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Reduced erythrocytic CHCHD2 mRNA is associated with brain pathology of Parkinson’s disease

Reduced erythrocytic CHCHD2 mRNA is associated with brain pathology of Parkinson’s disease Peripheral biomarkers indicative of brain pathology are critically needed for early detection of Parkinson’s disease (PD). In this study, using NanoString and digital PCR technologies, we began by screening for alterations in genes associated with PD or atypical Parkinsonism in erythrocytes of PD patients, in which PD-related changes have been reported, and which contain ~ 99% of blood α-synuclein. Erythrocytic CHCHD2 mRNA was significantly reduced even at the early stages of the disease. A significant reduction in protein and/or mRNA expression of CHCHD2 was confirmed in PD brains collected at autopsy as well as in the brains of a PD animal model overexpressing α-synuclein, in addition to seeing a reduction of CHCHD2 in erythrocytes of the same animals. Overexpression of α-synuclein in cellular models of PD also resulted in reduced CHCHD2, via mechanisms likely involving altered subcellular localiza- tion of p300 histone acetyltransferase. Finally, the utility of reduced CHCHD2 mRNA as a biomarker for detecting PD, including early-stage PD, was validated in a larger cohort of 205 PD patients and 135 normal controls, with a receiver operating characteristic analysis demonstrating > 80% sensitivity and specificity. Keywords: Parkinson’s disease, Mitochondria dysfunction, α-synuclein, CHCHD2 Introduction until a sustained therapeutic response is observed during Diagnosis of Parkinson’s disease (PD), a common neuro- additional years of follow-up. In other words, there is an degenerative disorder [28], is currently based on a combi- essential need to develop a method for accurate diagnosis nation of medical history, observation of cardinal motor of PD at early stage, where disease modifying therapies indicators, and response to pharmaceutical therapies are likely to be most effective. [42, 47]. However, motor symptoms often present after Much effort in the PD biomarker field has focused on greater than 50% of affected neurons have degenerated the discovery of biomarkers using the cerebrospinal fluid [4]. Further, a more definitive diagnosis cannot be made (CSF), with α-synuclein and its variants receiving most attention [32, 41, 45]. However, very few markers inves- tigated thus far demonstrate clinically useful power in *Correspondence: happyft@sina.com; zhangj@uw.edu; jzhang1989@zju.edu. detecting PD or following PD progression, and none has cn been widely validated. Alongside the challenges associ- Xiaodan Liu, Qilong Wang and Ying Yang have contributed equally to this work ated with these protein markers, CSF collection (via lum- Department of Pathology, School of Basic Medical Sciences, Peking bar puncture), often perceived as a high-risk and painful University Third Hospital, Peking University Health Science Center, procedure, is impractical for routine screening purposes. Beijing 100191, China Center for Movement Disorders, Department of Neurology, Beijing More recently, investigations on PD-related proteins in Tiantan Hospital, Capital Medical University, Beijing 100070, China blood have received more consideration; however, they Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. acta neuropathol commun (2021) 9:37 Page 2 of 16 have not yielded consistent results, largely because of these, only CHCHD2 mRNA consistently demonstrated the complexity of the samples [24]. In contrast, blood- significantly reduced expression in all PD groups com - based changes in mRNA expression present an additional pared to the control group [Fig.  1a, F(3, 4) = 52.0, one- and promising biomarker strategy for differentiating way ANOVA, n = 3; p < 0.01 for Early or Mid PD vs. PD patients from healthy controls [3, 31, 36]. One study control, p < 0.001 for Late PD vs. control]. This result was compared the transcriptional profile of PD patients with next confirmed by digital droplet PCR (ddPCR), which or without postural instability and found > 200 differen - also detected significantly reduced CHCHD2 mRNA in tially expressed genes, some of which were also found erythrocytes across all PD groups compared to controls to be dysregulated in a dopaminergic cell model of PD [Fig.  1b, F(3, 8) = 20.80, one-way ANOVA, n = 3; p < 0.01 [26], suggesting that altered gene expression in the blood or 0.001 for each PD group vs. the controls]. There were may reflect changes in the central nervous system (CNS). no differences, however, between PD groups with differ - Another group explored the genetic signature in blood ent disease severity. Having validated the mRNA result from a large cohort of PD patients and identified 87 genes independently, we next examined the protein expression which differentiated between patients with idiopathic of CHCHD2 in erythrocytes of PD patients. As shown PD and controls [36], further supporting the concept in Fig.  1c, the relative CHCHD2 protein levels were of altered gene expression within the blood as a useful decreased significantly from 1.01 ± 0.07 in pooled eryth- tool for predicting PD. Notably, both studies used RNA rocytes of controls to 0.43 ± 0.12 in those of PD patients extracted from whole blood cells, mainly leukocytes, (p < 0.05, Mann–Whitney U test, n = 3). which undergo nuclear and transcriptional changes dur- ing disease states. Compared to leukocytes (and most Reduced expression of CHCHD2 in the post‑mortem other blood cells), mature erythrocytes are uniquely substantia nigra slices of PD patients structured, lacking organelles and nuclei necessary for To probe whether expression of CHCHD2 is also replacing dysfunctional proteins. Thus, alterations in reduced in the central nervous system (CNS) of PD residual RNA and protein levels in erythrocytes likely patients, its expression was examined in the post-mor- reflect pathological rather than physiological changes tem substantia nigra slices obtained at autopsy from the [2]. Additionally, ~ 99% of blood α-synuclein is located in brains of PD patients, along with age-matched controls, erythrocytes, and pathological changes in erythrocytes as well as in the animal model. Firstly, we examined the have been described in PD patients in several independ- expression pattern of CHCHD2, both in the substantia ent investigations [1, 2, 14, 21, 23, 30, 40, 44]. Therefore, nigra of human and wild type C57BL mice. Co-labeling in this study, we tested the hypothesis that erythrocytes of CHCHD2 with a pan-neuronal marker (NeuN) and a carry biomarkers that reflect or correlate with brain dopaminergic neuronal marker (Tyrosine hydroxylase, pathology, and are capable of detecting PD at early stages. TH) revealed that CHCHD2 is expressed in nearly all neurons (> 90%, Fig.  2a for human, Fig. S2A for mice). CHCHD2 is also expressed in > 90% astrocytes (Fig.  2b Results for human, Fig. S2B for mice), but only in 10–30% micro- Reduced mRNA and protein expression of CHCHD2 glia cells (Fig.  2c for human, Fig. S2C for mice). Subcel- in erythrocytes of PD patients lularly, CHCHD2 is located in mitochondria, as indicated A NanoString multiplex gene expression method was by co-localization with Translocase of Outer Mitochon- used to screen for potential mRNA biomarkers for PD drial Membrane 20 (TOMM20) (Fig. S2D). Consistent within erythrocytes. A panel of 21 genes associated with with previous reports [19, 27], loss of dopaminergic neu- PD or atypical Parkinsonism (see “Materials and meth- rons, indicated by TH staining, was detected (Fig.  2e–g, ods” section for a complete list) chosen based on previ- 13.2 ± 1.3 in Ctl vs. 4.6 ± 0.6 in PD per field, p < 0.001, ous reports [6, 18, 20, 34, 43], was examined in a cohort multiple t-test). The number of CHCHD2 positive cells consisting of 48 participants separated according to four was also significantly lower, with 5.46 ± 0.6 per field in diagnostic classifications: control, early-stage PD (Early PD patients versus 15.2 ± 1.6 neurons in control (Fig. 2e– PD), middle-stage PD (Mid PD), or late-stage PD (Late g, p < 0.001, multiple t-test). Fluorescence intensity of PD) patients according to Unified Parkinson’s Disease CHCHD2 protein in the surviving neurons of substantia Rating Scale (UPDRS) score (Additional file  1: Table S1). nigra of PD patients also decreased markedly (Fig.  2e–f mRNA expression frequency of each gene in erythro- and h p < 0.001, Mann–Whitney U test). cytes of healthy control is shown in Additional file  1: We also detected the expression of CHCHD2 in Fig. S1, with SNCA, FBXO7, CHCHD2, PSEN1, LRRK2, post-mortem slices across different brain regions by VPS35, GATA1, MAPT, APOE, and PINK1 among the immunohistochemistry. Consistent with the results ten most highly expressed genes in erythrocytes. Of obtained by immunofluorescence in Fig.  2e–h, the Liu  et al. acta neuropathol commun (2021) 9:37 Page 3 of 16 Fig. 1 CHCHD2 mRNA and protein expression was reduced in erythrocytes of PD patients. a Genes associated with PD or atypical Parkinsonism in pooled erythrocyte samples from controls and PD patients at various stages were analyzed by the NanoString multiplex gene expression method. The samples were pooled according to Additional file 1: Table S1 [four comparison groups (n = 12 each) with each pooled into three sub-groups]. Ten genes were analyzed following removal of low expression genes. Significantly decreased CHCHD2 mRNA was detected in all PD groups compared to the control group [F (3, 4) = 52.0, one-way ANOVA, n = 3; p < 0.01 for Early or Mid PD vs. control, p < 0.001 for Late PD vs. control]. b Validation of the discovery cohort by ddPCR in the discovery set also identified decreased CHCHD2 mRNA in erythrocytes of all PD groups [F(3, 8) = 20.80, one-way ANOVA, n = 3; p < 0.01 or 0.001 for each PD group vs. the controls] compared to controls. No difference in CHCHD2 mRNA expression was observed between PD groups by Tukey’s multiple comparisons test. Left: Original representative ddPCR picture. FAM channel (Axis X) indicates CHCHD2, VIC channel (Axis Y ) indicates AHSP. Right: Statistical result. c Decreased CHCHD2 protein in erythrocytes of PD patients. Proteins were extracted from pooled erythrocytes of the same cohort as used in the NanoString and ddPCR studies. Compared to controls, CHCHD2 was decreased significantly from 1.01 ± 0.07 to 0.43 ± 0.12 (p < 0.05, Mann–Whitney U test, n = 3) Liu et al. acta neuropathol commun (2021) 9:37 Page 4 of 16 Fig. 2 Reduced expression of CHCHD2 in post-mortem substantia nigra slices of PD patients. a Representative images of co-staining of CHCHD2 (green) with neuronal marker NeuN (blue) and dopaminergic neuronal marker TH (red), showing expression of CHCHD2 in neurons. Scale bar: 10 μm. b Representative images of co-staining of CHCHD2 (green) with astrocyte marker GFAP (red), showing expression of CHCHD2 in astrocytes. Scale bar: 10 μm. c Representative images of co-staining of CHCHD2 (green) with microglial marker Iba1 (red), showing expression of CHCHD2 in a minority of microglia. Scale bar: 10 μm. d Quantification of the percentage of CHCHD2 positive cells by cell type in Iba1, GFAP or NeuN positive cells. e and f Immunofluorescence staining images of substantia nigra of control and PD brain slices probed with the antibody against CHCHD2 (green) + + and TH (red). Scale bar = 20 μm. g Quantification of TH and CHCHD2 cells in substantia nigra, demonstrating lower numbers of surviving neuronal cells and surviving CHCHD2 cells in PD patients. p < 0.001, multiple t-test, n = 3 each. h Quantification of immunofluorescent signal demonstrating reduced CHCHD2 immunofluorescent intensity in substantia nigra (p < 0.001, Mann–Whitney U test, n = 3) Liu  et al. acta neuropathol commun (2021) 9:37 Page 5 of 16 number of CHCHD2 positive cells (Fig.  3a, p < 0.0001, contrast, the expression of CHCHD2 in frontal cortex Mann–Whitney U test) and density of CHCHD2 in (Fig.  3c, p > 0.05, Mann–Whitney U test) and cerebel- the surviving cells (Fig.  3b, p < 0.01, Mann–Whit- lum (Fig.  3d, p > 0.05, Mann–Whitney U test) were not ney U test) of substantia nigra of PD patients were changed. These results suggested CHCHD2 was prefer - reduced significantly, compared with control. In