Elevated polyglutamylation and tau phosphorylation levels are associated with cognitive impairment at diagnosis in patients with primary central nervous system lymphoma

Elevated polyglutamylation and tau phosphorylation levels are associated with cognitive impairment at diagnosis in patients with primary central nervous system lymphoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Elevated polyglutamylation and tau phosphorylation levels are associated with cognitive impairment at diagnosis in patients with primary central nervous system lymphoma Yuki Takeshima, Naoki Shinojima, Kenji Fujimoto, Daiki Yoshii, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6983936/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Alzheimer's Research & Therapy → Version 1 posted 10 You are reading this latest preprint version Abstract Background Primary central nervous system lymphoma (PCNSL) often manifests with cognitive impairment or nonspecific symptoms, which can delay diagnosis and worsen prognosis. However, the mechanisms underlying these neurological manifestations remain poorly understood. Previous studies have shown that polyglutamylation, a posttranslational modification, is associated with better responses to methotrexate-based chemotherapy in patients with PCNSL. Moreover, excessive polyglutamylation in neurons has been implicated in neurodegeneration via phosphorylated tau accumulation. This study aimed to elucidate the relationship between polyglutamylation, phosphorylated tau, and cognitive impairment in PCNSL. Methods We retrospectively analyzed 140 patients with histologically confirmed PCNSL treated at our institution between 2001 and 2022. Cognitive status at hospital admission was assessed using the Clinical Dementia Rating (CDR) scale. Immunohistochemical analysis of tumor specimens was performed to quantify the polyglutamylation and phosphorylated tau levels. Furthermore, in vitro studies with PCNSL cell lines were conducted to investigate whether the pharmacological upregulation of polyglutamylation by a histone deacetylase inhibitor promotes tau phosphorylation. Statistical analyses examined associations among polyglutamylation status, cognitive impairment, tau phosphorylation, and clinical outcomes. Results High polyglutamylation levels were observed in 59% of tumor samples, and this factor was independently associated with cognitive impairment at diagnosis (odds ratio: 4.35, 95% confidence interval 1.47–12.9, p = 0.0080). Immunohistochemical analysis demonstrated that tumors with elevated polyglutamylation showed significantly higher phosphorylated tau levels. In vitro experiments confirmed that increased polyglutamylation levels in PCNSL cells led to enhanced tau phosphorylation in PCNSL cell lines. Conclusions High polyglutamylation levels in PCNSL were associated with cognitive impairment and increased tau phosphorylation at diagnosis. These findings suggest that polyglutamylation may contribute to neurocognitive symptoms by promoting tau pathology. Elucidating this mechanism may provide novel insights into PCNSL pathophysiology and may inform future studies on disease mechanisms and potential treatment targets. Polyglutamylation Cognitive impairment Primary central nervous system lymphoma Tau protein Figures Figure 1 Figure 2 Background Primary central nervous system lymphoma (PCNSL) is a highly aggressive non-Hodgkin lymphoma affecting the brain, eyes, leptomeninges, and spinal cord [ 1 ]. Excluding immunocompromised patients, PCNSL typically presents in older adults, and its incidence is rising in countries with aging populations [ 2 – 4 ]. The initial symptoms of PCNSL vary, as some patients present with focal neurological deficits or increased intracranial pressure, leading to headaches or vomiting. Importantly, PCNSL can also manifest as a rapidly progressive dementia [ 5 ], with up to 40% of patients exhibiting nonspecific cognitive decline and neuropsychiatric changes [ 1 , 6 ]. These insidious symptoms frequently delay diagnosis and treatment, leading to a poor prognosis [ 7 , 8 ]. Moreover, several cohort studies have shown that baseline cognitive impairment itself is an independent predictor of worse overall survival (OS) in patients with PCNSL [ 9 ]. Despite their clinical importance, the mechanisms underlying these changes in PCNSL remain unclear. Tauopathies, which are associated with tau protein dysfunction and misfolded tau accumulation in neurons, have been attracting considerable attention as a key contributor to neurodegenerative pathology [ 10 ]. Tau, a microtubule-associated protein, plays an essential role in maintaining neuronal integrity and intracellular transport under physiological conditions. However, pathological modifications of tau, including hyperphosphorylation, lead to its aggregation and the disruption of microtubular stability, ultimately resulting in neurodegeneration [ 11 ]. Beyond cognitive deficits, tauopathies can manifest with a range of symptoms, including motor dysfunction and neuropsychiatric abnormalities [ 12 ]. Polyglutamylation, a reversible posttranslational modification that adds glutamate chains to proteins, plays a role in stabilizing proteins like microtubules [ 13 ]. Notably, a recent study has demonstrated that aberrant C-terminal-specific tubulin polyglutamylation facilitates the aggregation of hyperphosphorylated tau in tauopathy models, providing a mechanistic link between these two pathological processes in neurodegenerative disorders [ 14 ]. Hyperglutamylation of microtubules in neurons has been implicated in neurodegeneration [ 15 – 17 ]. We previously reported that polyglutamylation levels in tumor specimens were elevated in more than half of the patients with PCNSL and were associated with responsiveness to methotrexate (MTX)-based chemotherapy [ 18 , 19 ]. However, despite its apparent benefit in progression-free survival (PFS), the polyglutamylation status was not correlated with OS in the entire cohort, although a trend toward improved OS was observed only in younger patients. These findings suggest that, in PCNSL, additional factors may offset the clinical benefits of increased polyglutamylation, particularly with regard to long-term outcomes. Given the high incidence and prognostic impact of cognitive decline in PCNSL, we hypothesized that polyglutamylation might contribute not only to chemosensitivity but also to neurocognitive impairment, potentially through mechanisms involving tau pathology. To explore this hypothesis, we conducted a series of investigations aimed at elucidating the relationship between polyglutamylation and tau-related pathology in PCNSL. First, we performed immunohistochemical analyses to evaluate the expression of polyglutamylation and tau in tumor specimens from patients with PCNSL, focusing on the potential correlation of expression levels with clinical symptoms including cognitive impairment. Next, we conducted in vitro experiments using PCNSL cell lines to investigate the effects of altered polyglutamylation on tau phosphorylation. Materials and methods Study design and participants A retrospective observational study was conducted at our institution. This study has been reported in line with the STROBE guidelines. In total, 207 patients were diagnosed with PCNSL between January 2001 and December 2022. Histopathological diagnoses were based on the 2021 World Health Organization Classification of Tumours of the Central Nervous System Tumours (5th edition) [ 20 ]. In cases of diffuse large B cell lymphomas (DLBCLs), the cell of origin was classified into germinal center B-cell-like (GCB) or activated B-cell-like (non-GCB) types according to the Hans algorithm [ 21 ]. Patients who were comatose or responded only to noxious stimuli at the time of hospital admission were excluded, as accurate baseline cognitive evaluation was not feasible. Moreover, cases in which tissue volume was not sufficient for immunohistological evaluation were excluded. Tumor involvement was defined as a contrast-enhanced area on T1-weighted contrast-enhanced magnetic resonance (MR) images. However, four cases were evaluated using contrast-enhanced computed tomography (CT), and two cases of non-contrast-enhanced lesions were assessed using MR images without contrast enhancement. The total number of involved brain regions was calculated by adding all individually affected regions. In cases of tumor involvement in multiple brain regions, histopathologic diagnoses were based on tissue samples from contrast-enhanced areas if a sufficient sample volume was safely available [ 19 ]. Patient evaluation of cognitive impairment Cognitive status at the time of hospital admission was evaluated using the Clinical Dementia Rating (CDR) scale [ 22 – 24 ]. A rater blinded to treatment and outcome data derived each global CDR score by systematically reviewing the complete electronic record—admission and nursing notes, social-work entries, neuro-psychiatric consultations, medication lists, bedside mental-status examinations, laboratory results, and all ancillary reports as previously described [ 25 ]. A global CDR score ≥ 0.5 was set to indicate cognitive impairment. Survival analysis As previously reported [ 19 ], OS was defined as the time from initial diagnosis to death, whereas PFS was defined as the time from diagnosis to the first disease progression. Patients who had an uncertain date of death or who were alive at the time of analysis were censored, with the time from the first diagnosis to the last clinic visit or contact used as the censoring time. For patients with an uncertain progressive disease (PD) date or no PD at the time of analysis, the time from diagnosis to the last MR imaging or CT evaluation was used as the censoring time. Histology and immunohistochemistry Immunohistochemistry was performed using formalin-fixed paraffin-embedded tumor specimens according to our previously published protocol, with verification using positive and negative controls [ 19 ]. The following primary antibodies were used: mouse anti-polyglutamylation (clone GT335, 1:2,000; AdipoGen AG), monoclonal rabbit anti-tau (clone D1M9X, 1:500; Cell Signaling Technology), recombinant monoclonal rabbit anti-tau (phospho S396) (clone EPR2731, 1:4,000; abcam), and mouse anti-β-amyloid (clone 6F/3D, 1:100; Dako). As we previously reported, samples with > 10% positive cells comprised the high-polyglutamylation group, and those with < 10% comprised the low-polyglutamylation group [ 19 ]. Image quantification Slide images were obtained using a digital slide scanner (NanoZoomer XR; HAMAMATSU, Hamamatsu, Japan). Total tau- and phosphorylated tau-positive areas were quantitatively evaluated using an integrated fluorescence microscope (BZ-X800) equipped with an image cytometer module (BZ-H4XI; both KEYENCE, Osaka, Japan). Cell lines and cell culture The human PCNSL-derived cell lines TK (RRID: CVCL_E941) and HKBML (RRID: CVCL_8161) were purchased from the JCRB Cell Bank (Osaka, Japan) and the RIKEN BioResource Center (Tsukuba, Japan), respectively. As previously described [ 18 , 19 ], each cell line was cultured in the appropriate medium for maintenance. For experiments, the cells were seeded into cell culture plates (100,000 cells/mL), and sodium butyrate (NaBu; Schircks Laboratories, Switzerland) was immediately added to the wells at a final concentration of 1 mM. Vehicle control was 0.1% dimethyl sulfoxide. Afterward, cells were incubated for 72 h and then used for various experiments. Western blotting The protocols for western blotting experiments were previously described [ 26 ]. The following primary antibodies were used: polyclonal rabbit anti-folylpolyglutamate synthase (FPGS) (1:1,000; Spring Bioscience), monoclonal rabbit anti-tau (clone D1M9X, 1:500; Cell Signaling Technology), monoclonal rabbit anti-phospho-tau Ser202/Thr205 (clone 4A6, 1:5,000; Proteintech), and mouse anti-α-tubulin (clone DM1A, 1:2,000; Cell Signaling Technology). Quantitative polymerase chain reaction (qPCR) Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hulsterweg, Netherlands). Total RNA (500 ng in a 20 µL reaction volume) was reverse-transcribed into complementary DNA using SuperScript Ⅳ Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. The subsequent qPCR analysis