 min washed with PBST for 10 min, blocked with Dako blocking reagent for 24 h, and incubated overnight with guinea pig anti-p62 (Progen, catalog no. GP62-C) and polyclonal anti-SPAST (Proteintech, catalog no. 22792) and 0.1% Triton-X 100; the antibodies were diluted 1:200 in DAKO antibody diluent (Agilent, S302283-2) overnight. The sections were washed three times with PBST for 10 min each and incubated with Alexa-conjugated secondary antibodies (Invitrogen, catalog nos. A-11075 and A32790) in detergent-supplemented DAKO antibody diluent buffer for 2 h in the dark. The sections were washed three times with PBST for 10 min each and mounted with DAPI Fluoromount-G Mounting Medium (Invitrogen). The total surface of stained brain sections from three mice per genotype group was scanned (Sanderson Center for Optical Experimentation) (SCOPE) (UMass Chan Medical School). Images from each channel were exported with Tissue-FACSL viewer software and processed in ImageJ. JACop plugin in Image J was used to calculate Mander's overlap coefficient [3,15]. P62 was considered as an aggregate marker to reveal the extent to which two signals occupy the same place. Manual thresholding was applied to exclude the background signals from all images. Representative figures were obtained with a confocal microscope (Leica SP8).

Results and discussion
The splicing site mutation in CHMP2B results in the production of a truncated protein missing the MIM domain, CHMP2B Intron5 (Fig. 1a), that is highly toxic when expressed in cultured cells and primary neurons [10,22,23]. To understand how mutant CHMP2B causes neurodegeneration through a gain-of-toxic function mechanism, we used immunoprecipitation (IP) and mass spectrometry to identify proteins that bind with greater affinity to CHMP2B Intron5 than to CHMP2B WT in HEK293 cells (Additional File 1: Table S1). Among the top 12 interacting proteins were CHMP5, CHMP1B, and CHMP1A (Additional File 1: Fig. S1), other subunits of the ESCRT-III complex. We reported previously that CHMP2B Intron5 blocks dissociation of ESCRT-III [10,11], thus, this result confirms the validity of this biochemical approach. Another protein that seems to associate with CHMP2B Intron5 stronger than to CHMP2B WT is spastin (Additional File 1: Table S1), a microtubule-severing protein whose loss-of-function mutations are the most common genetic cause of hereditary spastic paraplegias (HSP) [20,21]. We confirmed by IP and western blot analysis that spastin indeed binds with greater affinity to CHMP2B Intron5 than to CHMP2B WT (Fig. 1b), as 11 times more spastin was pulled down by CHMP2B Intron5 than by CHMP2B WT based on four independent experiments. This biochemical association was also confirmed by a reverse IP experiment in which spastin antibody pulled down 3.3 times more spastin-bound CHMP2B Intron5 than spastin-bound CHMP2B WT based on three independent experiments (Fig. 1c). The lack of MIM in CHMP2B Intron5 indicates that its enhanced association with spastin may be mediated through other ESCRT-III components. Expression of CHMP2B Intron5 , but not CHMP2B WT , in HeLa cells resulted in the formation of p62-positive puncta (Fig. 1d), consistent with our previous observation that the p62 level in the insoluble fraction is greatly increased in neurons of CHMP2B Intron5 transgenic mice [7]. Interestingly, EGFP-tagged spastin was recruited to these cytoplasmic aggregates (Additional File 2: Fig. S2). More importantly, endogenous spastin also colocalized Proteins that coimmunoprecipitated with FLAG antibody were analyzed by western blots with spastin antibody. The experiment was repeated 4 times. After normalizing against the relative abundance of CHMP2B WT versus CHMP2B Intron5 , 11 times more spastin was bound to CHMP2B Intron5 than CHMP2B WT . c Immunoprecipitation with spastin antibody followed by western blot analysis with FLAG antibody. d and e Co-immunostaining analysis shows colocalization of p62 d and endogenous spastin e with CHMP2B Intron5 aggregates with CHMP2B Intron5 in these aggregates (Fig. 1e), further confirming the enhanced biochemical association between these two disease proteins. The C-terminal tail of CHMP1B, another ESCRT-III protein, directly interacts with the microtubule interacting and trafficking (MIT) domain of spastin [24,27]. CHMP2B Intron5 prevents dissociation of ESCRT-III [10], thus, its enhanced associated with spastin may be mediated through other ESCRT-III components, such as CHMP1B. We speculate other ESCRT-III proteins that show an enhanced interaction with CHMP2B Intron5 versus CHMP2B WT (Additional File 4: Table S1) may be also sequestered in p62/spastinpositive aggregates.