entially reduced in the substantia nigra of PD patients. Fig. 3 CHCHD2 expression was reduced specifically in the substantia nigra of PD patients when examined by immunohistochemical staining. a Reduced number of CHCHD2 positive cells in substantia nigra of PD patients. Whole slice scanned images of control and PD patients were shown in left and middle panel. Scale bar: 1000 μm. Statistics data of the number of CHCHD2 positive cells was shown in right panel. p < 0.0001, Mann– Whitney U test. b The immunohistochemical staining intensity of CHCHD2 was reduced in substantia nigra of PD patients. p < 0.01, Mann–Whitney U test, n = 3. Scale bar: 50 μm. c CHCHD2 expression was not altered in frontal cortex. p > 0.05, Mann–Whitney U test, n = 3. Scale bar: 50 μm. d CHCHD2 expression was not altered in cerebellum. p > 0.05, Mann–Whitney U test, n = 3. Scale bar: 50 μm Liu et al. acta neuropathol commun (2021) 9:37 Page 6 of 16 Reduced mRNA and protein expression of CHCHD2 expression of CHCHD2 in erythrocytes was significantly +/+ in erythrocytes and brains of A53T α‑synuclein mice lower in A53T mice (0.27 ± 0.03 for mRNA, p < 0.01, Having observed both CNS and peripheral reduction Mann–Whitney U test, n = 6, Fig.  4a; and 0.29 ± 0.04 for of CHCHD2 expression in PD patients, we next sought protein, p < 0.01, Mann–Whitney U test, n = 3, Fig.  4b) to explore the potential underlying mechanisms in a compared to wild type control mice (1.11 ± 0.20 for PD mouse model. For this purpose, we used the A53T mRNA; 1.02 ± 0.14 for protein). In the substantia nigra, α-synuclein transgenic mouse model (M83 line), an real time PCR and Western blot results clearly showed extensively utilized model where progressive pathol- that the mRNA (Fig.  4c, p < 0.01, Mann–Whitney U test, ogy and behavior are driven by expression of mutated n = 6) and protein expression (Fig.  4d, p < 0.05, Mann– +/+ α-synuclein [8]. A53T mice were used at 10  months Whitney U test, n = 3) of CHCHD2 were significantly old, when neurologic defects are readily detectable. decreased. As shown in Fig.  4, the relative mRNA and protein Fig. 4 Reduced expression of CHCHD2 in the erythrocytes and substantia nigra of A53T α-synuclein transgenic mice. a Compared to non-transgenic wild type mice, normalized mRNA of CHCHD2 in erythrocytes of A53T α-synuclein transgenic mice was significantly decreased from 1.11 ± 0.20 to 0.27 ± 0.03 (p < 0.01, Mann–Whitney U test, n = 6). b Western blot results showed a significantly reduced protein expression of CHCHD2 in erythrocytes of transgenic mice (p < 0.01, Mann–Whitney U test, n = 3). Upper: representative Western blot images. β-actin was used as an internal control. Wt: non-transgenic wild type mice. Tg: A53T transgenic mice. Lower: quantification of Western blots. c Real-time qPCR also detected a reduced mRNA expression of CHCHD2 in substantia nigra of transgenic mice (p < 0.01, Mann–Whitney U test, n = 6). d CHCHD2 protein was decreased from 1.21 ± 0.21 in substantia nigra of wild type mice to 0.21 ± 0.10 in substantia nigra of transgenic mice (p < 0.05, Mann–Whitney U test, n = 3). Upper: representative Western blot images. β-tubulin was used as an internal control. Lower: quantification of Western blots. Of note, the sample number (N) is different because mRNA and proteins were measured in two different sets of animals Liu  et al. acta neuropathol commun (2021) 9:37 Page 7 of 16 We also detected a reduced expression of CHCHD2 U test, n = 3). A correlation analysis indicated a nega- by immunofluorescence across several brain regions, tive correlation between the expression of CHCHD2 including substantia nigra (Fig.  5a, b), the rest and the level of aggregated α-synuclein (r = − 0.978, of midbrain (Fig.  5c, d), frontal cortex (Fig.  5e, f) p < 0.001 in substantia nigra, Fig. S3A; r = − 0.943, +/+ and cerebellum (Fig.  5g, h) in A53T mice com- p < 0.01 in the rest of midbrain, Fig. S3B; r = − 0.952, pared to non-transgenic wild type mice (all p <0.05, p < 0.01 in frontal cortex, Fig. S3C; r = − 0.978, Mann–Whitney U test, n = 3). Consistent with previ- p < 0.001 in cerebellum, Fig. S3D). ous reports [8, 10, 35], aggregated α-synuclein was increased in these regions (all p < 0.05, Mann–Whitne y Fig. 5 Immunofluorescent staining of CHCHD2 and α-synuclein in different areas of mouse brain slices. a and b Decreased CHCHD2 (green) ## and increased α-synuclein (blue) in substantia nigra (***p < 0.001, p < 0.01, Mann–Whitney U test, n = 3). c and d Decreased CHCHD2 (green) ## and increased α-synuclein (blue) in rest of midbrain (**p < 0.01, p < 0.01, Mann–Whitney U test, n = 3). e and f Decreased CHCHD2 (green) and ## increased α-synuclein (blue) in frontal cortex (***p < 0.001, p < 0.01, Mann–Whitney U test, n = 3). g and h Decreased CHCHD2 (green) and increased α-synuclein (blue) in cerebellum (***p < 0.001, p < 0.05, Mann–Whitney U test, n = 3). Scale bar: 10 μm Liu et al. acta neuropathol commun (2021) 9:37 Page 8 of 16 Reduced mRNA and protein expression of CHCHD2 p300 histone acetyltransferase [13], we tested whether in a cellular model of PD there is a direct interaction between p300 and the pro- To further explore the potential mechanisms leading moter of CHCHD2. As shown in Fig. 6c, ChIP results dis- to decreased CHCHD2 expression in PD patients, wild tinctly showed that p300 can directly bind the promoter type and A53T α-synuclein plasmids were transfected of CHCHD2. Additionally, overexpression of wild type or in MN9D cells, a line that is derived from dopamin- mutant α-synuclein in MN9D cells reduced the interac- ergic cells and previously utilized in multiple in  vitro tion of p300 with the promoter of CHCHD2 (p < 0.001, F PD models [5, 15, 37]. As expected, transfection of (2, 6) = 73.44, one-way ANOVA, n = 3). the cells with α-synuclein vector resulted in increased We next examined the potential mechanisms by which mRNA [Fig. S4A, p < 0.01, F(2, 6) = 20.95, one-way α-synuclein expression alters the function of p300, by ANOVA, n = 3] and protein [Fig. S4B, F(2, 9) = 14.37, measuring the effect of α-synuclein overexpression one-way ANOVA, n = 4; p < 0.01 for A53T vs. Vec- on p300. Significant reductions in the mRNA (Fig.  6d, tor] expression of α-synuclein. The overexpression of p <0.001, F(2, 6) = 130.2, one-way ANOVA, n = 3) and α-synuclein also dramatically reduced the number of protein expression (Fig. 6e, p <0.001, F(2, 6) = 41.95, one- CHCHD2 mRNA transcripts, regardless of whether the way ANOVA, n = 3) of p300 were seen 48h after trans- vector was wild type or A53T α-synuclein [Fig.  6a F(2, fection of α-synuclein vector. Direct protein interaction 6) = 35.31, one-way ANOVA, n = 3; p < 0.001 for Wt between p300 and α-synuclein was also demonstrated α-syn vs. Vector, p < 0.01 for A53T α-syn vs. Vector]. by reciprocal co-immunoprecipitation experiments Consistent with the human data, the protein expres- (Additional file  1: Fig. S5B). Immunofluorescence results sion of CHCHD2 was also reduced markedly[Fig.  6b, showed that p300 was mainly expressed in the nucleus, F(2, 6) = 20.75, one-way ANOVA, n = 3; p < 0.05 for Wt while it was less expressed in the cytoplasm of vec- or A53T α-syn vs. Vector]. tor transfected Mn9D cells. However, overexpression of wild type or A53T α-synuclein significantly reduced α‑synuclein negatively regulates the expression the nuclear expression of p300 (Fig.  6f, p < 0.0001, F(2, of CHCHD2, possibly via modulation of P300 154) = 12.8, one-way ANOVA). Western blot of the pro- The results shown above suggest that reduced tein of isolated subcellular fractions showed an altered CHCHD2 protein is largely attributable to a decreased distribution, with a higher proportion of p300 in the level of mRNA, likely secondary to overexpression cytoplasm, compared to a lower amount in the nucleus of α-synuclein. To further investigate potential links after α-synuclein (A53T) overexpression compared to between α-synuclein overexpression and CHCHD2 vector-transfected cells (Fig.  6g, p < 0.05, Mann–Whit- level, we asked whether there is a direct interaction of ney U test, n = 3). All the above results suggested that α-synuclein with the promoter of CHCHD2. Initial chro- α-synuclein may inhibit the expression of CHCHD2, pos- matin immunoprecipitation (ChIP) experiments failed to sibly by reducing the expression and the nuclear distribu- demonstrate any direct interaction between α-synuclein tion of p300. and CHCHD2 promoter (Additional file  1: Fig. S5A). Therefore, we next investigated whether α-synuclein Exploration of erythrocytic CHCHD2 as a PD biomarker could negatively regulate the expression of CHCHD2 in a large cohort indirectly. Because it has previously been shown that Having demonstrated that expression of CHCHD2 is α-synuclein could negatively regulate protein kinase C significantly reduced in the substantia nigra as well as expression by reducing the expression and activity of in erythrocytes in PD patients, we turned our attention (See figure on next page.) Fig. 6 Overexpression of α-synuclein reduced CHCHD2 expression in MN9D cells, possibly by altering the expression and subcellular localization of p300. a Reduced mRNA expression of CHCHD2 in MN9D cells after overexpression of α-synuclein (F(2, 6) = 35.31, one-way ANOVA, n = 3; p < 0.001 for Wt α-syn vs. Vector, p < 0.01 for A53T α-syn vs. Vector). b Reduced protein expression of CHCHD2 in MN9D cells after overexpression of α-synuclein [F(2, 6) = 20.75, one-way ANOVA, n = 3; p < 0.05 for Wt or A53T α-syn vs. Vector]. c Direct interaction between p300 and CHCHD2 promoter as revealed by ChIP result. Overexpression of both wild type and A53T α-synuclein reduced the interaction of p300 and CHCHD2 promoter [F(2, 6) = 73.44,one-way ANOVA, n = 3; both p < 0.001, compared to Vector]. Histone 3 (H3) was used as positive control. d Decreased mRNA expression of p300 after overexpression of α-synuclein [F(2, 6) = 130.2, one-way ANOVA, n = 3; both p < 0.001, compared to Vector]. e Protein expression of p300 [F(2, 6) = 41.95, one-way ANOVA, n = 3; both p < 0.001, compared to Vector]. f Reduced nuclear distribution of p300 after overexpression of α-synuclein revealed by immunofluorescence (F (2, 154) = 12.8, one-way ANOVA, p < 0.05 or 0.01 or 0.0001). g Reduced localization of p300 in nucleus revealed by western blot (p < 0.05, Mann–Whitney U test, n = 3); Increased localization of p300 in cytoplasm (p < 0.05, Mann–Whitney U test, n = 3) Liu  et al. acta neuropathol commun (2021) 9:37 Page 9 of 16 Liu et al. acta neuropathol commun (2021) 9:37 Page 10 of 16 Fig. 7 Reduced CHCHD2 mRNA in erythrocytes predicts PD. a Reduced CHCHD2 mRNA in erythrocytes of PD patients was further confirmed in a larger, validation cohort of 135 normal controls, 73 Early PD, 98 Mid PD and 34 Late PD patients [p < 0.001 each, F (3, 332) = 26.11, one-way ANOVA]. No difference in CHCHD2 mRNA expression was determined among PD groups by Tukey’s multiple comparisons test. b ROC analysis revealed diagnostic values of 80.4% sensitivity and 81.1% specificity of CHCHD2 for PD diagnosis back to its utility as a more convenient biomarker for early PD versus healthy controls was 80.82%, yielding a PD diagnosis. To achieve this goal, we further exam- final diagnostic value of 85.38% for PD vs. controls (Addi - ined CHCHD2 mRNA in erythrocytes of individual PD tional file 1: Table S2 and Fig. 7b). patients in a cohort of 340 subjects including patients Finally, we compared the CHCHD2 mRNA levels in with early- (n = 73), middle- (n = 98), and late-stage erythrocytes with disease severity, including worsen- PD (n = 34) along with controls (n = 135). As shown in ing of motor symptoms and mild cognitive impairment Fig. 7a, CHCHD2 mRNA was reduced significantly in all (MCI), and disease duration by comparing CHCHD2 PD groups compared to controls, as detected by ddPCR mRNA to the UPDRS part III on-state motor scores, [F (3, 332) = 26.11, one-way ANOVA; p < 0.001 for each Montreal Cognitive Assessment (MoCA) score, and PD group vs. control], consistent with our discovery time following diagnosis in the validation cohorts of PD cohort results. patients (n = 205 total; cases < 50 years were not excluded Because PD is an age-related disorder, not evenly dis- due to the lack of age dependence as determined above). tributed by sex, we next investigated