was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). Reactions were performed on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Data were normalized to TBP levels, and the relative mRNA expression was calculated using the 2 −ΔΔCT method. The primer sequences are listed in Supplementary Table 1 (see Additional file 1). Statistical analysis All uni- and multivariate analyses were performed as previously reported [ 18 , 19 ]. Between-group differences in numerical values were tested using Student’s t-test and the Mann–Whitney U test for normally and non-normally distributed data, respectively. Categorical data were compared using Fisher’s exact test. Kaplan–Meier survival analysis and the log-rank test were performed to compare survival time between the low- and high-polyglutamylation groups. Multivariate logistic regression analysis was performed to identify independent factors associated with cognitive impairment. Variables with a p -value < 0.10 in univariate analyses, as well as clinically important variables regardless of their p -value, were included as candidate predictors in the multivariate model. Two-tailed p -values < 0.05 were considered statistically significant. Results Clinical study Of 207 consecutive patients with PCNSL, 140 met the inclusion criteria of this study. The clinical characteristics of these 140 patients are presented in Table 1. The median age was 68 years (range: 41–92). The cohort included 71 men and 69 women, and the median Karnofsky performance status (KPS) score was 50 (range: 20–100) at diagnosis. Histologically, 138 patients (98.6%) were diagnosed with DLBCL, one with Burkitt lymphoma, and one with low-grade B-cell lymphoma. Among the 138 DLBCLs, the cell of origin was assessed; 97 (70.3%) were categorized as non-GCB type. High levels of polyglutamylation were present in 83 (59.3%) patients. Additionally, 88 patients (62.9%) had multiple lesions. Table 1 Characteristics of the enrolled patients with PCNSL Overall Variables n = 140 Age Median (range) 68 (41–92) Sex (# of pts) Male/female 71/69 KPS score (%) Median (range) 50 (20–100) Cell of origin GCB/non-GCB 41/97 LDH (# of pts) High/normal range 45/95 Polyglutamylation (# of pts) High/low 83/57 Number of involved brain regions (# of pts) Single/multiple 52/88 Tumor involvement in each brain region (Right/left, # of pts) Frontal lobe 37/38 Temporal lobe (including the hippocampus) 16/10 Temporoparietal lobe (excluding the hippocampus) 19/20 Occipital lobe 10/6 Basal ganglia and/or thalamus 38/31 Corpus callosum and/or cingulate gyrus 41 Ventricle 20 Brainstem and/or cerebellum 35 Abbreviations: GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; PCNSL, primary central nervous system lymphoma; pts, patients. Next, we compared clinical characteristics between patients with and without cognitive impairment (Table 2). Cognitive impairment at diagnosis was present in 84 patients (60.0%). The two groups (with vs. without cognitive impairment) differed significantly in age, KPS score, and polyglutamylation status. Patients with cognitive impairment were significantly older ( p < 0.0001) and had significantly lower KPS scores ( p = 0.0005) than those without impairment. A higher proportion of cognitively impaired patients also exhibited high polyglutamylation levels ( p = 0.036). A comparison of tumor involvement in each brain region is shown in Fig. 1a, b and Supplementary Table 2 (see Additional file 1). Cognitive impairment was significantly associated with the involvement of the corpus callosum and/or cingulate gyrus, whereas brainstem and cerebellar lesions were more common in patients without cognitive impairment (all p < 0.0001; Fig. 1a, c). The proportions of patients with multiple lesions did not significantly differ between those with and those without cognitive impairment (Fig. 1b). In multivariate analysis, a high-polyglutamylation status emerged as an independent risk factor for cognitive impairment at diagnosis (odds ratio [OR] 4.35, 95% confidence interval [CI] 1.47–12.9, p = 0.0080, Table 3), even after controlling for age, KPS scores, and tumor location. Table 2 Comparison of the clinical characteristics between patients with and without cognitive impairment at diagnosis Cognitive impairment Yes No Variables n = 84 n = 56 p Age Median (range) 72 (43–92) 64 (41–85) <0.0001 Sex (# of pts) Male/female 41/43 30/26 0.61 KPS score (%) Median (range) 50 (20–100) 70 (20–100) 0.00050 Cell of origin GCB/non-GCB 22/60 19/37 0.44 LDH (# of pts) High/normal range 29/55 16/40 0.58 Polyglutamylation (# of pts) High/low 56/28 27/29 0.036 Abbreviations: GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; pts, patients. Table 3 Multivariate logistic regression analysis of the predictors of cognitive impairment at diagnosis Variables OR 95% CI p Age (y) 1.08 1.04–1.14 0.00057 KPS score (%) 0.960 0.935–0.986 0.0023 Polyglutamylation 4.35 1.47–12.9 0.0080 Number of involved brain regions 0.98 0.333–2.88 0.97 Corpus callosum and/or cingulate gyrus 3.39 1.03–11.2 0.045 Brainstem and/or cerebellum 0.084 0.0256–0.276 <0.0001 Ventricle 2.65 0.563–12.4 0.22 Abbreviations: CI, confidence interval; KPS, Karnofsky performance status; OR, odds ratio. To further explore the clinical implications of polyglutamylation, we compared patients according to polyglutamylation status. The high-polyglutamylation group exhibited a higher frequency of cognitive impairment ( p =0.036, Table 4), as observed in the overall cohort. The high-polyglutamylation group also showed significantly longer PFS ( p = 0.0046, Fig. 1d), whereas OS did not differ between groups ( p = 0.23, Fig. 1e). Table 4 Comparison of clinical characteristics between patients with high- and low-polyglutamylation status Polyglutamylation level High Low Variables n = 83 n = 57 p Age Median (range) 69 (41–92) 68 (46–87) 0.91 Sex (# of pts) Male/female 42/41 29/28 1 KPS score (%) Median (range) 60 (20–100) 50 (20–100) 0.026 Cell of origin GCB/non-GCB 28/53 13/44 0.18 LDH (# of pts) High/normal range 26/27 19/38 0.86 Cognitive impairment Yes/No 56/27 28/29 0.036 Abbreviations: GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; pts, patients. A high-polyglutamylation status was also more common in tumors involving the left frontal lobe (Supplementary Table 3 [see Additional file 1]). In the subset of patients with left frontal lobe involvement, 23 of 30 patients (77%) with high polyglutamylation levels had cognitive impairment, compared to 4 of 8 (50%) with low polyglutamylation levels. Although this difference was not significant ( p = 0.20, Supplementary Table 4 [see Additional file 1]), the trend was consistent with that observed in the overall cohort. In summary, these findings indicate that cognitive impairment in the high-polyglutamylation group cannot be solely explained by age, KPS score, or tumor location, suggesting that additional neurobiological mechanisms related to polyglutamylation may contribute to cognitive dysfunction in PCNSL. Histopathology and immunohistochemistry Next, patient samples were examined for the accumulation of phosphorylated tau and β-amyloid, two of the molecules most commonly associated with cognitive decline in Alzheimer’s disease [27]. Notably, the expression of total and phosphorylated tau coincided with that of polyglutamylation (Fig. 2a). To quantitatively assess this association, we compared the expression of tau and phosphorylated tau between the low- and high-polyglutamylation groups using a subset of patient samples with sufficient tissue volume (six samples in each group). The areas positive for total tau and phosphorylated tau were significantly larger in the high-polyglutamylation group than in the low-polyglutamylation group (Fig. 2b). Although phosphorylated tau was expressed, no β-amyloid deposits were observed in the samples of patients with PCNSL (Fig. 2c). Elevated polyglutamylation levels increase the levels of phosphorylated tau in PCNSL cells Subsequently, we evaluated whether the level of polyglutamylation correlates with that of phosphorylated tau in PCNSL cells. We used an in vitro model of treatment with histone deacetylase inhibitors (HDACIs) that we had previously used to control polyglutamylation levels in cells [18]. We treated cells with the HDACI NaBu to increase the polyglutamylation level by upregulating FPGS (Fig. 2d). First, we compared the levels of phosphorylated tau between cells with and without HDACI treatment. Immunoblots showed that NaBu treatment increased the expression levels of both FPGS and phosphorylated tau in two different cell lines (Fig. 2e). Tau phosphorylation is regulated by the balance between phosphorylation and dephosphorylation. Proline-directed kinases, such as glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (CDK5), mediate aberrant tau phosphorylation [28, 29]. In contrast, protein phosphatase 2A (PP2A) [30] and peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) [31] facilitate tau dephosphorylation. Therefore, we determined the gene expression levels of enzymes involved in tau phosphorylation and dephosphorylation in cells with and without HDACI treatment using qPCR. Notably, mRNA expression of CDK5 was consistently increased in PCNSL cell lines with high levels of polyglutamylation after NaBu treatment, but no significant changes were observed in the expression of tau dephosphorylation-related enzymes (Fig. 2f). These in vitro studies also confirmed that the extent of polyglutamylation is significantly correlated with that of tau phosphorylation. In summary, our results suggest that phosphorylated tau is involved in the association of polyglutamylation with cognitive impairment in patients with PCNSL. Discussion In the present study, we found for the first time that high polyglutamylation levels in PCNSL tumors are closely associated with cognitive impairment at diagnosis. Furthermore, our data suggest that this association is accompanied by the accumulation of phosphorylated tau, both in patient-derived tumor samples and PCNSL cell lines, supporting the hypothesis that polyglutamylation may contribute to tau-related neurodegenerative processes. While these findings suggest a possible mechanistic link between polyglutamylation and cognitive impairment in PCNSL, causality cannot be definitively established based on the results of the current study, warranting further investigation. Several preclinical studies have investigated the relationship between polyglutamylation and tau expression. For example, Hausrat et al. recently demonstrated in a mouse model that polyglutamylation plays a key role in tauopathy caused by the accumulation of phosphorylated tau [14]. Although our results are consistent with these experimental findings, the precise mechanism by which polyglutamylation in tumor cells may influence neuronal function in patients with PCNSL remains to be clarified. In tauopathies, it is thought that pathological tau produced within neurons is released by these cells and spreads into the parenchyma, where it can then be transferred to other cells [32-36]. A similar pathological process can occur in PCNSL, where tau is shed from tumor cells and affects neurons. Future studies using advanced imaging modalities such as Tau positron emission tomography (PET) [37], or autopsy-based investigations, may provide valuable insights into the pathological mechanisms underlying cognitive impairment in PCNSL. Regarding the localization of lesions, a previous report demonstrated that neuropsychological changes in PCNSL correlate with diffuse involvement of the periventricular white matter or corpus callosum [38], which aligns with our findings. Importantly, although high polyglutamylation is associated with longer PFS, no significant difference in OS was observed between high- and low- polyglutamylation groups. This may reflect the delayed diagnosis and therapy initiation in patients whose initial symptoms were subtle cognitive changes. Indeed, we encountered a case where the patient died before receiving initial treatment due to rapid disease progression following surgery, resulting in central cerebral herniation. This patient presented with cognitive changes at onset and had a highly polyglutamylated tumor, for which MTX treatment was anticipated to be effective. These findings underscore the