Like the p62 level in CHMP2B Intron5 mice, the spastin level in the insoluble fraction from cells expressing CHMP2B Intron5 was greatly increased than that in cells expressing CHMP2B WT (Fig. 2a, b). As a consequence, the spastin level in the soluble fraction was decreased (Fig. 2a, b). This decrease was not due to reduced expression of SPAST mRNA (Additional File 3: Fig. S3). In fact, SPAST mRNA is increased by about 45% (Additional File 3: Fig. S3), which is probably a compensatory mechanism and further highlighting the decrease of spastin protein level in the soluble fraction is a direct consequence of CHMP2B Intron5 interaction. Spastin was localized in both the cytoplasm and the nucleus (Fig. 1e), but the level of soluble spastin was decreased only in the cytoplasm, as shown by fractionation and western blot analyses (Fig. 2c,  d), consistent with the formation of cytoplasmic spastin aggregates (Fig. 1e). Thus, the increased aggregation of spastin and the decreased level of soluble spastin in the cytoplasm are novel pathological features of cellular toxicity induced by FTD3-associated mutant CHMP2B.
To further assess the functional significance of the biochemical interaction between CHMP2B Intron5 and spastin in vivo, we took advantage of our mouse model that expresses CHMP2B Intron5 specifically in forebrain excitatory neurons by CAMKII promoter controlled expression of tTA [7]. These mice exhibit FTD-like social behavioral deficits at 4 months, but not 2 months, of age, as well as cellular phenotypes such as ubiquitin-positive aggregates and astrogliosis [7]. We found that the level of soluble spastin was decreased in CHMP2B Intron5 mice as young as 2 months of age (Fig. 3a, b), suggesting an early disease phenotype, and this deficit was even more pronounced in older mice (Fig. 3a, b). In 12-month-old CHMP2B Intron5 mice, co-immunostaining analysis revealed the presence of spastin in p62-positive aggregates in mouse cortical neurons (Fig. 3c, d)-a novel pathological feature of FTD caused by CHMP2B mutations. Moreover, in a genetic interaction analysis in a Drosophila model of mutant CHMP2B toxicity [1], we found that RNAi knockdown of spastin with two different RNAi lines did not by itself cause retinal degeneration in the fly eye; however, it greatly increased CHMP2B Intron5 toxicity (Fig. 4), suggesting that partial loss of spastin function contributes to the toxicity of CHMP2B Intron5 in vivo. Loss-of-function mutations in SPAST cause spastic paraplegia 4 (SPG4) [20,21], the most common autosomal dominant form of HSP, which can be associated with clinical dementia [25]. SPAST mutations have also been reported in ALS [8]. The presence of spastin aggregates and the loss of soluble cytoplasmic spastin in FTD3 we identified in this study suggest that Fig. 3 Functional significance of the interaction between spastin and CHMP2B Intron5 in a mouse model of FTD3. a Western blot analysis of the effect of CHMP2B Intron5 on spastin in a mouse model of FTD caused by mutant CHMP2B. The double bands are presumably two isoforms of spastin and the lower band corresponds to the M87 isoform that is used for transfection experiments throughout this study. b Level of total soluble spastin in the cortex of tTA:CHMP2B Intron5 mice. Values are mean ± SEM from three western blot experiments. *p < 0.05, **p < 0.01 by two-sided t test. c Representative images from identical areas of the cortex double-stained for p62 (red) and spastin (green). P62 is known to co-aggregate with CHMP2B Intron5 that is specifically expressed in excitatory neurons in this mouse model. The squares indicate areas shown at higher magnification in the adjacent panels. Scale bar: 20 µm. d Fraction of spastin signal overlapping with p62, calculated with Mander's overlap coefficient. The analysis was done with the JACop plugin in Image J. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Bonferroni post hoc test for multiple comparisons