the impact of both Once again, as determined by linear regression analy- sex and age dependence on CHCHD2 mRNA levels iso- ses, no significant association could be determined lated from erythrocytes. Two-way ANOVA compari- between CHCHD2 mRNA levels and the disease severity sons of male and female control and PD patient samples (p = 0.49, R = − 0.05 for UPDRS motor), disease duration revealed no significant difference in CHCHD2 mRNA (p = 0.62, R = 0.04) or MOCA scores (p = 0.11, R = 0.11) levels between sexes [Additional file  1: Fig. S6A, p = 0.65, (Additional file 1: Fig. S6C–E). F (1, 326) = 0.2077], while linear regression analysis revealed no correlation between CHCHD2 mRNA levels and age in either group (Additional file  1: Fig. S6B, con- Discussion trol: p = 0.49, R = 0.06; PD: p = 0.35, R = − 0.07). The CHCHD2 gene is a recently discovered PD causa - We also evaluated CHCHD2 mRNA as a diagnostic tive gene that drives development of PD with autoso- marker in erythrocytes. Analysis of Receiver Operating mal dominant inheritance [6]. More recently, mutations Characteristic (ROC) curve was performed to evaluate in CHCHD2 have also been found in sporadic PD [29, the diagnostic accuracy of CHCHD2 mRNA levels in the 39, 46]. CHCHD2 protein is localized to mitochondria, total PD cohort as well as PD patients at early-stage. The which are intimately associated with PD pathogenesis results were similar for both analyses, and when the spec- [48]. It has been demonstrated that the loss of CHCHD2 ificity was anchored to ≥ 80%, sensitivity for detecting causes abnormal matrix structures and impaired oxygen Liu  et al. acta neuropathol commun (2021) 9:37 Page 11 of 16 respiration in mitochondria, leading to oxidative stress, remains to be determined. Moreover, as with other gene dopaminergic neuron loss and motor dysfunction in mutations associated with PD, e.g., SNCA and LRRK2, Drosophila [22]. Additionally, knockout of CHCHD2 though mutations occur systematically, dopaminer- or CHCHD10 in human induced pluripotent stem cells gic neurons are preferentially vulnerable. Why and how led to increased proton leakage and respiration, as well CHCHD2 mutations/reductions result specifically in as loss of synaptic function [11]. CHCHD2/CHCHD10 dopaminergic neurodegeneration needs to be investi- double knockout mice showed disrupted mitochondrial gated further. cristae [17]. Within affected brain regions, compared to healthy Two major observations of the current study are: (1) controls, both the number of CHCHD2 positive neu- the expression of CHCHD2 was reduced significantly in rons and its level of expression in surviving neurons human erythrocytes, substantia nigra of brain collected were reduced in the substantia nigra of PD patients at autopsy, and mice carrying transgenic mutant A53T (Figs. 2 and 3), suggesting the possibility of an interaction α-synuclein; and (2) the expression of CHCHD2 in eryth- between PD pathological processes (e.g., accumulation of rocytes, a readily accessible clinical sample source, could pathological α-synuclein species) and CHCHD2 reduc- be an early diagnostic marker of PD. tion. Consistent with this hypothesis, correlation analysis Using NanoString multiplex gene expression analysis, revealed a negative correlation between protein expres- among a total of 21 genes screened, CHCHD2 mRNA sion levels of α-synuclein and CHCHD2 in mice (Addi- was discovered to be consistently reduced in various tional file  1: Fig. S3), i.e., reduced CHCHD2 level may be stages of PD patients. The corresponding protein was driven by increased levels of pathogenic α-synuclein. also reduced in erythrocytes (Fig.  1). The mechanisms How might α-synuclein, especially pathogenic forms, responsible for the CHCHD2 reduction in the erythro- inhibit the expression of CHCHD2? A previous study cytes of PD patients are unknown, especially given that indicated that α-synuclein negatively regulates protein mature erythrocytes are incapable of transcriptional kinase C expression by reducing the expression and activ- regulation. One possibility is that the transcriptional reg- ity of p300 histone acetyltransferase [13]. Consistent with ulation occurred in bone marrow or at the stage of retic- this observation, overexpression of wild type and A53T ulocytes, a data point not collected in the current study. mutant α-synuclein in MN9D cells, a mouse dopamin- However, reticulocytes account for 0.5–2.5% of red blood ergic cell line, significantly reduced the mRNA and pro - cells [38], and there is no evidence to suggest that there tein expression of CHCHD2, with the effect being more is a difference between PD and controls in reticulocyte significant when A53T mutant α-synuclein was used counts. Therefore, future study needs to include investi - (Fig.  6). Additionally, direct interaction of p300, instead gations in bone marrow or at least to consider the ratio of of α-synuclein, with the promoter of CHCHD2 gene was erythrocytes/reticulocytes in various cohorts. detected by ChIP experiment whereas overexpression One surprising aspect of this study is that while of wild type or A53T mutant α-synuclein reduced their reduced CHCHD2 expression was observed in (and cor- interaction. However, α-synuclein and CHCHD2 may related between) both the substantia nigra (Figs.  2 and have a more complicated mutual interaction. For exam- 3) and periphery of human PD patients, the reduction ple, in a recent study, mutations in CHCHD2 resulted was not universal within the CNS, as it did not extend in α-synuclein aggregation [12]. Further, although a to the frontal cortex or cerebellum. In contrast, reduced link between the reduced expression of CHCHD2 and CHCHD2 mRNA and protein expression were detected nuclear distribution of p300 is clearly implicated (Fig. 6), in the substantia nigra, prefrontal cortex and cerebellum the direct involvement of p300 needs to be confirmed by of A53T α-synuclein transgenic mice (Figs. 4 and 5). The additional studies, e.g., knockdown of its expression in a discrepancy in affected brain region between human and cellular model of PD. mice studies may have been due to the different expres - Regardless of the mechanism(s) underlying CHCHD2 sion patterns of pathological α-synuclein species in expression in PD patients, reduced CHCHD2 mRNA human and mice; specifically, the PD patients used in our in all stages of PD was validated by ddPCR in a larger study were at Braak Stage 3, a stage at which Lewy body cohort, suggesting that levels of CHCHD2 mRNA may pathology is present in the substantia nigra, but not the act as a biomarker for detecting PD using patient blood, cortex or cerebellum. In contrast, transgenic α-synuclein even at early disease stage, with a sensitivity and specific - is widely expressed in the central nervous system in the ity of 80 and 81%, respectively (Fig.  7). Indeed, the level mouse model (M83 line), without the progressive spread of CHCHD2 was not correlated with disease severity, observed in human patients. Whether lower expres- motor or cognition, suggesting a possible flooring effect, sion of CHCHD2 in other brain regions becomes more possibly because the changes occur early during the dis- widespread in the human brain as the disease progresses ease process. In addition, no significant correlation was Liu et al. acta neuropathol commun (2021) 9:37 Page 12 of 16 observed between CHCHD2 mRNA levels and sex or age Laboratory. Non-transgenic mice with the same back- (Additional file  1: Fig. S6). The result is quite significant ground were used as controls. All mice were housed in in that reduced CHCHD2 mRNA could be a potential separate cages with free access to food and water. The biomarker for early PD, when clinical diagnosis is most room temperature was kept at 24 ± 1  °C under natural difficult due to overlapping clinical phenotypes in related light–dark cycle. All animal experimental procedures diseases. However, to truly test this biomarker, future val- were approved by the Animal Care and Use Committee idation studies should include a reasonably large cohort of Peking University. of patients with multiple system atrophy (MSA) and/ or progressive supranuclear palsy (PSP). Additionally, RNA isolation from erythrocytes moving forward, the biomarker should be tested in the To separate erythrocytes from plasma and buffy coat, premotor stage when nigrostriatal degeneration can be fresh whole blood samples were immediately centri- confirmed by DAT or PET imaging. fuged after phlebotomy at 2000  g for 10  min. 200  μl of In summary, reduction in protein and mRNA expres- pure erythrocytes were then used to extract total RNA sion of CHCHD2 is widespread in PD patients, from using Trizol reagent (Invitrogen, USA) according to the CNS to peripheral erythrocytes. The underlying mecha - manufacturer’s protocol. RNA was assessed for quantity nisms, though they remain to be investigated, are likely using Nanodrop 2000, and for quality using the 2100 Bio- related to synucleinopathy and involvement of p300. analyzer (Agilent Technologies, Canada). RNA was either CHCHD2 mRNA in erythrocytes, which is easily acces- used immediately or stored at − 80 ℃ consistently within sible, may serve as a convenient yet robust biomarker of experiments. PD, particularly for diagnosis of PD at early stages. NanoString nCounter method Materials and methods The nCounter Analysis System (NanoString Technolo - Participants gies, Seattle, WA) allows for multiplexed digital mRNA This study was approved by the Institutional Review Boards profiling without amplification or generation of cDNA of all participating institutions. Subjects for both the dis- [7]. A total of 21 PD related genes [APOE (NM_000041.2), covery (48 in total; 12 healthy controls, 36 PD patients) APP (NM_000484.3), ATP13A2 (NM_001141974.1), and validation (340 in total; 135 healthy controls, and 205 CHCHD2 (NM_016139.2), EIF4G1 (NM_004953.3), PD patients) studies were recruited from Beijing Tiantan FBXO7 (NM_001033024.1), GATA1 (NM_002049.2), Hospital between 2016 and 2018. All participants provided GBA (NM_001005742.2), LRRK2 (NM_198578.3), informed consent and underwent a neurologist-conducted MAPT (NM_016834.3), PARK2 (NM_004562.2), PARK7 evaluation that consisted of a structured interview, neuro- (NM_001123377.1), PINK1 (NM_032409.2), PLA2G6 logical examination, laboratory tests, and neuropsycho- (NM_001199562.1), PSEN1 (NM_000021.2), PSEN2 logical assessments. All control subjects were community (NM_000447.2), SNCA (NM_000345.2), SNCAIP volunteers who had Mini Mental State Exam Scores > 24, (NM_001242935.2), TPPP (NM_007030.2), UCHL1 paragraph recall scores > 6, no history of neurological dis- (NM_004181.3), VPS35 (NM_018206.4)] were screened. ease, and no history or evidence of cognitive or functional Total RNA (100  ng) was hybridized with the Tagset decline. All PD patients met UKPD Society Brain Bank probes and loaded into the nCounter prep-station, and clinical diagnostic criteria for PD [9]. PD patient samples then quantified using the nCounter Digital Analyzer. The were further categorized based on UPDRS Part III on-state NanoString platform includes negative control probes motor scores to approximate disease stage, according to the (not complementary to any endogenous mRNA) to assess method we reported previously [16]. That is, patients with background noise associated with the fluorescent bar - UPDRS scores < 15 were defined as having early-stage PD, code optical recognition system. Raw probe counts were those with scores ranging from 15 to 30 were classified as normalized to a panel of three endogenous control genes middle-stage PD, while those with scores > 30 were clas- [AHSP (NM_016633.2), β-actin (NM_001101.2), and sified as late-stage PD patients. Demographic data for all GAPDH (NM_002046.3)] by taking the ratios of each subjects and UPDRS scores for all PD patients used in the gene’s counts per sample to the average across all samples study are listed in Additional file 1 : Tables S1 and S2. and scaling by the median of these ratios in each sample. This normalization factor was also applied to the negative Animals control probes counts. A detection threshold was defined Transgenic mice expressing A53T (M83 line) human for each sample as five times the mean of the negative α-synuclein