need for increased clinical awareness and early intervention in PCNSL patients presenting with cognitive or behavioral symptoms, particularly in the context of highly polyglutamylated PCNSL. As wearable AI and other digital technologies become more widespread in daily life, it may become possible to detect cognitive and behavioral changes earlier and more continuously [39, 40]. This study has several limitations. First, its retrospective nature and reliance on chart reviews to assess cognitive status may introduce bias. However, recent literature supports the feasibility and reliability of retrospective CDR scoring based on comprehensive chart reviews and collateral histories [25]. To minimize bias, CDR scoring in this study was performed systematically and blinded to outcomes. Prospective studies incorporating comprehensive and objective neuropsychological evaluations are needed to confirm our findings. Second, the sample size in some subgroups (e.g., patients with left frontal lobe involvement) was limited, which may have reduced the statistical power to detect significant associations between polyglutamylation status and cognitive impairment. Further studies with larger cohorts are warranted to validate these findings. Third, our histological analyses were restricted to samples from the main tumor mass, precluding the assessment of tau pathology in surrounding brain parenchyma. Advanced imaging modalities, such as tau PET, or post-mortem studies, may provide additional insights into the distribution and impact of tau pathology in PCNSL. Fourth, our in vitro experiments used cell lines and chemical modulation to mimic polyglutamylation; although informative, these models cannot fully recapitulate the complex in vivo tumor microenvironment. Despite these limitations, our findings offer new perspectives on the pathophysiology of PCNSL. They highlight a potential role for polyglutamylation in cognitive dysfunction, suggest directions for further mechanistic and translational research, and underscore the clinical need for early recognition of subtle cognitive changes in PCNSL. Conclusions This study demonstrated that PCNSL tumors with high polyglutamylation levels were associated with cognitive impairment at diagnosis. Additionally, a significant correlation was observed between polyglutamylation and phosphorylated tau accumulation, suggesting a potential role in the underlying pathophysiology of PCNSL. Recognizing PCNSL as a potential cause of rapidly progressive cognitive impairment is essential for timely diagnosis and treatment, which may lead to improved patient outcomes. These findings provide new insights into the pathological mechanisms of PCNSL and may inform future research on disease progression and therapeutic strategies. Abbreviations CDK5 cyclin-dependent kinase 5 CDR Clinical Dementia Rating CI confidence interval CT computed tomography DLBCL diffuse large B cell lymphoma FPGS folylpolyglutamate synthase GCB germinal center B-cell-like GSK3 glycogen synthase kinase 3 HDACI histone deacetylase inhibitor KPS Karnofsky performance status MTX methotrexate MR magnetic resonance NaBu sodium butyrate OR odds ratio OS overall survival PCNSL primary central nervous lymphoma PD progressive disease PET positron emission tomography PFS progression-free survival PIN1 peptidylprolyl cis/trans isomerase, NIMA-interacting 1 PP2A protein phosphatase 2A qPCR quantitative polymerase chain reaction Declarations Ethics approval and consent to participate All study procedures involving human participants conformed to the ethical standards of the institutional and/or national research committee and to the Helsinki Declaration of 1964 and its subsequent amendments or comparable ethical standards. Informed consent was obtained from all individual participants enrolled in this study or their families. The study protocol was approved by the Institutional Review Board (IRB) of Kumamoto University (Genome No. 231). Consent for publication Not applicable. All MR images included in this manuscript have been carefully reviewed, fully anonymized, and stripped of any direct or indirect identifiers to ensure patient privacy. No identifiable personal information is presented. Availability of data and materials All original data from this manuscript are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by a Grant-in-Aid for Scientific Research (19K09485) from the Japanese Society for the Promotion of Science. Author contributions Study conception and design: YT, NS. Data acquisition: YT, NS, KF, DY, YH, MO, MT, YM, HU, TH. Data analysis and interpretation: YT, NS, KF, AM. Drafting of the manuscript: YT, NS. Review of the manuscript: NS. Critical revision: TH, AM. Acknowledgements During the preparation stage of this work, the authors used DeepL, an AI-assisted technology, to improve readability and expressiveness. Subsequently, they used the English editing service Editage. Afterward, the authors reviewed and edited the content as necessary, and they take full responsibility for the content of this publication. References C. Grommes, J.L. Rubenstein, L.M. DeAngelis, A.J.M. Ferreri, T.T. Batchelor, Comprehensive approach to diagnosis and treatment of newly diagnosed primary CNS lymphoma. Neuro Oncol 2019;21:296-305. K. Makino, H. Nakamura, T. Kino, H. Takeshima, J. Kuratsu, Rising incidence of primary central nervous system lymphoma in Kumamoto, Japan. Surg Neurol 2006;66:503-6. H. Nakamura, K. Makino, S. Yano, J. Kuratsu, Kumamoto Brain Tumor Research Group. Epidemiological study of primary intracranial tumors: a regional survey in Kumamoto prefecture in southern Japan—20-year study. Int J Clin Oncol 2011;16:314-21. J.S. Mendez, Q.T. Ostrom, H. Gittleman, C. Kruchko, L.M. DeAngelis, J.S. Barnholtz-Sloan, et al, The elderly left behind-changes in survival trends of primary central nervous system lymphoma over the past 4 decades. Neuro Oncol 2018;20:687-94. P. Hermann, I. Zerr, Rapidly progressive dementias – aetiologies, diagnosis and management. Nat Rev Neurol 2022;18:363-76. L.M. DeAngelis, Brain tumors. N Engl J Med 2001;344:114-23. R. Velasco, S. Mercadal, N. Vidal, M. Alañá, M.I. Barceló, M.J. Ibáñez-Juliá , et al, Diagnostic delay and outcome in immunocompetent patients with primary central nervous system lymphoma in Spain: a multicentric study. J Neurooncol 2020;148:545-54. R.M.H. Lim, J.Y. Chan, Absence of meaningful neurocognitive recovery in comatose patients with primary central nervous system lymphoma despite an effective response to chemotherapy: Case reports. Mol Clin Oncol 2021;14:44. M. van der Meulen, L. Dirven, K. Bakunina, M.J. van den Bent, S. Issa, J.K. Doorduijn, et al, MMSE is an independent prognostic factor for survival in primary central nervous system lymphoma. J Neurooncol 2021;152:357-62. N. Samudra, C. Lane-Donovan, L. VandeVrede, A.L. Boxer, Tau pathology in neurodegenerative disease: disease mechanisms and therapeutic avenues. J Clin Invest 2023;133:e168553. K. Iqbal, F. Liu, C.X. Gong, Tau and neurodegenerative disease: the story so far. Nat Rev Neurol 2016;12:15-27. N. Olfati, A. Shoeibi, I. Litvan, Clinical spectrum of tauopathies. Front Neurol 2022;13:944806. D. Boucher, J.C. Larcher, F. Gros, P. Denoulet, Polyglutamylation of tubulin as a progressive regulator of in vitro interactions between the microtubule-associated protein Tau and tubulin. Biochemistry 1994;33:12471-7. T.J. Hausrat, P.C. Janiesch, P. Breiden, D. Lutz, S. Hoffmeister-Ullerich, I. Hermans-Borgmeyer, et al, Disruption of tubulin-alpha4a polyglutamylation prevents aggregation of hyper-phosphorylated tau and microglia activation in mice. Nat Commun 2022;13:4192. K. Rogowski, J. van Dijk, M.M. Magiera, C. Bosc, J.C. Deloulme, A. Bosson, et al, A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 2010;143:564-78. M.M. Magiera, S. Bodakuntla, J. Žiak, S. Lacomme, P. Marques Sousa, S. Leboucher, et al, Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport. EMBO J 2018;37:e100440. S. Bodakuntla, A. Schnitzler, C. Villablanca, C. Gonzalez-Billault, I. Bieche, C. Janke, et al, Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons. J Cell Sci 2020;133:jcs241802. K. Fujimoto, N. Shinojima, M. Hayashi, T. Nakano, K. Ichimura, A. Mukasa, Histone deacetylase inhibition enhances the therapeutic effects of methotrexate on primary central nervous system lymphoma. Neurooncol Adv 2020;2:vdaa084. N. Shinojima, K. Fujimoto, K. Makino, K. Todaka, K. Yamada, Y. Mikami, et al, Clinical significance of polyglutamylation in primary central nervous system lymphoma. Acta Neuropathol Commun 2018;6:15. D.N. Louis, A. Perry, P. Wesseling, D.J. Brat, I.A. Cree, D. Figarella-Branger, et al, The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro Oncol 2021;23:1231-51. C.P. Hans, D.D. Weisenburger, T.C. Greiner, R.D. Gascoyne, J. Delabie, G. Ott, et al, Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004;103:275-82. J.C. Morris, The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology 1993;43:2412-4. C.P. Hughes, L. Berg, W.L. Danziger, L.A. Coben, R.L. Martin, A new clinical scale for the staging of dementia. Br J Psychiatry 1982;140:566-72. V. Haroutunian, D.P. Purohit, D.P. Perl, D. Marin, K. Khan, M. Lantz, et al, Neurofibrillary tangles in nondemented elderly subjects and mild Alzheimer disease. Arch Neurol 1999;56:713-8. V. Dauphinot, S. Calvi, C. Moutet, J. Xie, S. Dautricourt, A. Batsavanis, et al, Reliability of the assessment of the clinical dementia rating scale from the analysis of medical records in comparison with the reference method. Alzheimers Res Ther 2024;16:198. N. Shinojima, A. Hossain, T. Takezaki, J. Fueyo, J. Gumin, F. Gao, et al, TGF-β mediates homing of bone marrow-derived human mesenchymal stem cells to glioma stem cells. Cancer Res 2013;73:2333-44. B.J. Hanseeuw, R.A. Betensky, H.I.L. Jacobs, A.P. Schultz, J. Sepulcre, J.A. Becker, et al, Association of amyloid and tau with cognition in preclinical Alzheimer disease: a longitudinal study. JAMA Neurol 2019;76:915-24. F. Plattner, M. Angelo, K.P. Giese, The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation. J Biol Chem 2006;281:25457-65. W. Noble, V. Olm, K. Takata, E. Casey, O. Mary, J. Meyerson, et al, Cdk5 is a key factor in tau aggregation and tangle formation in vivo. Neuron 2003;38:555-65. F. Liu, I. Grundke-Iqbal, K. Iqbal, C.X. Gong, Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur J Neurosci 2005;22:1942-50. T. Kimura, K. Tsutsumi, M. Taoka, T. Saito, M. Masuda-Suzukake, K. Ishiguro, et al, Isomerase Pin1 stimulates dephosphorylation of tau protein at cyclin-dependent kinase (Cdk5)-dependent Alzheimer phosphorylation sites. J Biol Chem 2013;288:7968-77. F. Clavaguera, T. Bolmont, R.A. Crowther, D. Abramowski, S. Frank, A. Probst, et al, Transmission and spreading of tauopathy in transgenic mouse brain. Nat Cell Biol 2009;11:909-13. N. Annadurai, J.B. De Sanctis, M. Hajdúch, V. Das, Tau secretion and propagation: Perspectives for potential preventive interventions in Alzheimer’s disease and other tauopathies. Exp Neurol 2021;343:113756. P.T. Nelson, I. Alafuzoff, E.H. Bigio, C. Bouras, H. Braak, N.J. Cairns, et al, Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. J Neuropathol Exp Neurol 2012;71:362-81. A. Seitkazina, K.H. Kim, E. Fagan, Y. Sung, Y.K. Kim, S. Lim, The fate of tau aggregates between clearance and transmission. Front Aging Neurosci, 2022;14:932541. Y. Wang, E. Mandelkow, Tau in physiology and pathology. Nat Rev Neurosci 2016;17:5-21. R. Ossenkoppele, A. Pichet Binette, C. Groot, R. Smith, O. Strandberg, S. Palmqvist, et al, Amyloid and tau PET-positive cognitively unimpaired individuals are at high risk for future cognitive decline. Nat Med 2022;28:2381-7. W. Küker, T. Nägele, A. Korfel, S. Heckl, E. Thiel, M. Bamberg, et al, Primary central nervous system lymphomas (PCNSL): MRI features at presentation in 100 patients. J Neurooncol 2005;72:169-77. A.Y. DuBord, E.W. Paolillo, A.M. Staffaroni, Remote digital technologies for the early detection and monitoring of cognitive decline in patients with type 2 diabetes: insights from studies of neurodegenerative diseases. J Diabetes Sci Technol 2024;18:1489-99. R.L. Nosheny, D. Yen, T. Howell, M. Camacho, K. Moulder, S. Gummadi, et al, Evaluation of the Electronic Clinical Dementia Rating for dementia screening. JAMA Netw Open 2023;6:e2333786. Additional Declarations No competing interests reported. Supplementary Files Additionalfile120250620.docx Additional file 1 Supplementary Table 1 Primer sequences for quantitative polymerase chain reaction Supplementary Table 2 Comparison of tumor involvement between patients with and without cognitive impairment at diagnosis Supplementary Table 3 Comparison of tumor involvement between patients with high- and low-polyglutamylation status Supplementary Table 4 Association between polyglutamylation levels and cognitive impairment at diagnosis in cases with left frontal lobe involvement Additionalfile2.pdf Additional file 2 Supplementary Figure 1 Original western blots. a Original western blots of Fig. 2d. b Original western blots of Fig. 2e. Abbreviations: IB, immunoblot; NaBu, sodium butyrate; FPGS, folylpolyglutamate synthase Cite Share Download PDF Status: Published Journal Publication published 02 Dec, 2025 Read the published version in Alzheimer's Research & Therapy → Version 1 posted Editorial decision: Revision requested 01 Sep, 2025 Reviews received at journal 13 Aug, 2025 Reviews received at journal 11 Aug, 2025 Reviewers agreed at journal 22 Jul, 2025 Reviewers agreed at journal 22 Jul, 2025 Reviewers agreed at journal 22 Jul, 2025 Reviewers invited by journal 14 Jul, 2025 Editor assigned by journal 27 Jun, 2025 Submission checks completed at journal 27 Jun, 2025 First submitted to journal 26 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6983936","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485327475,"identity":"c3cb731d-3f34-49a4-90c0-c8f184517f5c","order_by":0,"name":"Yuki Takeshima","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Takeshima","suffix":""},{"id":485327477,"identity":"43e48784-6b24-4a18-8481-dc627f7d1e08","order_by":1,"name":"Naoki Shinojima","email":"data:image/png;base64,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","orcid":"","institution":"Kumamoto University","correspondingAuthor":true,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Shinojima","suffix":""},{"id":485327478,"identity":"14b0063a-25da-4410-a8f4-de6a3f616321","order_by":2,"name":"Kenji Fujimoto","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Fujimoto","suffix":""},{"id":485327480,"identity":"46f6ac5c-cc7a-404d-8d49-31245a9cd558","order_by":3,"name":"Daiki Yoshii","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Yoshii","suffix":""},{"id":485327482,"identity":"1f6cac26-a769-4ef0-8636-51c67236414d","order_by":4,"name":"Yasushi Hayakata","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Hayakata","suffix":""},{"id":485327486,"identity":"40c40d8d-03b1-4272-b952-b929f8784ec1","order_by":5,"name":"Masafumi Oya","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Masafumi","middleName":"","lastName":"Oya","suffix":""},{"id":485327489,"identity":"bd3821c9-106e-428f-865d-d56433421714","order_by":6,"name":"Masayoshi Tasaki","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Masayoshi","middleName":"","lastName":"Tasaki","suffix":""},{"id":485327492,"identity":"d62f0ca6-486b-4ec3-8374-63b8d3c8091b","order_by":7,"name":"Yoshiki Mikami","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Yoshiki","middleName":"","lastName":"Mikami","suffix":""},{"id":485327493,"identity":"80481dd8-5aad-4090-9d85-37d533331f33","order_by":8,"name":"Hiroyuki Uetani","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Uetani","suffix":""},{"id":485327496,"identity":"e5bc77fe-25bc-447a-9874-ed2c1e4d24ae","order_by":9,"name":"Toshinori Hirai","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Toshinori","middleName":"","lastName":"Hirai","suffix":""},{"id":485327497,"identity":"6af030ae-6dc5-407e-bd71-d243e0d24ed4","order_by":10,"name":"Akitake Mukasa","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Akitake","middleName":"","lastName":"Mukasa","suffix":""}],"badges":[],"createdAt":"2025-06-26 13:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6983936/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6983936/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13195-025-01927-z","type":"published","date":"2025-12-02T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87044571,"identity":"ebc8de57-d70c-4f79-aeae-1a234d1325e1","added_by":"auto","created_at":"2025-07-18 14:23:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1781844,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of tumor involvement between patients with and without cognitive impairment. \u003cstrong\u003ea\u003c/strong\u003e Each bar represents the number of patients whose tumors involve the indicated brain region. Data are grouped by “Cognitive impairment = Yes” (black bars) versus “Cognitive impairment = No” (gray bars). Note that tumor involvement was counted separately for each involved brain region within the same patient. \u003cstrong\u003eb\u003c/strong\u003e Each bar represents the number of patients whose tumors involve multiple brain regions. \u003cstrong\u003ec\u003c/strong\u003eRepresentative contrast-enhanced magnetic resonance images of lesions in the corpus callosum or cingulate gyrus (left), lesions in the brainstem or cerebellum (middle), and lesions involving multiple brain regions (right). \u003cstrong\u003ed, e\u003c/strong\u003e Kaplan–Meier curves for (\u003cstrong\u003ed\u003c/strong\u003e) progression-free survival and (\u003cstrong\u003ee\u003c/strong\u003e) overall survival based on the polyglutamylation status in 140 patients with primary central nervous system lymphoma.\u003cstrong\u003e Abbreviations:\u003c/strong\u003e Lt, left; OS, overall survival; PCNSL, primary central nervous system lymphoma; PFS, progression-free survival; PG, polyglutamylation; Rt, right\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6983936/v1/7b0f83a11ff98c5bf6376ebb.png"},{"id":87044570,"identity":"82ccf6f2-678e-4edb-bd0d-bba051f89cd7","added_by":"auto","created_at":"2025-07-18 14:23:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2761719,"visible":true,"origin":"","legend":"\u003cp\u003ePolyglutamylation in PCNSL correlates with phosphorylated tau accumulation. \u003cstrong\u003ea\u003c/strong\u003e Findings of immunohistochemical stainings. Each upper and lower subset of panels shows high- and low-polyglutamylated tumor specimens, respectively. The left panels show immunohistochemical stainings for polyglutamylation, the middle panels for total tau, and the right panels for tau (phospho S396). Scale bars: 50 µm.\u003cstrong\u003e b \u003c/strong\u003eQuantification of tau-positive areas in the polyglutamylated and non-polyglutamylated groups (6 samples per group). The ratios (percentages) of total tau- and tau (phospho S396)-positive areas were compared between the two groups using the Mann–Whitney U test (**\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01). \u003cstrong\u003ec \u003c/strong\u003eRepresentative figures of immunohistochemistry for β-amyloid. A brain section with Alzheimer’s disease was used as a positive control. \u003cstrong\u003ed–f \u003c/strong\u003eCorrelations between polyglutamylation and tau phosphorylation in the human PCNSL cell lines TK and HKBML. PCNSL cells were treated for 72 h with 0.1% dimethyl sulfoxide (control) or 1 mM NaBu. a-tubulin was used as a loading control. \u003cstrong\u003ed \u003c/strong\u003eImmunoblots showing the FPGS expression in TK and HKBML cells with or without NaBu treatment. \u003cstrong\u003ee\u003c/strong\u003e Immunoblots showing the expression of phosphorylated (p-tau) and total tau (tau) in TK and HKBML cells with or without NaBu treatment. The blots in \u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e are cutouts due to incubation with different secondary antibodies. The entire blots are presented in Supplementary Figure 1 (see Additional file 2). \u003cstrong\u003ef \u003c/strong\u003eQuantitative polymerase chain reaction analysis of \u003cem\u003ePIN1\u003c/em\u003e, \u003cem\u003ePPP2R5A \u003c/em\u003e(protein phosphatase 2, regulatory subunit B', alpha isoform), \u003cem\u003eCDK5\u003c/em\u003e, and \u003cem\u003eGSK3b\u003c/em\u003eexpression in TK and HKBML cells. Data are expressed as the mean ± standard error of the mean (n = 3) and were analyzed using Student’s t-test (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001). \u003cstrong\u003eAbbreviations: \u003c/strong\u003eFPGS, folylpolyglutamate synthase; IB, immunoblot; NaBu, sodium butyrate; ns, not significant; PCNSL, primary central nervous system lymphoma; PG, polyglutamylation\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6983936/v1/8d75d11956f4ae2f58157a30.png"},{"id":97724587,"identity":"58cb6111-ff35-4467-8499-318352c9ccbf","added_by":"auto","created_at":"2025-12-08 16:12:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5305312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6983936/v1/e3a1f1ac-783e-488b-ba4b-445afb3ced76.pdf"},{"id":87044622,"identity":"15f2b8a7-4e36-4392-9bf5-15ecef87b099","added_by":"auto","created_at":"2025-07-18 14:23:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26991,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1\u003c/p\u003e\n\u003cp\u003eSupplementary Table 1 Primer sequences for quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eSupplementary Table 2 Comparison of tumor involvement between patients with and without cognitive impairment at diagnosis\u003c/p\u003e\n\u003cp\u003eSupplementary Table 3 Comparison of tumor involvement between patients with high- and low-polyglutamylation status\u003c/p\u003e\n\u003cp\u003eSupplementary Table 4 Association between polyglutamylation levels and cognitive impairment at diagnosis in cases with left frontal lobe involvement\u003c/p\u003e","description":"","filename":"Additionalfile120250620.docx","url":"https://assets-eu.researchsquare.com/files/rs-6983936/v1/1c610d45bcb6af67dcf35564.docx"},{"id":87044560,"identity":"e706f70b-9202-4694-8a3a-debb94bb5374","added_by":"auto","created_at":"2025-07-18 14:23:45","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5244212,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 2\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 1 Original western blots. \u003cstrong\u003ea\u003c/strong\u003e Original western blots of Fig. 2d. \u003cstrong\u003eb \u003c/strong\u003eOriginal western blots of Fig. 2e. \u003cstrong\u003eAbbreviations: \u003c/strong\u003eIB, immunoblot;\u003cstrong\u003e \u003c/strong\u003eNaBu, sodium butyrate;\u003cstrong\u003e \u003c/strong\u003eFPGS, folylpolyglutamate synthase\u003c/p\u003e","description":"","filename":"Additionalfile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6983936/v1/5244e432a7a735e9215a319f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elevated polyglutamylation and tau phosphorylation levels are associated with cognitive impairment at diagnosis in patients with primary central nervous system lymphoma","fulltext":[{"header":"Background","content":"\u003cp\u003ePrimary central nervous system lymphoma (PCNSL) is a highly aggressive non-Hodgkin lymphoma affecting the brain, eyes, leptomeninges, and spinal cord [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Excluding immunocompromised patients, PCNSL typically presents in older adults, and its incidence is rising in countries with aging populations [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The initial symptoms of PCNSL vary, as some patients present with focal neurological deficits or increased intracranial pressure, leading to headaches or vomiting. Importantly, PCNSL can also manifest as a rapidly progressive dementia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], with up to 40% of patients exhibiting nonspecific cognitive decline and neuropsychiatric changes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These insidious symptoms frequently delay diagnosis and treatment, leading to a poor prognosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, several cohort studies have shown that baseline cognitive impairment itself is an independent predictor of worse overall survival (OS) in patients with PCNSL [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite their clinical importance, the mechanisms underlying these changes in PCNSL remain unclear.