protein under the control of the prion pro- control probe normalized counts. tein promoter [8] were purchased from The Jackson Liu  et al. acta neuropathol commun (2021) 9:37 Page 13 of 16 Digital droplet PCR quantification the manufacturer’s protocol (Takara Inc, Japan). Oligo- For absolute quantification of mRNA of CHCHD2 in nucleotide primers corresponding to cDNA for mouse erythrocytes, we used the recently developed digital CHCHD2/α-synuclein/p300/GAPDH can be seen in droplet PCR [25]. TaqMan probes (250 nM final concen - Additional file  1: Table S3. The specificity for each primer tration) and related primers set for CHCHD2 (labeled set was confirmed by both electrophoresis of the PCR with a FAM at the 5′ end, 900  nM primers at final con - products on a 2.0% agarose gel and analyzing the melting centration) and AHSP (labeled with a VIC at the 5′ end, (dissociation) curve using a 7500 ABI PRISM Sequence 150  nM primers at final concentration) were purchased Detector System according to the manufacturer’s instruc- from Thermo Fisher Scientific, Inc. (USA). AHSP, which tions (Applied Biosystems) after each real-time PCR is expressed mainly in erythrocytes and did not change reaction. The relative amount of transcripts was calcu - −ΔΔCT in PD based on our NanoString data, was chosen as an lated using the 2 method [33] and normalized to ™ ™ internal control. We used SuperScript III Platinum the endogenous reference gene GAPDH. One-Step qRT-PCR Kit (#11732020, Thermo Scientific, USA) for cDNA synthesis and PCR amplification per - Immunofluorescence staining of human and mouse brain formed in a single tube following the instructions of the tissues manufacturer. The RainDrop Source emulsion genera - The post-mortem human brain material was obtained tor (RainDance Technologies, Inc.) was used to generate from China National Health and Disease Human Brain emulsified micro droplets. Each 25  μl reaction system Tissue Resource Center (Hangzhou). All materials consisted of the 24  μl TaqMan gene expression ddPCR have been collected from donors who provided writ- system and 1  μl of 25 × droplet stabilizer. After emul- ten informed consent for a brain autopsy and permit- sion, the tube was directly sealed and put into an ABI ting the use of the material and clinical information for ProFlex thermalcycler (Thermo Fisher Scientific, Inc. research purposes. Demographic data for all subjects USA). PCR amplification program was as follows: 50  ℃, used in the study are listed in Additional file  1: Table S4. 15 min; 95 ℃, 4 min; 45 cycles of (95 ℃, 15 s; 60 ℃, 45 s). The paraffin-fixed formaldehyde-embedded human or The heating and cooling rates of the thermalcycler were frozen mouse brain tissues were cut into 6–10  μm sec- adjusted to 0.6 ℃/s for better PCR amplification in mil - tions. The immunofluorescence staining of brain tissues lions of micro droplets. After amplification, the tube was was performed in a double-blinded manner. Brain slices transferred to Raindrop Sense machine and the numbers were incubated overnight at 4 °C with primary antibod- of amplified droplets with suitable targets were analyzed ies diluted in blocking solution. Brain slices were then with RainDrop Analyst v3 software. washed with washing buffer (0.1% Tween in PBS) and incubated with corresponding secondary antibodies Western blot diluted in PBS containing 0.3% of Triton X-100 for 3  h. Proteins were extracted from erythrocytes, cell lines, After washing with PBS or washing buffer, brain slides or tissues using SDS lysis buffer (2% SDS, 10% glycerol, were embedded in Vectashield medium or Vectashield 0.1 mM dithiothreitol and 0.2 M Tris–HCl, pH 6.8). Pro- medium with DAPI. Immunofluorescence images were tein samples were resolved by SDS–polyacrylamide gel captured at room temperature using a Zeiss Confocal electrophoresis, transferred to polyvinylidene difluoride Microscope under 20× or 40× magnification. Mouse membrane and blotted with respective primary antibod- monoclonal antibody to CHCHD2(66302–1-Ig, Protein- ies at 4 ℃ over night. The blots were washed in TBST and tech, 1:50), rabbit monoclonal antibodies [EPR12763] then incubated in horseradish peroxidase–conjugated to NeuN (ab177487, Abcam, 1:500), chicken poly- goat anti-rabbit/mouse IgG secondary antibody. Protein clonal antibodies to GFAP (AB5541, Merck Millipore, bands were visualized using an enhanced chemilumines- 1:500), rabbit monoclonal antibodies [EPR16588] to cence detection kit followed by autoradiography using Iba1(ab178846, Abcam, 1:500), rabbit monoclonal anti- Hyperfilm MP. bodies [EPR15581-54] to TOMM20 (ab186735, Abcam, 1:250), chicken polyclonal antibodies to Tyrosine Real‑time PCR Hydroxylase (ab76442, Abcam, 1:500) and rabbit mono- The levels of mRNA for CHCHD2, α-synuclein or p300 clonal [MJFR-14-6-4-2] antibodies to α-synuclein aggre- in mice or cell lines were measured by real-time PCR. gate (ab209538, Abcam, 1:200) were used in IF. Alexa Briefly, total RNA was isolated from brain tissues or cell Fluor 405, 488, 555 or 633 conjugated secondary anti- lines using Trizol (Life technologies, USA). Synthesis of bodies used in IF were purchased from Thermo Fisher first-strand cDNA was performed by reverse transcrip - Scientific, Inc. IF signals were quantified using Image J tion of 1.0  μg total RNA using RT-PCR kit according to software (NIH). Liu et al. acta neuropathol commun (2021) 9:37 Page 14 of 16 Immunohistochemistry staining of human brain tissues by qPCR. Primers can be seen in Additional file  1: For histochemical analysis of post-mortem brain slices Table S3. of normal subjects and PD patients, paraffin-embedded sections (6 µm) were stained with anti-CHCHD2 (66302- 1-Ig, Proteintech, 1:500) or anti-TH (ab76442, Abcam, Statistical analysis 1:500) antibodies. Quantitation of immunostaining was All analyses were performed with Prism 8.0 (GraphPad). conducted using NIH image J software. The same image Linear regression analysis was used to determine the exposure times and threshold settings were used for sec- relationships between age, PD severity, MOCA, disease tions from all groups. The immunohistochemical staining duration and CHCHD2. One-way analysis of variance of brain tissues and quantification were performed in a (ANOVA) followed by Tukey’s post-hoc test or two-way double-blinded manner. ANOVA followed by Bonferroni post-hoc test was used for multiple comparisons. F values with their associated degrees of freedom (treatment, time, interaction and Cell culture and transfection residual) were expressed as F The MN9D dopaminergic neuronal cell line (American (df of treatment, time, interaction/resid- = F values (treatment, time, interaction) in two-way Type Culture Collection, Manassas, Va., USA) was cul- ual) ANOVA, and F = F values. Please note tured in DMEM high glucose medium containing 10% (df of treatment, residual) that a non-parametric test was used whenever the sample fetal bovine serum (Gibco, Carlsbad, CA, USA) and 100 numbers were too small (N = 3–6) to test for normality. U/mL of penicillin (Invitrogen) and 100 μg/mL of strep- Nonparametric Mann–Whitney U test was used for the tomycin (Invitrogen). The cells were incubated at 37  °C comparison of the mean values between two groups. The in an incubator with a humidified atmosphere of 95% multiple t-test analysis was used to analyze "matched" or air and 5% CO . MN9D cells were transfected with wild "paired" data from the Grouped format data table. Addi- type or A53T human α-synuclein cDNA in the pGV219 tionally, relationships between the analytes and age, sex, vector. Since α-synuclein expression and oligomeriza- and UPDRS motor score were analyzed with bivariate tion are dynamic in these clones, comparisons were made correlation using Pearson’s correlation coefficients. Val - between clones of wild type and A53T that were main- ues with p < 0.05 were regarded as significant. Receiver tained in parallel from DNA transfection. operating characteristic (ROC) curves were used to cal- culate the relationship between sensitivity and specificity Immunofluorescence (IF) for cultured cells for PD disease group versus healthy control. The “opti - Cultured cells were fixed with 4% paraformaldehyde mum” cutoff value from the ROC curve is determined by for 10  min followed by permeabilization by 0.2% Tri- anchoring the specificity (or sensitivity) to be ≥ 80%. ton X-100 for 3 min before staining. Subsequently, cells were incubated with a primary antibody at 4  °C over- Supplementary Information night and a secondary antibody conjugated with Alexa The online version contains supplementary material available at https ://doi. Fluor (Thermo Fisher Scientific) was added for detec - org/10.1186/s4047 8-021-01133 -6. tion. Cell nuclei were counterstained with DAPI dye. Fluorescence microscopy was performed with a Zeiss Additional file 1.Supplementary data Confocal Microscope. IF signals were quantitated using the Image J software (NIH). Acknowledgements We sincerely thank the China National Health and Disease Human Brain Tissue Resource Center for providing post-mortem human brain tissues, and all the Chromatin immunoprecipitation community volunteers and PD patients for the donation of blood samples. Control, wild type, or A53T human α-synuclein- Authors’ contributions overexpressing MN9D cells were collected for ChIP JZ and TF conceived and supervised the project; XDL designed the project using a SimpleChIP Enzymatic Chromatin IP Kit with JZ, performed all of the NanoString and PCR experiments, part of Immunofluorescence, statistical analyses, and drafted the manuscript; QLW (Magnetic Beads) (#9003; Cell Signaling), according to performed part of Immunofluorescence, Western blot and ChIP experiments the manufacturer’s instructions. In brief, MN9D cells and assisted in statistical analyses; YY performed part of Immunofluorescence were first fixed with 1% formaldehyde to cross-link experiments in human and mice brain slices. DS and ET contributed to the immunofluorescence experiments in human brain slices. TS, MS, EMC, ZRL and protein and DNA. Cell lysates were then subjected to YRH contributed to data interpretation and preparation of the manuscript; sonication for ChIP. ChIP was done using anti-p300 GLL were responsible for patient characterization and sample collection; all antibody (ab14984; Abcam) or control IgG (sc-2027; authors critically reviewed the manuscript. Santa Cruz Technology). Precipitated DNA fragments Funding containing gene promoter of CHCHD2 were detected This research was supported by National Key R&D Program of China (No. 2016YFC1306500), National Natural Science Foundation of China (No. Liu  et al. acta neuropathol commun (2021) 9:37 Page 15 of 16 81571226, No. 81671187), Beijing Municipal Science and Technology Com- 10. Hansen C, Angot E, Bergstrom AL, Steiner JA, Pieri L, Paul G, Outeiro TF, mission (No. Z17110700100000, Z151100003915117, No. Z151100003915150, Melki R, Kallunki P, Fog K et al (2011) alpha-Synuclein propagates from Z161100000216150 and Z171100000117013), Natural Science Foundation of mouse brain to grafted dopaminergic neurons and seeds aggrega- Beijing Municipality (No. 7164254) and Science Fund for Creative Research tion in cultured human cells. J Clin Investig 121:715–725. https ://doi. Groups of the National Natural Science Foundation of China (No. 81521002).org/10.1172/JCI43 366 11. Harjuhaahto S, Rasila TS, Molchanova SM, Woldegebriel R, Kvist J, Availability of data and materials Konovalova S, Sainio MT, Pennonen J, Torregrosa-Munumer R, Ibrahim H All data needed to evaluate the conclusions in the paper are present in the et al (2020) ALS and Parkinson’s disease genes CHCHD10 and CHCHD2 paper and the Additional file are available from authors upon request. modify synaptic transcriptomes in human iPSC-derived motor neurons. Neurobiol Dis 141:104940. https ://doi.org/10.1016/j.nbd.2020.10494 0 Competing interests 12. Ikeda A, Nishioka K, Meng H, Takanashi M, Inoshita T, Shiba-Fukushima The authors declare that they have no competing interests. K, Li Y, Yoshino H, Mori A, Okuzumi A et al (2019) Mutations in CHCHD2 cause alpha-synuclein aggregation. Hum Mol Genet. https ://doi. Author detailsorg/10.1093/hmg/ddz24 1 Department of Pathology, School of Basic Medical Sciences, Peking Univer- 13. 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Reduced erythrocytic CHCHD2 mRNA is associated with brain pathology of Parkinson’s disease