\u003c/p\u003e\u003cp\u003eTauopathies, which are associated with tau protein dysfunction and misfolded tau accumulation in neurons, have been attracting considerable attention as a key contributor to neurodegenerative pathology [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Tau, a microtubule-associated protein, plays an essential role in maintaining neuronal integrity and intracellular transport under physiological conditions. However, pathological modifications of tau, including hyperphosphorylation, lead to its aggregation and the disruption of microtubular stability, ultimately resulting in neurodegeneration [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Beyond cognitive deficits, tauopathies can manifest with a range of symptoms, including motor dysfunction and neuropsychiatric abnormalities [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePolyglutamylation, a reversible posttranslational modification that adds glutamate chains to proteins, plays a role in stabilizing proteins like microtubules [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notably, a recent study has demonstrated that aberrant C-terminal-specific tubulin polyglutamylation facilitates the aggregation of hyperphosphorylated tau in tauopathy models, providing a mechanistic link between these two pathological processes in neurodegenerative disorders [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Hyperglutamylation of microtubules in neurons has been implicated in neurodegeneration [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe previously reported that polyglutamylation levels in tumor specimens were elevated in more than half of the patients with PCNSL and were associated with responsiveness to methotrexate (MTX)-based chemotherapy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, despite its apparent benefit in progression-free survival (PFS), the polyglutamylation status was not correlated with OS in the entire cohort, although a trend toward improved OS was observed only in younger patients. These findings suggest that, in PCNSL, additional factors may offset the clinical benefits of increased polyglutamylation, particularly with regard to long-term outcomes. Given the high incidence and prognostic impact of cognitive decline in PCNSL, we hypothesized that polyglutamylation might contribute not only to chemosensitivity but also to neurocognitive impairment, potentially through mechanisms involving tau pathology. To explore this hypothesis, we conducted a series of investigations aimed at elucidating the relationship between polyglutamylation and tau-related pathology in PCNSL. First, we performed immunohistochemical analyses to evaluate the expression of polyglutamylation and tau in tumor specimens from patients with PCNSL, focusing on the potential correlation of expression levels with clinical symptoms including cognitive impairment. Next, we conducted in vitro experiments using PCNSL cell lines to investigate the effects of altered polyglutamylation on tau phosphorylation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eStudy design and participants\u003c/p\u003e\u003cp\u003eA retrospective observational study was conducted at our institution. This study has been reported in line with the STROBE guidelines. In total, 207 patients were diagnosed with PCNSL between January 2001 and December 2022. Histopathological diagnoses were based on the 2021 World Health Organization Classification of Tumours of the Central Nervous System Tumours (5th edition) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In cases of diffuse large B cell lymphomas (DLBCLs), the cell of origin was classified into germinal center B-cell-like (GCB) or activated B-cell-like (non-GCB) types according to the Hans algorithm [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Patients who were comatose or responded only to noxious stimuli at the time of hospital admission were excluded, as accurate baseline cognitive evaluation was not feasible. Moreover, cases in which tissue volume was not sufficient for immunohistological evaluation were excluded. Tumor involvement was defined as a contrast-enhanced area on T1-weighted contrast-enhanced magnetic resonance (MR) images. However, four cases were evaluated using contrast-enhanced computed tomography (CT), and two cases of non-contrast-enhanced lesions were assessed using MR images without contrast enhancement. The total number of involved brain regions was calculated by adding all individually affected regions. In cases of tumor involvement in multiple brain regions, histopathologic diagnoses were based on tissue samples from contrast-enhanced areas if a sufficient sample volume was safely available [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePatient evaluation of cognitive impairment\u003c/p\u003e\u003cp\u003eCognitive status at the time of hospital admission was evaluated using the Clinical Dementia Rating (CDR) scale [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A rater blinded to treatment and outcome data derived each global CDR score by systematically reviewing the complete electronic record\u0026mdash;admission and nursing notes, social-work entries, neuro-psychiatric consultations, medication lists, bedside mental-status examinations, laboratory results, and all ancillary reports as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A global CDR score\u0026thinsp;\u0026ge;\u0026thinsp;0.5 was set to indicate cognitive impairment.\u003c/p\u003e\u003cp\u003eSurvival analysis\u003c/p\u003e\u003cp\u003eAs previously reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], OS was defined as the time from initial diagnosis to death, whereas PFS was defined as the time from diagnosis to the first disease progression. Patients who had an uncertain date of death or who were alive at the time of analysis were censored, with the time from the first diagnosis to the last clinic visit or contact used as the censoring time. For patients with an uncertain progressive disease (PD) date or no PD at the time of analysis, the time from diagnosis to the last MR imaging or CT evaluation was used as the censoring time.\u003c/p\u003e\u003cp\u003eHistology and immunohistochemistry\u003c/p\u003e\u003cp\u003eImmunohistochemistry was performed using formalin-fixed paraffin-embedded tumor specimens according to our previously published protocol, with verification using positive and negative controls [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The following primary antibodies were used: mouse anti-polyglutamylation (clone GT335, 1:2,000; AdipoGen AG), monoclonal rabbit anti-tau (clone D1M9X, 1:500; Cell Signaling Technology), recombinant monoclonal rabbit anti-tau (phospho S396) (clone EPR2731, 1:4,000; abcam), and mouse anti-β-amyloid (clone 6F/3D, 1:100; Dako). As we previously reported, samples with \u0026gt;\u0026thinsp;10% positive cells comprised the high-polyglutamylation group, and those with \u0026lt;\u0026thinsp;10% comprised the low-polyglutamylation group [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImage quantification\u003c/p\u003e\u003cp\u003eSlide images were obtained using a digital slide scanner (NanoZoomer XR; HAMAMATSU, Hamamatsu, Japan). Total tau- and phosphorylated tau-positive areas were quantitatively evaluated using an integrated fluorescence microscope (BZ-X800) equipped with an image cytometer module (BZ-H4XI; both KEYENCE, Osaka, Japan).\u003c/p\u003e\u003cp\u003eCell lines and cell culture\u003c/p\u003e\u003cp\u003eThe human PCNSL-derived cell lines TK (RRID: CVCL_E941) and HKBML (RRID: CVCL_8161) were purchased from the JCRB Cell Bank (Osaka, Japan) and the RIKEN BioResource Center (Tsukuba, Japan), respectively. As previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], each cell line was cultured in the appropriate medium for maintenance. For experiments, the cells were seeded into cell culture plates (100,000 cells/mL), and sodium butyrate (NaBu; Schircks Laboratories, Switzerland) was immediately added to the wells at a final concentration of 1 mM. Vehicle control was 0.1% dimethyl sulfoxide. Afterward, cells were incubated for 72 h and then used for various experiments.\u003c/p\u003e\u003cp\u003eWestern blotting\u003c/p\u003e\u003cp\u003eThe protocols for western blotting experiments were previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The following primary antibodies were used: polyclonal rabbit anti-folylpolyglutamate synthase (FPGS) (1:1,000; Spring Bioscience), monoclonal rabbit anti-tau (clone D1M9X, 1:500; Cell Signaling Technology), monoclonal rabbit anti-phospho-tau Ser202/Thr205 (clone 4A6, 1:5,000; Proteintech), and mouse anti-α-tubulin (clone DM1A, 1:2,000; Cell Signaling Technology).\u003c/p\u003e\u003cp\u003eQuantitative polymerase chain reaction (qPCR)\u003c/p\u003e\u003cp\u003eTotal RNA was extracted using an RNeasy Mini Kit (Qiagen, Hulsterweg, Netherlands). Total RNA (500 ng in a 20 \u0026micro;L reaction volume) was reverse-transcribed into complementary DNA using SuperScript Ⅳ Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer\u0026rsquo;s instructions. The subsequent qPCR analysis was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). Reactions were performed on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Data were normalized to \u003cem\u003eTBP\u003c/em\u003e levels, and the relative mRNA expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primer sequences are listed in Supplementary Table\u0026nbsp;1 (see Additional file 1).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll uni- and multivariate analyses were performed as previously reported [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Between-group differences in numerical values were tested using Student\u0026rsquo;s t-test and the Mann\u0026ndash;Whitney U test for normally and non-normally distributed data, respectively. Categorical data were compared using Fisher\u0026rsquo;s exact test. Kaplan\u0026ndash;Meier survival analysis and the log-rank test were performed to compare survival time between the low- and high-polyglutamylation groups. Multivariate logistic regression analysis was performed to identify independent factors associated with cognitive impairment. Variables with a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.10 in univariate analyses, as well as clinically important variables regardless of their \u003cem\u003ep\u003c/em\u003e-value, were included as candidate predictors in the multivariate model. Two-tailed \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003eClinical study\u003c/h2\u003e\n\u003cp\u003eOf 207 consecutive patients with PCNSL, 140 met the inclusion criteria of this study. The clinical characteristics of these 140 patients are presented in Table 1. The median age was 68 years (range: 41\u0026ndash;92). The cohort included 71 men and 69 women, and the median Karnofsky performance status (KPS) score was 50 (range: 20\u0026ndash;100) at diagnosis. Histologically, 138 patients (98.6%) were diagnosed with DLBCL, one with Burkitt lymphoma, and one with low-grade B-cell lymphoma. Among the 138 DLBCLs, the cell of origin was assessed; 97 (70.3%) were categorized as non-GCB type. High levels of polyglutamylation were present in 83 (59.3%) patients. Additionally, 88 patients (62.9%) had multiple lesions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eCharacteristics of the enrolled patients with PCNSL\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"522\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOverall\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003en = 140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e68 (41\u0026ndash;92)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eSex (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e71/69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eKPS score (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e50 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eCell of origin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eGCB/non-GCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e41/97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eLDH (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eHigh/normal range\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e45/95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003ePolyglutamylation (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eHigh/low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e83/57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eNumber of involved brain regions (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eSingle/multiple\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e52/88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eTumor involvement in each brain region (Right/left, # of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eFrontal lobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e37/38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eTemporal lobe (including the hippocampus)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e16/10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eTemporoparietal lobe (excluding the hippocampus)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e19/20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eOccipital lobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e10/6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eBasal ganglia and/or thalamus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e38/31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eCorpus callosum and/or cingulate gyrus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eVentricle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 373px;\"\u003e\n \u003cp\u003eBrainstem and/or cerebellum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 149px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; PCNSL, primary central nervous system lymphoma; pts, patients.