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Abstract

Peripheral biomarkers indicative of brain pathology are critically needed for early detection of Parkinson’s disease (PD). In this study, using NanoString and digital PCR technologies, we began by screening for alterations in genes associated with PD or atypical Parkinsonism in erythrocytes of PD patients, in which PD-related changes have been reported, and which contain ~ 99% of blood α-synuclein. Erythrocytic CHCHD2 mRNA was significantly reduced even at the early stages of the disease. A significant reduction in protein and/or mRNA expression of CHCHD2 was confirmed in PD brains collected at autopsy as well as in the brains of a PD animal model overexpressing α-synuclein, in addition to seeing a reduction of CHCHD2 in erythrocytes of the same animals. Overexpression of α-synuclein in cellular models of PD also resulted in reduced CHCHD2, via mechanisms likely involving altered subcellular localiza- tion of p300 histone acetyltransferase. Finally, the utility of reduced CHCHD2 mRNA as a biomarker for detecting PD, including early-stage PD, was validated in a larger cohort of 205 PD patients and 135 normal controls, with a receiver operating characteristic analysis demonstrating > 80% sensitivity and specificity. Keywords: Parkinson’s disease, Mitochondria dysfunction, α-synuclein, CHCHD2 Introduction until a sustained therapeutic response is observed during Diagnosis of Parkinson’s disease (PD), a common neuro- additional years of follow-up. In other words, there is an degenerative disorder [28], is currently based on a combi- essential need to develop a method for accurate diagnosis nation of medical history, observation of cardinal motor of PD at early stage, where disease modifying therapies indicators, and response to pharmaceutical therapies are likely to be most effective. [42, 47]. However, motor symptoms often present after Much effort in the PD biomarker field has focused on greater than 50% of affected neurons have degenerated the discovery of biomarkers using the cerebrospinal fluid [4]. Further, a more definitive diagnosis cannot be made (CSF), with α-synuclein and its variants receiving most attention [32, 41, 45]. However, very few markers inves- tigated thus far demonstrate clinically useful power in *Correspondence: happyft@sina.com; zhangj@uw.edu; jzhang1989@zju.edu. detecting PD or following PD progression, and none has cn been widely validated. Alongside the challenges associ- Xiaodan Liu, Qilong Wang and Ying Yang have contributed equally to this work ated with these protein markers, CSF collection (via lum- Department of Pathology, School of Basic Medical Sciences, Peking bar puncture), often perceived as a high-risk and painful University Third Hospital, Peking University Health Science Center, procedure, is impractical for routine screening purposes. Beijing 100191, China Center for Movement Disorders, Department of Neurology, Beijing More recently, investigations on PD-related proteins in Tiantan Hospital, Capital Medical University, Beijing 100070, China blood have received more consideration; however, they Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. acta neuropathol commun (2021) 9:37 Page 2 of 16 have not yielded consistent results, largely because of these, only CHCHD2 mRNA consistently demonstrated the complexity of the samples [24]. In contrast, blood- significantly reduced expression in all PD groups com - based changes in mRNA expression present an additional pared to the control group [Fig.  1a, F(3, 4) = 52.0, one- and promising biomarker strategy for differentiating way ANOVA, n = 3; p < 0.01 for Early or Mid PD vs. PD patients from healthy controls [3, 31, 36]. One study control, p < 0.001 for Late PD vs. control]. This result was compared the transcriptional profile of PD patients with next confirmed by digital droplet PCR (ddPCR), which or without postural instability and found > 200 differen - also detected significantly reduced CHCHD2 mRNA in tially expressed genes, some of which were also found erythrocytes across all PD groups compared to controls to be dysregulated in a dopaminergic cell model of PD [Fig.  1b, F(3, 8) = 20.80, one-way ANOVA, n = 3; p < 0.01 [26], suggesting that altered gene expression in the blood or 0.001 for each PD group vs. the controls]. There were may reflect changes in the central nervous system (CNS). no differences, however, between PD groups with differ - Another group explored the genetic signature in blood ent disease severity. Having validated the mRNA result from a large cohort of PD patients and identified 87 genes independently, we next examined the protein expression which differentiated between patients with idiopathic of CHCHD2 in erythrocytes of PD patients. As shown PD and controls [36], further supporting the concept in Fig.  1c, the relative CHCHD2 protein levels were of altered gene expression within the blood as a useful decreased significantly from 1.01 ± 0.07 in pooled eryth- tool for predicting PD. Notably, both studies used RNA rocytes of controls to 0.43 ± 0.12 in those of PD patients extracted from whole blood cells, mainly leukocytes, (p < 0.05, Mann–Whitney U test, n = 3). which undergo nuclear and transcriptional changes dur- ing disease states. Compared to leukocytes (and most Reduced expression of CHCHD2 in the post‑mortem other blood cells), mature erythrocytes are uniquely substantia nigra slices of PD patients structured, lacking organelles and nuclei necessary for To probe whether expression of CHCHD2 is also replacing dysfunctional proteins. Thus, alterations in reduced in the central nervous system (CNS) of PD residual RNA and protein levels in erythrocytes likely patients, its expression was examined in the post-mor- reflect pathological rather than physiological changes tem substantia nigra slices obtained at autopsy from the [2]. Additionally, ~ 99% of blood α-synuclein is located in brains of PD patients, along with age-matched controls, erythrocytes, and pathological changes in erythrocytes as well as in the animal model. Firstly, we examined the have been described in PD patients in several independ- expression pattern of CHCHD2, both in the substantia ent investigations [1, 2, 14, 21, 23, 30, 40, 44]. Therefore, nigra of human and wild type C57BL mice. Co-labeling in this study, we tested the hypothesis that erythrocytes of CHCHD2 with a pan-neuronal marker (NeuN) and a carry biomarkers that reflect or correlate with brain dopaminergic neuronal marker (Tyrosine hydroxylase, pathology, and are capable of detecting PD at early stages. TH) revealed that CHCHD2 is expressed in nearly all neurons (> 90%, Fig.  2a for human, Fig. S2A for mice). CHCHD2 is also expressed in > 90% astrocytes (Fig.  2b Results for human, Fig. S2B for mice), but only in 10–30% micro- Reduced mRNA and protein expression of CHCHD2 glia cells (Fig.  2c for human, Fig. S2C for mice). Subcel- in erythrocytes of PD patients lularly, CHCHD2 is located in mitochondria, as indicated A NanoString multiplex gene expression method was by co-localization with Translocase of Outer Mitochon- used to screen for potential mRNA biomarkers for PD drial Membrane 20 (TOMM20) (Fig. S2D). Consistent within erythrocytes. A panel of 21 genes associated with with previous reports [19, 27], loss of dopaminergic neu- PD or atypical Parkinsonism (see “Materials and meth- rons, indicated by TH staining, was detected (Fig.  2e–g, ods” section for a complete list) chosen based on previ- 13.2 ± 1.3 in Ctl vs. 4.6 ± 0.6 in PD per field, p < 0.001, ous reports [6, 18, 20, 34, 43], was examined in a cohort multiple t-test). The number of CHCHD2 positive cells consisting of 48 participants separated according to four was also significantly lower, with 5.46 ± 0.6 per field in diagnostic classifications: control, early-stage PD (Early PD patients versus 15.2 ± 1.6 neurons in control (Fig. 2e– PD), middle-stage PD (Mid PD), or late-stage PD (Late g, p < 0.001, multiple t-test). Fluorescence intensity of PD) patients according to Unified Parkinson’s Disease CHCHD2 protein in the surviving neurons of substantia Rating Scale (UPDRS) score (Additional file  1: Table S1). nigra of PD patients also decreased markedly (Fig.  2e–f mRNA expression frequency of each gene in erythro- and h p < 0.001, Mann–Whitney U test). cytes of healthy control is shown in Additional file  1: We also detected the expression of CHCHD2 in Fig. S1, with SNCA, FBXO7, CHCHD2, PSEN1, LRRK2, post-mortem slices across different brain regions by VPS35, GATA1, MAPT, APOE, and PINK1 among the immunohistochemistry. Consistent with the results ten most highly expressed genes in erythrocytes. Of obtained by immunofluorescence in Fig.  2e–h, the Liu  et al. acta neuropathol commun (2021) 9:37 Page 3 of 16 Fig. 1 CHCHD2 mRNA and protein expression was reduced in erythrocytes of PD patients. a Genes associated with PD or atypical Parkinsonism in pooled erythrocyte samples from controls and PD patients at various stages were analyzed by the NanoString multiplex gene expression method. The samples were pooled according to Additional file 1: Table S1 [four comparison groups (n = 12 each) with each pooled into three sub-groups]. Ten genes were analyzed following removal of low expression genes. Significantly decreased CHCHD2 mRNA was detected in all PD groups compared to the control group [F (3, 4) = 52.0, one-way ANOVA, n = 3; p < 0.01 for Early or Mid PD vs. control, p < 0.001 for Late PD vs. control]. b Validation of the discovery cohort by ddPCR in the discovery set also identified decreased CHCHD2 mRNA in erythrocytes of all PD groups [F(3, 8) = 20.80, one-way ANOVA, n = 3; p < 0.01 or 0.001 for each PD group vs. the controls] compared to controls. No difference in CHCHD2 mRNA expression was observed between PD groups by Tukey’s multiple comparisons test. Left: Original representative ddPCR picture. FAM channel (Axis X) indicates CHCHD2, VIC channel (Axis Y ) indicates AHSP. Right: Statistical result. c Decreased CHCHD2 protein in erythrocytes of PD patients. Proteins were extracted from pooled erythrocytes of the same cohort as used in the NanoString and ddPCR studies. Compared to controls, CHCHD2 was decreased significantly from 1.01 ± 0.07 to 0.43 ± 0.12 (p < 0.05, Mann–Whitney U test, n = 3) Liu et al. acta neuropathol commun (2021) 9:37 Page 4 of 16 Fig. 2 Reduced expression of CHCHD2 in post-mortem substantia nigra slices of PD patients. a Representative images of co-staining of CHCHD2 (green) with neuronal marker NeuN (blue) and dopaminergic neuronal marker TH (red), showing expression of CHCHD2 in neurons. Scale bar: 10 μm. b Representative images of co-staining of CHCHD2 (green) with astrocyte marker GFAP (red), showing expression of CHCHD2 in astrocytes. Scale bar: 10 μm. c Representative images of co-staining of CHCHD2 (green) with microglial marker Iba1 (red), showing expression of CHCHD2 in a minority of microglia. Scale bar: 10 μm. d Quantification of the percentage of CHCHD2 positive cells by cell type in Iba1, GFAP or NeuN positive cells. e and f Immunofluorescence staining images of substantia nigra of control and PD brain slices probed with the antibody against CHCHD2 (green) + + and TH (red). Scale bar = 20 μm. g Quantification of TH and CHCHD2 cells in substantia nigra, demonstrating lower numbers of surviving neuronal cells and surviving CHCHD2 cells in PD patients. p < 0.001, multiple t-test, n = 3 each. h Quantification of immunofluorescent signal demonstrating reduced CHCHD2 immunofluorescent intensity in substantia nigra (p < 0.001, Mann–Whitney U test, n = 3) Liu  et al. acta neuropathol commun (2021) 9:37 Page 5 of 16 number of CHCHD2 positive cells (Fig.  3a, p < 0.0001, contrast, the expression of CHCHD2 in frontal cortex Mann–Whitney U test) and density of CHCHD2 in (Fig.  3c, p > 0.05, Mann–Whitney U test) and cerebel- the surviving cells (Fig.  3b, p < 0.01, Mann–Whit- lum (Fig.  3d, p > 0.05, Mann–Whitney U test) were not ney U test) of substantia nigra of PD patients were changed. These results suggested CHCHD2 was prefer - reduced significantly, compared with control. In entially reduced in the substantia nigra of