\u003c/p\u003e\n\u003cp\u003eNext, we compared clinical characteristics between patients with and without cognitive impairment (Table 2). Cognitive impairment at diagnosis was present in 84 patients (60.0%). The two groups (with vs. without cognitive impairment) differed significantly in age, KPS score, and polyglutamylation status. Patients with cognitive impairment were significantly older (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) and had significantly lower KPS scores (\u003cem\u003ep\u003c/em\u003e = 0.0005) than those without impairment. A higher proportion of cognitively impaired patients also exhibited high polyglutamylation levels (\u003cem\u003ep\u003c/em\u003e = 0.036). A comparison of tumor\u003cem\u003e\u0026nbsp;\u003c/em\u003einvolvement in each brain region is shown in Fig. 1a, b and Supplementary Table 2 (see Additional file 1). Cognitive impairment was significantly associated with the involvement of the corpus callosum and/or cingulate gyrus, whereas brainstem and cerebellar lesions were more common in patients without cognitive impairment (all \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; Fig. 1a, c). The proportions of patients with multiple lesions did not significantly differ between those with and those without cognitive impairment (Fig. 1b). In multivariate analysis, a high-polyglutamylation status emerged as an independent risk factor for cognitive impairment at diagnosis (odds ratio [OR] 4.35, 95% confidence interval [CI] 1.47\u0026ndash;12.9, \u003cem\u003ep\u003c/em\u003e = 0.0080, Table 3), even after controlling for age, KPS scores, and tumor location.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eComparison of the clinical characteristics between patients with and without cognitive impairment at diagnosis\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"561\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 283px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCognitive impairment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003en = 84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003en = 56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e72 (43\u0026ndash;92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e64 (41\u0026ndash;85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eSex (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e41/43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e30/26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eKPS score (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e50 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e70 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.00050\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eCell of origin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eGCB/non-GCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e22/60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e19/37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eLDH (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eHigh/normal range\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e29/55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e16/40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003ePolyglutamylation (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 198px;\"\u003e\n \u003cp\u003eHigh/low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003e56/28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e27/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.036\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; pts, patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eMultivariate logistic regression analysis of the predictors of cognitive impairment at diagnosis\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003eAge (y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e1.04\u0026ndash;1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.00057\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003eKPS score (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e0.935\u0026ndash;0.986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.0023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003ePolyglutamylation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e4.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e1.47\u0026ndash;12.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.0080\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003eNumber of involved brain regions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e0.333\u0026ndash;2.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003eCorpus callosum and/or cingulate gyrus\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e3.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e1.03\u0026ndash;11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003eBrainstem and/or cerebellum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e0.0256\u0026ndash;0.276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 276px;\"\u003e\n \u003cp\u003eVentricle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e0.563\u0026ndash;12.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e CI, confidence interval; KPS, Karnofsky performance status; OR, odds ratio.\u003c/p\u003e\n\u003cp\u003eTo further explore the clinical implications of polyglutamylation, we compared patients according to polyglutamylation status. The high-polyglutamylation group exhibited a higher frequency of cognitive impairment (\u003cem\u003ep\u003c/em\u003e =0.036, Table 4), as observed in the overall cohort. The high-polyglutamylation group also showed significantly longer PFS (\u003cem\u003ep\u003c/em\u003e = 0.0046, Fig. 1d), whereas OS did not differ between groups (\u003cem\u003ep\u003c/em\u003e = 0.23, Fig. 1e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eComparison of clinical characteristics between patients with high- and low-polyglutamylation status\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 302px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePolyglutamylation level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003en = 83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003en = 57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e69 (41\u0026ndash;92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e68 (46\u0026ndash;87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eSex (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e42/41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e29/28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eKPS score (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e60 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e50 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eCell of origin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eGCB/non-GCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e28/53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e13/44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eLDH (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eHigh/normal range\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e26/27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e19/38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eCognitive impairment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 178px;\"\u003e\n \u003cp\u003eYes/No\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155px;\"\u003e\n \u003cp\u003e56/27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e28/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.036\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; pts, patients.\u003c/p\u003e\n\u003cp\u003eA high-polyglutamylation status was also more common in tumors involving the left frontal lobe (Supplementary Table 3\u0026nbsp;[see Additional file 1]). In the subset of patients with left frontal lobe involvement, 23 of 30 patients (77%) with high polyglutamylation levels had cognitive impairment, compared to 4 of 8 (50%) with low polyglutamylation levels. Although this difference was not significant (\u003cem\u003ep\u003c/em\u003e = 0.20, Supplementary Table 4 [see Additional file 1]), the trend was consistent with that observed in the overall cohort.\u003c/p\u003e\n\u003cp\u003eIn summary, these findings indicate that cognitive impairment in the high-polyglutamylation group cannot be solely explained by age, KPS score, or tumor location, suggesting that additional neurobiological mechanisms related to polyglutamylation may contribute to cognitive dysfunction in PCNSL.\u003c/p\u003e\n\u003ch2\u003eHistopathology and immunohistochemistry\u003c/h2\u003e\n\u003cp\u003eNext, patient samples were examined for the accumulation of phosphorylated tau and \u0026beta;-amyloid, two of the molecules most commonly associated with cognitive decline in Alzheimer\u0026rsquo;s disease [27]. Notably, the expression of total and phosphorylated tau coincided with that of polyglutamylation (Fig. 2a). To quantitatively assess this association, we compared the expression of tau and phosphorylated tau between the low- and high-polyglutamylation groups using a subset of patient samples with sufficient tissue volume (six samples in each group). The areas positive for total tau and phosphorylated tau were significantly larger in the high-polyglutamylation group than in the low-polyglutamylation group (Fig. 2b). Although phosphorylated tau was expressed, no \u0026beta;-amyloid deposits were observed in the samples of patients with PCNSL (Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eElevated polyglutamylation levels increase the levels of phosphorylated tau in PCNSL cells\u003c/h2\u003e\n\u003cp\u003eSubsequently, we evaluated whether the level of polyglutamylation correlates with that of phosphorylated tau in PCNSL cells. We used an in vitro model of treatment with histone deacetylase inhibitors (HDACIs) that we had previously used to control polyglutamylation levels in cells [18]. We treated cells with the HDACI NaBu to increase the polyglutamylation level by upregulating FPGS (Fig. 2d).\u003c/p\u003e\n\u003cp\u003eFirst, we compared the levels of phosphorylated tau between cells with and without HDACI treatment. Immunoblots showed that NaBu treatment increased the expression levels of both FPGS and phosphorylated tau in two different cell lines (Fig. 2e).