PD patients. Fig. 3 CHCHD2 expression was reduced specifically in the substantia nigra of PD patients when examined by immunohistochemical staining. a Reduced number of CHCHD2 positive cells in substantia nigra of PD patients. Whole slice scanned images of control and PD patients were shown in left and middle panel. Scale bar: 1000 μm. Statistics data of the number of CHCHD2 positive cells was shown in right panel. p < 0.0001, Mann– Whitney U test. b The immunohistochemical staining intensity of CHCHD2 was reduced in substantia nigra of PD patients. p < 0.01, Mann–Whitney U test, n = 3. Scale bar: 50 μm. c CHCHD2 expression was not altered in frontal cortex. p > 0.05, Mann–Whitney U test, n = 3. Scale bar: 50 μm. d CHCHD2 expression was not altered in cerebellum. p > 0.05, Mann–Whitney U test, n = 3. Scale bar: 50 μm Liu et al. acta neuropathol commun (2021) 9:37 Page 6 of 16 Reduced mRNA and protein expression of CHCHD2 expression of CHCHD2 in erythrocytes was significantly +/+ in erythrocytes and brains of A53T α‑synuclein mice lower in A53T mice (0.27 ± 0.03 for mRNA, p < 0.01, Having observed both CNS and peripheral reduction Mann–Whitney U test, n = 6, Fig.  4a; and 0.29 ± 0.04 for of CHCHD2 expression in PD patients, we next sought protein, p < 0.01, Mann–Whitney U test, n = 3, Fig.  4b) to explore the potential underlying mechanisms in a compared to wild type control mice (1.11 ± 0.20 for PD mouse model. For this purpose, we used the A53T mRNA; 1.02 ± 0.14 for protein). In the substantia nigra, α-synuclein transgenic mouse model (M83 line), an real time PCR and Western blot results clearly showed extensively utilized model where progressive pathol- that the mRNA (Fig.  4c, p < 0.01, Mann–Whitney U test, ogy and behavior are driven by expression of mutated n = 6) and protein expression (Fig.  4d, p < 0.05, Mann– +/+ α-synuclein [8]. A53T mice were used at 10  months Whitney U test, n = 3) of CHCHD2 were significantly old, when neurologic defects are readily detectable. decreased. As shown in Fig.  4, the relative mRNA and protein Fig. 4 Reduced expression of CHCHD2 in the erythrocytes and substantia nigra of A53T α-synuclein transgenic mice. a Compared to non-transgenic wild type mice, normalized mRNA of CHCHD2 in erythrocytes of A53T α-synuclein transgenic mice was significantly decreased from 1.11 ± 0.20 to 0.27 ± 0.03 (p < 0.01, Mann–Whitney U test, n = 6). b Western blot results showed a significantly reduced protein expression of CHCHD2 in erythrocytes of transgenic mice (p < 0.01, Mann–Whitney U test, n = 3). Upper: representative Western blot images. β-actin was used as an internal control. Wt: non-transgenic wild type mice. Tg: A53T transgenic mice. Lower: quantification of Western blots. c Real-time qPCR also detected a reduced mRNA expression of CHCHD2 in substantia nigra of transgenic mice (p < 0.01, Mann–Whitney U test, n = 6). d CHCHD2 protein was decreased from 1.21 ± 0.21 in substantia nigra of wild type mice to 0.21 ± 0.10 in substantia nigra of transgenic mice (p < 0.05, Mann–Whitney U test, n = 3). Upper: representative Western blot images. β-tubulin was used as an internal control. Lower: quantification of Western blots. Of note, the sample number (N) is different because mRNA and proteins were measured in two different sets of animals Liu  et al. acta neuropathol commun (2021) 9:37 Page 7 of 16 We also detected a reduced expression of CHCHD2 U test, n = 3). A correlation analysis indicated a nega- by immunofluorescence across several brain regions, tive correlation between the expression of CHCHD2 including substantia nigra (Fig.  5a, b), the rest and the level of aggregated α-synuclein (r = − 0.978, of midbrain (Fig.  5c, d), frontal cortex (Fig.  5e, f) p < 0.001 in substantia nigra, Fig. S3A; r = − 0.943, +/+ and cerebellum (Fig.  5g, h) in A53T mice com- p < 0.01 in the rest of midbrain, Fig. S3B; r = − 0.952, pared to non-transgenic wild type mice (all p <0.05, p < 0.01 in frontal cortex, Fig. S3C; r = − 0.978, Mann–Whitney U test, n = 3). Consistent with previ- p < 0.001 in cerebellum, Fig. S3D). ous reports [8, 10, 35], aggregated α-synuclein was increased in these regions (all p < 0.05, Mann–Whitne y Fig. 5 Immunofluorescent staining of CHCHD2 and α-synuclein in different areas of mouse brain slices. a and b Decreased CHCHD2 (green) ## and increased α-synuclein (blue) in substantia nigra (***p < 0.001, p < 0.01, Mann–Whitney U test, n = 3). c and d Decreased CHCHD2 (green) ## and increased α-synuclein (blue) in rest of midbrain (**p < 0.01, p < 0.01, Mann–Whitney U test, n = 3). e and f Decreased CHCHD2 (green) and ## increased α-synuclein (blue) in frontal cortex (***p < 0.001, p < 0.01, Mann–Whitney U test, n = 3). g and h Decreased CHCHD2 (green) and increased α-synuclein (blue) in cerebellum (***p < 0.001, p < 0.05, Mann–Whitney U test, n = 3). Scale bar: 10 μm Liu et al. acta neuropathol commun (2021) 9:37 Page 8 of 16 Reduced mRNA and protein expression of CHCHD2 p300 histone acetyltransferase [13], we tested whether in a cellular model of PD there is a direct interaction between p300 and the pro- To further explore the potential mechanisms leading moter of CHCHD2. As shown in Fig. 6c, ChIP results dis- to decreased CHCHD2 expression in PD patients, wild tinctly showed that p300 can directly bind the promoter type and A53T α-synuclein plasmids were transfected of CHCHD2. Additionally, overexpression of wild type or in MN9D cells, a line that is derived from dopamin- mutant α-synuclein in MN9D cells reduced the interac- ergic cells and previously utilized in multiple in  vitro tion of p300 with the promoter of CHCHD2 (p < 0.001, F PD models [5, 15, 37]. As expected, transfection of (2, 6) = 73.44, one-way ANOVA, n = 3). the cells with α-synuclein vector resulted in increased We next examined the potential mechanisms by which mRNA [Fig. S4A, p < 0.01, F(2, 6) = 20.95, one-way α-synuclein expression alters the function of p300, by ANOVA, n = 3] and protein [Fig. S4B, F(2, 9) = 14.37, measuring the effect of α-synuclein overexpression one-way ANOVA, n = 4; p < 0.01 for A53T vs. Vec- on p300. Significant reductions in the mRNA (Fig.  6d, tor] expression of α-synuclein. The overexpression of p <0.001, F(2, 6) = 130.2, one-way ANOVA, n = 3) and α-synuclein also dramatically reduced the number of protein expression (Fig. 6e, p <0.001, F(2, 6) = 41.95, one- CHCHD2 mRNA transcripts, regardless of whether the way ANOVA, n = 3) of p300 were seen 48h after trans- vector was wild type or A53T α-synuclein [Fig.  6a F(2, fection of α-synuclein vector. Direct protein interaction 6) = 35.31, one-way ANOVA, n = 3; p < 0.001 for Wt between p300 and α-synuclein was also demonstrated α-syn vs. Vector, p < 0.01 for A53T α-syn vs. Vector]. by reciprocal co-immunoprecipitation experiments Consistent with the human data, the protein expres- (Additional file  1: Fig. S5B). Immunofluorescence results sion of CHCHD2 was also reduced markedly[Fig.  6b, showed that p300 was mainly expressed in the nucleus, F(2, 6) = 20.75, one-way ANOVA, n = 3; p < 0.05 for Wt while it was less expressed in the cytoplasm of vec- or A53T α-syn vs. Vector]. tor transfected Mn9D cells. However, overexpression of wild type or A53T α-synuclein significantly reduced α‑synuclein negatively regulates the expression the nuclear expression of p300 (Fig.  6f, p < 0.0001, F(2, of CHCHD2, possibly via modulation of P300 154) = 12.8, one-way ANOVA). Western blot of the pro- The results shown above suggest that reduced tein of isolated subcellular fractions showed an altered CHCHD2 protein is largely attributable to a decreased distribution, with a higher proportion of p300 in the level of mRNA, likely secondary to overexpression cytoplasm, compared to a lower amount in the nucleus of α-synuclein. To further investigate potential links after α-synuclein (A53T) overexpression compared to between α-synuclein overexpression and CHCHD2 vector-transfected cells (Fig.  6g, p < 0.05, Mann–Whit- level, we asked whether there is a direct interaction of ney U test, n = 3). All the above results suggested that α-synuclein with the promoter of CHCHD2. Initial chro- α-synuclein may inhibit the expression of CHCHD2, pos- matin immunoprecipitation (ChIP) experiments failed to sibly by reducing the expression and the nuclear distribu- demonstrate any direct interaction between α-synuclein tion of p300. and CHCHD2 promoter (Additional file  1: Fig. S5A). Therefore, we next investigated whether α-synuclein Exploration of erythrocytic CHCHD2 as a PD biomarker could negatively regulate the expression of CHCHD2 in a large cohort indirectly. Because it has previously been shown that Having demonstrated that expression of CHCHD2 is α-synuclein could negatively regulate protein kinase C significantly reduced in the substantia nigra as well as expression by reducing the expression and activity of in erythrocytes in PD patients, we turned our attention (See figure on next page.) Fig. 6 Overexpression of α-synuclein reduced CHCHD2 expression in MN9D cells, possibly by altering the expression and subcellular localization of p300. a Reduced mRNA expression of CHCHD2 in MN9D cells after overexpression of α-synuclein (F(2, 6) = 35.31, one-way ANOVA, n = 3; p < 0.001 for Wt α-syn vs. Vector, p < 0.01 for A53T α-syn vs. Vector). b Reduced protein expression of CHCHD2 in MN9D cells after overexpression of α-synuclein [F(2, 6) = 20.75, one-way ANOVA, n = 3; p < 0.05 for Wt or A53T α-syn vs. Vector]. c Direct interaction between p300 and CHCHD2 promoter as revealed by ChIP result. Overexpression of both wild type and A53T α-synuclein reduced the interaction of p300 and CHCHD2 promoter [F(2, 6) = 73.44,one-way ANOVA, n = 3; both p < 0.001, compared to Vector]. Histone 3 (H3) was used as positive control. d Decreased mRNA expression of p300 after overexpression of α-synuclein [F(2, 6) = 130.2, one-way ANOVA, n = 3; both p < 0.001, compared to Vector]. e Protein expression of p300 [F(2, 6) = 41.95, one-way ANOVA, n = 3; both p < 0.001, compared to Vector]. f Reduced nuclear distribution of p300 after overexpression of α-synuclein revealed by immunofluorescence (F (2, 154) = 12.8, one-way ANOVA, p < 0.05 or 0.01 or 0.0001). g Reduced localization of p300 in nucleus revealed by western blot (p < 0.05, Mann–Whitney U test, n = 3); Increased localization of p300 in cytoplasm (p < 0.05, Mann–Whitney U test, n = 3) Liu  et al. acta neuropathol commun (2021) 9:37 Page 9 of 16 Liu et al. acta neuropathol commun (2021) 9:37 Page 10 of 16 Fig. 7 Reduced CHCHD2 mRNA in erythrocytes predicts PD. a Reduced CHCHD2 mRNA in erythrocytes of PD patients was further confirmed in a larger, validation cohort of 135 normal controls, 73 Early PD, 98 Mid PD and 34 Late PD patients [p < 0.001 each, F (3, 332) = 26.11, one-way ANOVA]. No difference in CHCHD2 mRNA expression was determined among PD groups by Tukey’s multiple comparisons test. b ROC analysis revealed diagnostic values of 80.4% sensitivity and 81.1% specificity of CHCHD2 for PD diagnosis back to its utility as a more convenient biomarker for early PD versus healthy controls was 80.82%, yielding a PD diagnosis. To achieve this goal, we further exam- final diagnostic value of 85.38% for PD vs. controls (Addi - ined CHCHD2 mRNA in erythrocytes of individual PD tional file 1: Table S2 and Fig. 7b). patients in a cohort of 340 subjects including patients Finally, we compared the CHCHD2 mRNA levels in with early- (n = 73), middle- (n = 98), and late-stage erythrocytes with disease severity, including worsen- PD (n = 34) along with controls (n = 135). As shown in ing of motor symptoms and mild cognitive impairment Fig. 7a, CHCHD2 mRNA was reduced significantly in all (MCI), and disease duration by comparing CHCHD2 PD groups compared to controls, as detected by ddPCR mRNA to the UPDRS part III on-state motor scores, [F (3, 332) = 26.11, one-way ANOVA; p < 0.001 for each Montreal Cognitive Assessment (MoCA) score, and PD group vs. control], consistent with our discovery time following diagnosis in the validation cohorts of PD cohort results. patients (n = 205 total; cases < 50 years were not excluded Because PD is an age-related disorder, not evenly dis- due to the lack of age dependence as determined above). tributed by sex, we next investigated the impact of both Once again, as