\u003c/p\u003e\n\u003cp\u003eTau phosphorylation is regulated by the balance between phosphorylation and dephosphorylation. Proline-directed kinases, such as glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (CDK5), mediate aberrant tau phosphorylation [28, 29]. In contrast, protein phosphatase 2A (PP2A) [30]\u0026nbsp;and peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) [31]\u0026nbsp;facilitate tau dephosphorylation. Therefore, we determined the gene expression levels of enzymes involved in tau phosphorylation and dephosphorylation in cells with and without HDACI treatment using qPCR. Notably, mRNA expression of \u003cem\u003eCDK5\u003c/em\u003e was consistently increased in PCNSL cell lines with high levels of polyglutamylation after NaBu treatment, but no significant changes were observed in the expression of tau dephosphorylation-related enzymes (Fig. 2f). These in vitro studies also confirmed that the extent of polyglutamylation is significantly correlated with that of tau phosphorylation. In summary, our results suggest that phosphorylated tau is involved in the association of polyglutamylation with cognitive impairment in patients with PCNSL.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we found for the first time that high polyglutamylation levels in PCNSL tumors are closely associated with cognitive impairment at diagnosis. Furthermore, our data suggest that this association is accompanied by the accumulation of phosphorylated tau, both in patient-derived tumor samples and PCNSL cell lines, supporting the hypothesis that polyglutamylation may contribute to tau-related neurodegenerative processes. While these findings suggest a possible mechanistic link between polyglutamylation and cognitive impairment in PCNSL, causality cannot be definitively established based on the results of the current study, warranting further investigation.\u003c/p\u003e\n\u003cp\u003eSeveral preclinical studies have investigated the relationship between polyglutamylation and tau expression. For example, Hausrat et al. recently demonstrated in a mouse model that polyglutamylation plays a key role in tauopathy caused by the accumulation of phosphorylated tau [14]. Although our results are consistent with these experimental findings, the precise mechanism by which polyglutamylation in tumor cells may influence neuronal function in patients with PCNSL remains to be clarified. In tauopathies, it is thought that pathological tau produced within neurons is released by these cells and spreads into the parenchyma, where it can then be transferred to other cells [32-36]. A similar pathological process can occur in PCNSL, where tau is shed from tumor cells and affects neurons. Future studies using advanced imaging modalities such as Tau positron emission tomography (PET)\u0026nbsp;[37], or autopsy-based investigations, may provide valuable insights into the pathological mechanisms underlying cognitive impairment in PCNSL.\u003c/p\u003e\n\u003cp\u003eRegarding the localization of lesions, a previous report demonstrated that neuropsychological changes in PCNSL correlate with diffuse involvement of the periventricular white matter or corpus callosum [38], which aligns with our findings. Importantly,\u0026nbsp;although high polyglutamylation is associated with longer PFS, no significant difference in\u0026nbsp;OS was\u0026nbsp;observed between high- and low- polyglutamylation groups.\u0026nbsp;This may reflect the delayed diagnosis and therapy initiation in patients whose initial symptoms were subtle cognitive changes. Indeed, we encountered a case where the patient died before receiving initial treatment due to rapid disease progression following surgery, resulting in central cerebral herniation. This patient presented with cognitive changes at onset and had a highly polyglutamylated tumor, for which MTX treatment was anticipated to be effective. These findings underscore the need for increased clinical awareness and early intervention in PCNSL patients presenting with cognitive or behavioral symptoms, particularly in the context of highly polyglutamylated PCNSL. As wearable AI and other digital technologies become more widespread in daily life, it may become possible to detect cognitive and behavioral changes earlier and more continuously [39, 40].\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. First, its retrospective nature and reliance on chart reviews to assess cognitive status may introduce bias. However, recent literature supports the feasibility and reliability of retrospective CDR scoring based on comprehensive chart reviews and collateral histories [25]. To minimize bias, CDR scoring in this study was performed systematically and blinded to outcomes. Prospective studies incorporating comprehensive and objective neuropsychological evaluations are needed to confirm our findings. Second, the sample size in some subgroups (e.g., patients with left frontal lobe involvement) was limited, which may have reduced the statistical power to detect significant associations between polyglutamylation status and cognitive impairment. Further studies with larger cohorts are warranted to validate these findings. Third, our histological analyses were restricted to samples from the main tumor mass, precluding the assessment of tau pathology in surrounding brain parenchyma. Advanced imaging modalities, such as tau PET, or post-mortem studies, may provide additional insights into the distribution and impact of tau pathology in PCNSL. Fourth, our in vitro experiments used cell lines and chemical modulation to mimic polyglutamylation; although informative, these models cannot fully recapitulate the complex in vivo tumor microenvironment.\u003c/p\u003e\n\u003cp\u003eDespite these limitations, our findings offer new perspectives on the pathophysiology of PCNSL. They highlight a potential role for polyglutamylation in cognitive dysfunction, suggest directions for further mechanistic and translational research, and underscore the clinical need for early recognition of subtle cognitive changes in PCNSL.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated that PCNSL tumors with high polyglutamylation levels were associated with cognitive impairment at diagnosis. Additionally, a significant correlation was observed between polyglutamylation and phosphorylated tau accumulation, suggesting a potential role in the underlying pathophysiology of PCNSL. Recognizing PCNSL as a potential cause of rapidly progressive cognitive impairment is essential for timely diagnosis and treatment, which may lead to improved patient outcomes. These findings provide new insights into the pathological mechanisms of PCNSL and may inform future research on disease progression and therapeutic strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDK5\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecyclin-dependent kinase 5\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eClinical Dementia Rating\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003econfidence interval\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecomputed tomography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDLBCL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ediffuse large B cell lymphoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFPGS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efolylpolyglutamate synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGCB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egerminal center B-cell-like\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSK3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eglycogen synthase kinase 3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHDACI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehistone deacetylase inhibitor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKarnofsky performance status\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMTX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emethotrexate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emagnetic resonance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNaBu\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003esodium butyrate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eodds ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eoverall survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCNSL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprimary central nervous lymphoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprogressive disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePET\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epositron emission tomography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprogression-free survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePIN1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epeptidylprolyl cis/trans isomerase, NIMA-interacting 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePP2A\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprotein phosphatase 2A\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003equantitative polymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll study procedures involving human participants conformed to the ethical standards of the institutional and/or national research committee and to the Helsinki Declaration of 1964 and its subsequent amendments or comparable ethical standards. Informed consent was obtained from all individual participants enrolled in this study or their families. The study protocol was approved by the Institutional Review Board (IRB) of Kumamoto University (Genome No. 231).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable. All MR images included in this manuscript have been carefully reviewed, fully anonymized, and stripped of any direct or indirect identifiers to ensure patient privacy. No identifiable personal information is presented.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAll original data from this manuscript are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by a Grant-in-Aid for Scientific Research (19K09485) from the Japanese Society for the Promotion of Science.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eStudy conception and design: YT, NS. Data acquisition: YT, NS, KF, DY, YH, MO, MT, YM, HU, TH. Data analysis and interpretation: YT, NS, KF, AM. Drafting of the manuscript: YT, NS. Review of the manuscript: NS. Critical revision: TH, AM.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eDuring the preparation stage of this work, the authors used DeepL, an AI-assisted technology, to improve readability and expressiveness. Subsequently, they used the English editing service Editage. Afterward, the authors reviewed and edited the content as necessary, and they take full responsibility for the content of this publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eC. Grommes, J.L. Rubenstein, L.M. DeAngelis, A.J.M. Ferreri, T.T. Batchelor, Comprehensive approach to diagnosis and treatment of newly diagnosed primary CNS lymphoma. Neuro Oncol 2019;21:296-305.\u003c/li\u003e\n\u003cli\u003eK. Makino, H. Nakamura, T. Kino, H. Takeshima, J. Kuratsu, Rising incidence of primary central nervous system lymphoma in Kumamoto, Japan. Surg Neurol 2006;66:503-6.\u003c/li\u003e\n\u003cli\u003eH. Nakamura, K. Makino, S. Yano, J. Kuratsu, Kumamoto Brain Tumor Research Group. Epidemiological study of primary intracranial tumors: a regional survey in Kumamoto prefecture in southern Japan\u0026mdash;20-year study. Int J Clin Oncol 2011;16:314-21.\u003c/li\u003e\n\u003cli\u003eJ.S. Mendez, Q.T. Ostrom, H. Gittleman, C. Kruchko, L.M. DeAngelis, J.S. Barnholtz-Sloan, et al, The elderly left behind-changes in survival trends of primary central nervous system lymphoma over the past 4 decades. Neuro Oncol 2018;20:687-94.\u003c/li\u003e\n\u003cli\u003eP. Hermann, I. Zerr, Rapidly progressive dementias \u0026ndash; aetiologies, diagnosis and management. Nat Rev Neurol 2022;18:363-76.\u003c/li\u003e\n\u003cli\u003eL.M. DeAngelis, Brain tumors. N Engl J Med 2001;344:114-23.\u003c/li\u003e\n\u003cli\u003eR. Velasco, S. Mercadal, N. Vidal, M. Ala\u0026ntilde;\u0026aacute;, M.I. Barcel\u0026oacute;, M.J. Ib\u0026aacute;\u0026ntilde;ez-Juli\u0026aacute; , et al, Diagnostic delay and outcome in immunocompetent patients with primary central nervous system lymphoma in Spain: a multicentric study. J Neurooncol 2020;148:545-54.\u003c/li\u003e\n\u003cli\u003eR.M.H. Lim, J.Y. Chan, Absence of meaningful neurocognitive recovery in comatose patients with primary central nervous system lymphoma despite an effective response to chemotherapy: Case reports. Mol Clin Oncol 2021;14:44.