determined by linear regression analy- sex and age dependence on CHCHD2 mRNA levels iso- ses, no significant association could be determined lated from erythrocytes. Two-way ANOVA compari- between CHCHD2 mRNA levels and the disease severity sons of male and female control and PD patient samples (p = 0.49, R = − 0.05 for UPDRS motor), disease duration revealed no significant difference in CHCHD2 mRNA (p = 0.62, R = 0.04) or MOCA scores (p = 0.11, R = 0.11) levels between sexes [Additional file  1: Fig. S6A, p = 0.65, (Additional file 1: Fig. S6C–E). F (1, 326) = 0.2077], while linear regression analysis revealed no correlation between CHCHD2 mRNA levels and age in either group (Additional file  1: Fig. S6B, con- Discussion trol: p = 0.49, R = 0.06; PD: p = 0.35, R = − 0.07). The CHCHD2 gene is a recently discovered PD causa - We also evaluated CHCHD2 mRNA as a diagnostic tive gene that drives development of PD with autoso- marker in erythrocytes. Analysis of Receiver Operating mal dominant inheritance [6]. More recently, mutations Characteristic (ROC) curve was performed to evaluate in CHCHD2 have also been found in sporadic PD [29, the diagnostic accuracy of CHCHD2 mRNA levels in the 39, 46]. CHCHD2 protein is localized to mitochondria, total PD cohort as well as PD patients at early-stage. The which are intimately associated with PD pathogenesis results were similar for both analyses, and when the spec- [48]. It has been demonstrated that the loss of CHCHD2 ificity was anchored to ≥ 80%, sensitivity for detecting causes abnormal matrix structures and impaired oxygen Liu  et al. acta neuropathol commun (2021) 9:37 Page 11 of 16 respiration in mitochondria, leading to oxidative stress, remains to be determined. Moreover, as with other gene dopaminergic neuron loss and motor dysfunction in mutations associated with PD, e.g., SNCA and LRRK2, Drosophila [22]. Additionally, knockout of CHCHD2 though mutations occur systematically, dopaminer- or CHCHD10 in human induced pluripotent stem cells gic neurons are preferentially vulnerable. Why and how led to increased proton leakage and respiration, as well CHCHD2 mutations/reductions result specifically in as loss of synaptic function [11]. CHCHD2/CHCHD10 dopaminergic neurodegeneration needs to be investi- double knockout mice showed disrupted mitochondrial gated further. cristae [17]. Within affected brain regions, compared to healthy Two major observations of the current study are: (1) controls, both the number of CHCHD2 positive neu- the expression of CHCHD2 was reduced significantly in rons and its level of expression in surviving neurons human erythrocytes, substantia nigra of brain collected were reduced in the substantia nigra of PD patients at autopsy, and mice carrying transgenic mutant A53T (Figs. 2 and 3), suggesting the possibility of an interaction α-synuclein; and (2) the expression of CHCHD2 in eryth- between PD pathological processes (e.g., accumulation of rocytes, a readily accessible clinical sample source, could pathological α-synuclein species) and CHCHD2 reduc- be an early diagnostic marker of PD. tion. Consistent with this hypothesis, correlation analysis Using NanoString multiplex gene expression analysis, revealed a negative correlation between protein expres- among a total of 21 genes screened, CHCHD2 mRNA sion levels of α-synuclein and CHCHD2 in mice (Addi- was discovered to be consistently reduced in various tional file  1: Fig. S3), i.e., reduced CHCHD2 level may be stages of PD patients. The corresponding protein was driven by increased levels of pathogenic α-synuclein. also reduced in erythrocytes (Fig.  1). The mechanisms How might α-synuclein, especially pathogenic forms, responsible for the CHCHD2 reduction in the erythro- inhibit the expression of CHCHD2? A previous study cytes of PD patients are unknown, especially given that indicated that α-synuclein negatively regulates protein mature erythrocytes are incapable of transcriptional kinase C expression by reducing the expression and activ- regulation. One possibility is that the transcriptional reg- ity of p300 histone acetyltransferase [13]. Consistent with ulation occurred in bone marrow or at the stage of retic- this observation, overexpression of wild type and A53T ulocytes, a data point not collected in the current study. mutant α-synuclein in MN9D cells, a mouse dopamin- However, reticulocytes account for 0.5–2.5% of red blood ergic cell line, significantly reduced the mRNA and pro - cells [38], and there is no evidence to suggest that there tein expression of CHCHD2, with the effect being more is a difference between PD and controls in reticulocyte significant when A53T mutant α-synuclein was used counts. Therefore, future study needs to include investi - (Fig.  6). Additionally, direct interaction of p300, instead gations in bone marrow or at least to consider the ratio of of α-synuclein, with the promoter of CHCHD2 gene was erythrocytes/reticulocytes in various cohorts. detected by ChIP experiment whereas overexpression One surprising aspect of this study is that while of wild type or A53T mutant α-synuclein reduced their reduced CHCHD2 expression was observed in (and cor- interaction. However, α-synuclein and CHCHD2 may related between) both the substantia nigra (Figs.  2 and have a more complicated mutual interaction. For exam- 3) and periphery of human PD patients, the reduction ple, in a recent study, mutations in CHCHD2 resulted was not universal within the CNS, as it did not extend in α-synuclein aggregation [12]. Further, although a to the frontal cortex or cerebellum. In contrast, reduced link between the reduced expression of CHCHD2 and CHCHD2 mRNA and protein expression were detected nuclear distribution of p300 is clearly implicated (Fig. 6), in the substantia nigra, prefrontal cortex and cerebellum the direct involvement of p300 needs to be confirmed by of A53T α-synuclein transgenic mice (Figs. 4 and 5). The additional studies, e.g., knockdown of its expression in a discrepancy in affected brain region between human and cellular model of PD. mice studies may have been due to the different expres - Regardless of the mechanism(s) underlying CHCHD2 sion patterns of pathological α-synuclein species in expression in PD patients, reduced CHCHD2 mRNA human and mice; specifically, the PD patients used in our in all stages of PD was validated by ddPCR in a larger study were at Braak Stage 3, a stage at which Lewy body cohort, suggesting that levels of CHCHD2 mRNA may pathology is present in the substantia nigra, but not the act as a biomarker for detecting PD using patient blood, cortex or cerebellum. In contrast, transgenic α-synuclein even at early disease stage, with a sensitivity and specific - is widely expressed in the central nervous system in the ity of 80 and 81%, respectively (Fig.  7). Indeed, the level mouse model (M83 line), without the progressive spread of CHCHD2 was not correlated with disease severity, observed in human patients. Whether lower expres- motor or cognition, suggesting a possible flooring effect, sion of CHCHD2 in other brain regions becomes more possibly because the changes occur early during the dis- widespread in the human brain as the disease progresses ease process. In addition, no significant correlation was Liu et al. acta neuropathol commun (2021) 9:37 Page 12 of 16 observed between CHCHD2 mRNA levels and sex or age Laboratory. Non-transgenic mice with the same back- (Additional file  1: Fig. S6). The result is quite significant ground were used as controls. All mice were housed in in that reduced CHCHD2 mRNA could be a potential separate cages with free access to food and water. The biomarker for early PD, when clinical diagnosis is most room temperature was kept at 24 ± 1  °C under natural difficult due to overlapping clinical phenotypes in related light–dark cycle. All animal experimental procedures diseases. However, to truly test this biomarker, future val- were approved by the Animal Care and Use Committee idation studies should include a reasonably large cohort of Peking University. of patients with multiple system atrophy (MSA) and/ or progressive supranuclear palsy (PSP). Additionally, RNA isolation from erythrocytes moving forward, the biomarker should be tested in the To separate erythrocytes from plasma and buffy coat, premotor stage when nigrostriatal degeneration can be fresh whole blood samples were immediately centri- confirmed by DAT or PET imaging. fuged after phlebotomy at 2000  g for 10  min. 200  μl of In summary, reduction in protein and mRNA expres- pure erythrocytes were then used to extract total RNA sion of CHCHD2 is widespread in PD patients, from using Trizol reagent (Invitrogen, USA) according to the CNS to peripheral erythrocytes. The underlying mecha - manufacturer’s protocol. RNA was assessed for quantity nisms, though they remain to be investigated, are likely using Nanodrop 2000, and for quality using the 2100 Bio- related to synucleinopathy and involvement of p300. analyzer (Agilent Technologies, Canada). RNA was either CHCHD2 mRNA in erythrocytes, which is easily acces- used immediately or stored at − 80 ℃ consistently within sible, may serve as a convenient yet robust biomarker of experiments. PD, particularly for diagnosis of PD at early stages. NanoString nCounter method Materials and methods The nCounter Analysis System (NanoString Technolo - Participants gies, Seattle, WA) allows for multiplexed digital mRNA This study was approved by the Institutional Review Boards profiling without amplification or generation of cDNA of all participating institutions. Subjects for both the dis- [7]. A total of 21 PD related genes [APOE (NM_000041.2), covery (48 in total; 12 healthy controls, 36 PD patients) APP (NM_000484.3), ATP13A2 (NM_001141974.1), and validation (340 in total; 135 healthy controls, and 205 CHCHD2 (NM_016139.2), EIF4G1 (NM_004953.3), PD patients) studies were recruited from Beijing Tiantan FBXO7 (NM_001033024.1), GATA1 (NM_002049.2), Hospital between 2016 and 2018. All participants provided GBA (NM_001005742.2), LRRK2 (NM_198578.3), informed consent and underwent a neurologist-conducted MAPT (NM_016834.3), PARK2 (NM_004562.2), PARK7 evaluation that consisted of a structured interview, neuro- (NM_001123377.1), PINK1 (NM_032409.2), PLA2G6 logical examination, laboratory tests, and neuropsycho- (NM_001199562.1), PSEN1 (NM_000021.2), PSEN2 logical assessments. All control subjects were community (NM_000447.2), SNCA (NM_000345.2), SNCAIP volunteers who had Mini Mental State Exam Scores > 24, (NM_001242935.2), TPPP (NM_007030.2), UCHL1 paragraph recall scores > 6, no history of neurological dis- (NM_004181.3), VPS35 (NM_018206.4)] were screened. ease, and no history or evidence of cognitive or functional Total RNA (100  ng) was hybridized with the Tagset decline. All PD patients met UKPD Society Brain Bank probes and loaded into the nCounter prep-station, and clinical diagnostic criteria for PD [9]. PD patient samples then quantified using the nCounter Digital Analyzer. The were further categorized based on UPDRS Part III on-state NanoString platform includes negative control probes motor scores to approximate disease stage, according to the (not complementary to any endogenous mRNA) to assess method we reported previously [16]. That is, patients with background noise associated with the fluorescent bar - UPDRS scores < 15 were defined as having early-stage PD, code optical recognition system. Raw probe counts were those with scores ranging from 15 to 30 were classified as normalized to a panel of three endogenous control genes middle-stage PD, while those with scores > 30 were clas- [AHSP (NM_016633.2), β-actin (NM_001101.2), and sified as late-stage PD patients. Demographic data for all GAPDH (NM_002046.3)] by taking the ratios of each subjects and UPDRS scores for all PD patients used in the gene’s counts per sample to the average across all samples study are listed in Additional file 1 : Tables S1 and S2. and scaling by the median of these ratios in each sample. This normalization factor was also applied to the negative Animals control probes counts. A detection threshold was defined Transgenic mice expressing A53T (M83 line) human for each sample as five times the mean of the negative α-synuclein protein under the control of the prion