\u003c/li\u003e\n\u003cli\u003eM. van der Meulen, L. Dirven, K. Bakunina, M.J. van den Bent, S. Issa, J.K. Doorduijn, et al, MMSE is an independent prognostic factor for survival in primary central nervous system lymphoma. J Neurooncol 2021;152:357-62.\u003c/li\u003e\n\u003cli\u003eN. Samudra, C. Lane-Donovan, L. VandeVrede, A.L. Boxer, Tau pathology in neurodegenerative disease: disease mechanisms and therapeutic avenues. J Clin Invest 2023;133:e168553.\u003c/li\u003e\n\u003cli\u003eK. Iqbal, F. Liu, C.X. Gong, Tau and neurodegenerative disease: the story so far. Nat Rev Neurol 2016;12:15-27.\u003c/li\u003e\n\u003cli\u003eN. Olfati, A. Shoeibi, I. Litvan, Clinical spectrum of tauopathies. Front Neurol 2022;13:944806.\u003c/li\u003e\n\u003cli\u003eD. Boucher, J.C. Larcher, F. Gros, P. Denoulet, Polyglutamylation of tubulin as a progressive regulator of in vitro interactions between the microtubule-associated protein Tau and tubulin. Biochemistry 1994;33:12471-7.\u003c/li\u003e\n\u003cli\u003eT.J. Hausrat, P.C. Janiesch, P. Breiden, D. Lutz, S. Hoffmeister-Ullerich, I. Hermans-Borgmeyer, et al, Disruption of tubulin-alpha4a polyglutamylation prevents aggregation of hyper-phosphorylated tau and microglia activation in mice. Nat Commun 2022;13:4192.\u003c/li\u003e\n\u003cli\u003eK. Rogowski, J. van Dijk, M.M. Magiera, C. Bosc, J.C. Deloulme, A. Bosson, et al, A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 2010;143:564-78.\u003c/li\u003e\n\u003cli\u003eM.M. Magiera, S. Bodakuntla, J. Žiak, S. Lacomme, P. Marques Sousa, S. Leboucher, et al, Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport. EMBO J 2018;37:e100440.\u003c/li\u003e\n\u003cli\u003eS. Bodakuntla, A. Schnitzler, C. Villablanca, C. Gonzalez-Billault, I. Bieche, C. Janke, et al, Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons. J Cell Sci 2020;133:jcs241802.\u003c/li\u003e\n\u003cli\u003eK. Fujimoto, N. Shinojima, M. Hayashi, T. Nakano, K. Ichimura, A. Mukasa, Histone deacetylase inhibition enhances the therapeutic effects of methotrexate on primary central nervous system lymphoma. Neurooncol Adv 2020;2:vdaa084.\u003c/li\u003e\n\u003cli\u003eN. Shinojima, K. Fujimoto, K. Makino, K. Todaka, K. Yamada, Y. Mikami, et al, Clinical significance of polyglutamylation in primary central nervous system lymphoma. Acta Neuropathol Commun 2018;6:15.\u003c/li\u003e\n\u003cli\u003eD.N. Louis, A. Perry, P. Wesseling, D.J. Brat, I.A. Cree, D. Figarella-Branger, et al, The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro Oncol 2021;23:1231-51.\u003c/li\u003e\n\u003cli\u003eC.P. Hans, D.D. Weisenburger, T.C. Greiner, R.D. Gascoyne, J. Delabie, G. Ott, et al, Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004;103:275-82.\u003c/li\u003e\n\u003cli\u003eJ.C. Morris, The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology 1993;43:2412-4.\u003c/li\u003e\n\u003cli\u003eC.P. Hughes, L. Berg, W.L. Danziger, L.A. Coben, R.L. Martin, A new clinical scale for the staging of dementia. Br J Psychiatry 1982;140:566-72.\u003c/li\u003e\n\u003cli\u003eV. Haroutunian, D.P. Purohit, D.P. Perl, D. Marin, K. Khan, M. Lantz, et al, Neurofibrillary tangles in nondemented elderly subjects and mild Alzheimer disease. Arch Neurol 1999;56:713-8.\u003c/li\u003e\n\u003cli\u003eV. Dauphinot, S. Calvi, C. Moutet, J. Xie, S. Dautricourt, A. Batsavanis, et al, Reliability of the assessment of the clinical dementia rating scale from the analysis of medical records in comparison with the reference method. Alzheimers Res Ther 2024;16:198.\u003c/li\u003e\n\u003cli\u003eN. Shinojima, A. Hossain, T. Takezaki, J. Fueyo, J. Gumin, F. Gao, et al, TGF-\u0026beta; mediates homing of bone marrow-derived human mesenchymal stem cells to glioma stem cells. Cancer Res 2013;73:2333-44.\u003c/li\u003e\n\u003cli\u003eB.J. Hanseeuw, R.A. Betensky, H.I.L. Jacobs, A.P. Schultz, J. Sepulcre, J.A. Becker, et al, Association of amyloid and tau with cognition in preclinical Alzheimer disease: a longitudinal study. JAMA Neurol 2019;76:915-24.\u003c/li\u003e\n\u003cli\u003eF. Plattner, M. Angelo, K.P. Giese, The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation. J Biol Chem 2006;281:25457-65.\u003c/li\u003e\n\u003cli\u003eW. Noble, V. Olm, K. Takata, E. Casey, O. Mary, J. Meyerson, et al, Cdk5 is a key factor in tau aggregation and tangle formation in vivo. Neuron 2003;38:555-65.\u003c/li\u003e\n\u003cli\u003eF. Liu, I. Grundke-Iqbal, K. Iqbal, C.X. Gong, Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur J Neurosci 2005;22:1942-50.\u003c/li\u003e\n\u003cli\u003eT. Kimura, K. Tsutsumi, M. Taoka, T. Saito, M. Masuda-Suzukake, K. Ishiguro, et al, Isomerase Pin1 stimulates dephosphorylation of tau protein at cyclin-dependent kinase (Cdk5)-dependent Alzheimer phosphorylation sites. J Biol Chem 2013;288:7968-77.\u003c/li\u003e\n\u003cli\u003eF. Clavaguera, T. Bolmont, R.A. Crowther, D. Abramowski, S. Frank, A. Probst, et al, Transmission and spreading of tauopathy in transgenic mouse brain. Nat Cell Biol 2009;11:909-13.\u003c/li\u003e\n\u003cli\u003eN. Annadurai, J.B. De Sanctis, M. Hajd\u0026uacute;ch, V. Das, Tau secretion and propagation: Perspectives for potential preventive interventions in Alzheimer\u0026rsquo;s disease and other tauopathies. Exp Neurol 2021;343:113756.\u003c/li\u003e\n\u003cli\u003eP.T. Nelson, I. Alafuzoff, E.H. Bigio, C. Bouras, H. Braak, N.J. Cairns, et al, Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. J Neuropathol Exp Neurol 2012;71:362-81.\u003c/li\u003e\n\u003cli\u003eA. Seitkazina, K.H. Kim, E. Fagan, Y. Sung, Y.K. Kim, S. Lim, The fate of tau aggregates between clearance and transmission. Front Aging Neurosci, 2022;14:932541.\u003c/li\u003e\n\u003cli\u003eY. Wang, E. Mandelkow, Tau in physiology and pathology. Nat Rev Neurosci 2016;17:5-21.\u003c/li\u003e\n\u003cli\u003eR. Ossenkoppele, A. Pichet Binette, C. Groot, R. Smith, O. Strandberg, S. Palmqvist, et al, Amyloid and tau PET-positive cognitively unimpaired individuals are at high risk for future cognitive decline. Nat Med 2022;28:2381-7.\u003c/li\u003e\n\u003cli\u003eW. K\u0026uuml;ker, T. N\u0026auml;gele, A. Korfel, S. Heckl, E. Thiel, M. Bamberg, et al, Primary central nervous system lymphomas (PCNSL): MRI features at presentation in 100 patients. J Neurooncol 2005;72:169-77.\u003c/li\u003e\n\u003cli\u003eA.Y. DuBord, E.W. Paolillo, A.M. Staffaroni, Remote digital technologies for the early detection and monitoring of cognitive decline in patients with type 2 diabetes: insights from studies of neurodegenerative diseases. J Diabetes Sci Technol 2024;18:1489-99.\u003c/li\u003e\n\u003cli\u003eR.L. Nosheny, D. Yen, T. Howell, M. Camacho, K. Moulder, S. Gummadi, et al, Evaluation of the Electronic Clinical Dementia Rating for dementia screening. JAMA Netw Open 2023;6:e2333786.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"alzheimers-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"azrt","sideBox":"Learn more about [Alzheimer's Research and Therapy](http://alzres.biomedcentral.com/)","snPcode":"13195","submissionUrl":"https://submission.nature.com/new-submission/13195/3","title":"Alzheimer's Research \u0026 Therapy","twitterHandle":"@AlzheimersRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Polyglutamylation, Cognitive impairment, Primary central nervous system lymphoma, Tau protein","lastPublishedDoi":"10.21203/rs.3.rs-6983936/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6983936/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003ePrimary central nervous system lymphoma (PCNSL) often manifests with cognitive impairment or nonspecific symptoms, which can delay diagnosis and worsen prognosis. However, the mechanisms underlying these neurological manifestations remain poorly understood. Previous studies have shown that polyglutamylation, a posttranslational modification, is associated with better responses to methotrexate-based chemotherapy in patients with PCNSL. Moreover, excessive polyglutamylation in neurons has been implicated in neurodegeneration via phosphorylated tau accumulation. This study aimed to elucidate the relationship between polyglutamylation, phosphorylated tau, and cognitive impairment in PCNSL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eWe retrospectively analyzed 140 patients with histologically confirmed PCNSL treated at our institution between 2001 and 2022. Cognitive status at hospital admission was assessed using the Clinical Dementia Rating (CDR) scale. Immunohistochemical analysis of tumor specimens was performed to quantify the polyglutamylation and phosphorylated tau levels. Furthermore, in vitro studies with PCNSL cell lines were conducted to investigate whether the pharmacological upregulation of polyglutamylation by a histone deacetylase inhibitor promotes tau phosphorylation. Statistical analyses examined associations among polyglutamylation status, cognitive impairment, tau phosphorylation, and clinical outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eHigh polyglutamylation levels were observed in 59% of tumor samples, and this factor was independently associated with cognitive impairment at diagnosis (odds ratio: 4.35, 95% confidence interval 1.47–12.9, \u003cem\u003ep\u003c/em\u003e = 0.0080). Immunohistochemical analysis demonstrated that tumors with elevated polyglutamylation showed significantly higher phosphorylated tau levels. In vitro experiments confirmed that increased polyglutamylation levels in PCNSL cells led to enhanced tau phosphorylation in PCNSL cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eHigh polyglutamylation levels in PCNSL were associated with cognitive impairment and increased tau phosphorylation at diagnosis. These findings suggest that polyglutamylation may contribute to neurocognitive symptoms by promoting tau pathology. Elucidating this mechanism may provide novel insights into PCNSL pathophysiology and may inform future studies on disease mechanisms and potential treatment targets.\u003c/p\u003e","manuscriptTitle":"Elevated polyglutamylation and tau phosphorylation levels are associated with cognitive impairment at diagnosis in patients with primary central nervous system lymphoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 14:23:20","doi":"10.21203/rs.3.rs-6983936/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-01T11:38:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-13T16:45:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T21:43:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84518624711407689931795270301978538408","date":"2025-07-23T03:08:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15400144525480670813610589315340970076","date":"2025-07-22T16:45:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33478124230832486586523672628938970162","date":"2025-07-22T16:42:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T17:47:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-27T06:15:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-27T06:12:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Alzheimer's Research \u0026 Therapy","date":"2025-06-26T13:32:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"alzheimers-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"azrt","sideBox":"Learn more about [Alzheimer's Research and Therapy](http://alzres.biomedcentral.com/)","snPcode":"13195","submissionUrl":"https://submission.nature.com/new-submission/13195/3","title":"Alzheimer's Research \u0026 Therapy","twitterHandle":"@AlzheimersRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fe0edd32-6907-4173-9023-b4284fc65fce","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:09:16+00:00","versionOfRecord":{"articleIdentity":"rs-6983936","link":"https://doi.org/10.1186/s13195-025-01927-z","journal":{"identity":"alzheimers-research-and-therapy","isVorOnly":false,"title":"Alzheimer's Research \u0026 Therapy"},"publishedOn":"2025-12-02 15:58:02","publishedOnDateReadable":"December 2nd, 2025"},"versionCreatedAt":"2025-07-18 14:23:20","video":"","vorDoi":"10.1186/s13195-025-01927-z","vorDoiUrl":"https://doi.org/10.1186/s13195-025-01927-z","workflowStages":[]},"version":"v1","identity":"rs-6983936","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6983936","identity":"rs-6983936","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}