pro- control probe normalized counts. tein promoter [8] were purchased from The Jackson Liu  et al. acta neuropathol commun (2021) 9:37 Page 13 of 16 Digital droplet PCR quantification the manufacturer’s protocol (Takara Inc, Japan). Oligo- For absolute quantification of mRNA of CHCHD2 in nucleotide primers corresponding to cDNA for mouse erythrocytes, we used the recently developed digital CHCHD2/α-synuclein/p300/GAPDH can be seen in droplet PCR [25]. TaqMan probes (250 nM final concen - Additional file  1: Table S3. The specificity for each primer tration) and related primers set for CHCHD2 (labeled set was confirmed by both electrophoresis of the PCR with a FAM at the 5′ end, 900  nM primers at final con - products on a 2.0% agarose gel and analyzing the melting centration) and AHSP (labeled with a VIC at the 5′ end, (dissociation) curve using a 7500 ABI PRISM Sequence 150  nM primers at final concentration) were purchased Detector System according to the manufacturer’s instruc- from Thermo Fisher Scientific, Inc. (USA). AHSP, which tions (Applied Biosystems) after each real-time PCR is expressed mainly in erythrocytes and did not change reaction. The relative amount of transcripts was calcu - −ΔΔCT in PD based on our NanoString data, was chosen as an lated using the 2 method [33] and normalized to ™ ™ internal control. We used SuperScript III Platinum the endogenous reference gene GAPDH. One-Step qRT-PCR Kit (#11732020, Thermo Scientific, USA) for cDNA synthesis and PCR amplification per - Immunofluorescence staining of human and mouse brain formed in a single tube following the instructions of the tissues manufacturer. The RainDrop Source emulsion genera - The post-mortem human brain material was obtained tor (RainDance Technologies, Inc.) was used to generate from China National Health and Disease Human Brain emulsified micro droplets. Each 25  μl reaction system Tissue Resource Center (Hangzhou). All materials consisted of the 24  μl TaqMan gene expression ddPCR have been collected from donors who provided writ- system and 1  μl of 25 × droplet stabilizer. After emul- ten informed consent for a brain autopsy and permit- sion, the tube was directly sealed and put into an ABI ting the use of the material and clinical information for ProFlex thermalcycler (Thermo Fisher Scientific, Inc. research purposes. Demographic data for all subjects USA). PCR amplification program was as follows: 50  ℃, used in the study are listed in Additional file  1: Table S4. 15 min; 95 ℃, 4 min; 45 cycles of (95 ℃, 15 s; 60 ℃, 45 s). The paraffin-fixed formaldehyde-embedded human or The heating and cooling rates of the thermalcycler were frozen mouse brain tissues were cut into 6–10  μm sec- adjusted to 0.6 ℃/s for better PCR amplification in mil - tions. The immunofluorescence staining of brain tissues lions of micro droplets. After amplification, the tube was was performed in a double-blinded manner. Brain slices transferred to Raindrop Sense machine and the numbers were incubated overnight at 4 °C with primary antibod- of amplified droplets with suitable targets were analyzed ies diluted in blocking solution. Brain slices were then with RainDrop Analyst v3 software. washed with washing buffer (0.1% Tween in PBS) and incubated with corresponding secondary antibodies Western blot diluted in PBS containing 0.3% of Triton X-100 for 3  h. Proteins were extracted from erythrocytes, cell lines, After washing with PBS or washing buffer, brain slides or tissues using SDS lysis buffer (2% SDS, 10% glycerol, were embedded in Vectashield medium or Vectashield 0.1 mM dithiothreitol and 0.2 M Tris–HCl, pH 6.8). Pro- medium with DAPI. Immunofluorescence images were tein samples were resolved by SDS–polyacrylamide gel captured at room temperature using a Zeiss Confocal electrophoresis, transferred to polyvinylidene difluoride Microscope under 20× or 40× magnification. Mouse membrane and blotted with respective primary antibod- monoclonal antibody to CHCHD2(66302–1-Ig, Protein- ies at 4 ℃ over night. The blots were washed in TBST and tech, 1:50), rabbit monoclonal antibodies [EPR12763] then incubated in horseradish peroxidase–conjugated to NeuN (ab177487, Abcam, 1:500), chicken poly- goat anti-rabbit/mouse IgG secondary antibody. Protein clonal antibodies to GFAP (AB5541, Merck Millipore, bands were visualized using an enhanced chemilumines- 1:500), rabbit monoclonal antibodies [EPR16588] to cence detection kit followed by autoradiography using Iba1(ab178846, Abcam, 1:500), rabbit monoclonal anti- Hyperfilm MP. bodies [EPR15581-54] to TOMM20 (ab186735, Abcam, 1:250), chicken polyclonal antibodies to Tyrosine Real‑time PCR Hydroxylase (ab76442, Abcam, 1:500) and rabbit mono- The levels of mRNA for CHCHD2, α-synuclein or p300 clonal [MJFR-14-6-4-2] antibodies to α-synuclein aggre- in mice or cell lines were measured by real-time PCR. gate (ab209538, Abcam, 1:200) were used in IF. Alexa Briefly, total RNA was isolated from brain tissues or cell Fluor 405, 488, 555 or 633 conjugated secondary anti- lines using Trizol (Life technologies, USA). Synthesis of bodies used in IF were purchased from Thermo Fisher first-strand cDNA was performed by reverse transcrip - Scientific, Inc. IF signals were quantified using Image J tion of 1.0  μg total RNA using RT-PCR kit according to software (NIH). Liu et al. acta neuropathol commun (2021) 9:37 Page 14 of 16 Immunohistochemistry staining of human brain tissues by qPCR. Primers can be seen in Additional file  1: For histochemical analysis of post-mortem brain slices Table S3. of normal subjects and PD patients, paraffin-embedded sections (6 µm) were stained with anti-CHCHD2 (66302- 1-Ig, Proteintech, 1:500) or anti-TH (ab76442, Abcam, Statistical analysis 1:500) antibodies. Quantitation of immunostaining was All analyses were performed with Prism 8.0 (GraphPad). conducted using NIH image J software. The same image Linear regression analysis was used to determine the exposure times and threshold settings were used for sec- relationships between age, PD severity, MOCA, disease tions from all groups. The immunohistochemical staining duration and CHCHD2. One-way analysis of variance of brain tissues and quantification were performed in a (ANOVA) followed by Tukey’s post-hoc test or two-way double-blinded manner. ANOVA followed by Bonferroni post-hoc test was used for multiple comparisons. F values with their associated degrees of freedom (treatment, time, interaction and Cell culture and transfection residual) were expressed as F The MN9D dopaminergic neuronal cell line (American (df of treatment, time, interaction/resid- = F values (treatment, time, interaction) in two-way Type Culture Collection, Manassas, Va., USA) was cul- ual) ANOVA, and F = F values. Please note tured in DMEM high glucose medium containing 10% (df of treatment, residual) that a non-parametric test was used whenever the sample fetal bovine serum (Gibco, Carlsbad, CA, USA) and 100 numbers were too small (N = 3–6) to test for normality. U/mL of penicillin (Invitrogen) and 100 μg/mL of strep- Nonparametric Mann–Whitney U test was used for the tomycin (Invitrogen). The cells were incubated at 37  °C comparison of the mean values between two groups. The in an incubator with a humidified atmosphere of 95% multiple t-test analysis was used to analyze "matched" or air and 5% CO . MN9D cells were transfected with wild "paired" data from the Grouped format data table. Addi- type or A53T human α-synuclein cDNA in the pGV219 tionally, relationships between the analytes and age, sex, vector. Since α-synuclein expression and oligomeriza- and UPDRS motor score were analyzed with bivariate tion are dynamic in these clones, comparisons were made correlation using Pearson’s correlation coefficients. Val - between clones of wild type and A53T that were main- ues with p < 0.05 were regarded as significant. Receiver tained in parallel from DNA transfection. operating characteristic (ROC) curves were used to cal- culate the relationship between sensitivity and specificity Immunofluorescence (IF) for cultured cells for PD disease group versus healthy control. The “opti - Cultured cells were fixed with 4% paraformaldehyde mum” cutoff value from the ROC curve is determined by for 10  min followed by permeabilization by 0.2% Tri- anchoring the specificity (or sensitivity) to be ≥ 80%. ton X-100 for 3 min before staining. Subsequently, cells were incubated with a primary antibody at 4  °C over- Supplementary Information night and a secondary antibody conjugated with Alexa The online version contains supplementary material available at https ://doi. Fluor (Thermo Fisher Scientific) was added for detec - org/10.1186/s4047 8-021-01133 -6. tion. Cell nuclei were counterstained with DAPI dye. Fluorescence microscopy was performed with a Zeiss Additional file 1.Supplementary data Confocal Microscope. IF signals were quantitated using the Image J software (NIH). Acknowledgements We sincerely thank the China National Health and Disease Human Brain Tissue Resource Center for providing post-mortem human brain tissues, and all the Chromatin immunoprecipitation community volunteers and PD patients for the donation of blood samples. Control, wild type, or A53T human α-synuclein- Authors’ contributions overexpressing MN9D cells were collected for ChIP JZ and TF conceived and supervised the project; XDL designed the project using a SimpleChIP Enzymatic Chromatin IP Kit with JZ, performed all of the NanoString and PCR experiments, part of Immunofluorescence, statistical analyses, and drafted the manuscript; QLW (Magnetic Beads) (#9003; Cell Signaling), according to performed part of Immunofluorescence, Western blot and ChIP experiments the manufacturer’s instructions. In brief, MN9D cells and assisted in statistical analyses; YY performed part of Immunofluorescence were first fixed with 1% formaldehyde to cross-link experiments in human and mice brain slices. DS and ET contributed to the immunofluorescence experiments in human brain slices. TS, MS, EMC, ZRL and protein and DNA. Cell lysates were then subjected to YRH contributed to data interpretation and preparation of the manuscript; sonication for ChIP. ChIP was done using anti-p300 GLL were responsible for patient characterization and sample collection; all antibody (ab14984; Abcam) or control IgG (sc-2027; authors critically reviewed the manuscript. Santa Cruz Technology). Precipitated DNA fragments Funding containing gene promoter of CHCHD2 were detected This research was supported by National Key R&D Program of China (No. 2016YFC1306500), National Natural Science Foundation of China (No. Liu  et al. acta neuropathol commun (2021) 9:37 Page 15 of 16 81571226, No. 81671187), Beijing Municipal Science and Technology Com- 10. Hansen C, Angot E, Bergstrom AL, Steiner JA, Pieri L, Paul G, Outeiro TF, mission (No. Z17110700100000, Z151100003915117, No. Z151100003915150, Melki R, Kallunki P, Fog K et al (2011) alpha-Synuclein propagates from Z161100000216150 and Z171100000117013), Natural Science Foundation of mouse brain to grafted dopaminergic neurons and seeds aggrega- Beijing Municipality (No. 7164254) and Science Fund for Creative Research tion in cultured human cells. J Clin Investig 121:715–725. https ://doi. Groups of the National Natural Science Foundation of China (No. 81521002).org/10.1172/JCI43 366 11. Harjuhaahto S, Rasila TS, Molchanova SM, Woldegebriel R, Kvist J, Availability of data and materials Konovalova S, Sainio MT, Pennonen J, Torregrosa-Munumer R, Ibrahim H All data needed to evaluate the conclusions in the paper are present in the et al (2020) ALS and Parkinson’s disease genes CHCHD10 and CHCHD2 paper and the Additional file are available from authors upon request. modify synaptic transcriptomes in human iPSC-derived motor neurons. Neurobiol Dis 141:104940. https ://doi.org/10.1016/j.nbd.2020.10494 0 Competing interests 12. Ikeda A, Nishioka K, Meng H, Takanashi M, Inoshita T, Shiba-Fukushima The authors declare that they have no competing interests. K, Li Y, Yoshino H, Mori A, Okuzumi A et al (2019) Mutations in CHCHD2 cause alpha-synuclein aggregation. Hum Mol Genet. https ://doi. Author detailsorg/10.1093/hmg/ddz24 1 Department of Pathology, School of Basic Medical Sciences, Peking Univer- 13. 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