Elevated polyglutamylation and tau phosphorylation levels in patients with primary central nervous system lymphoma are related to cognitive decline and neuropsychiatric changes at disease onset

Elevated polyglutamylation and tau phosphorylation levels in patients with primary central nervous system lymphoma are related to cognitive decline and neuropsychiatric changes at disease onset | 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 in patients with primary central nervous system lymphoma are related to cognitive decline and neuropsychiatric changes at disease onset Yuki Takeshima, Naoki Shinojima, Kenji Fujimoto, Daiki Yoshii, and 7 more This is a preprint; it has not been peer reviewed by a journal.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-5685172/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Primary central nervous system lymphoma (PCNSL) often presents with nonspecific cognitive decline and neuropsychiatric changes at onset, yet the underlying mechanisms remain poorly understood. Polyglutamylation, a posttranslational modification, is associated with better responses to methotrexate-based chemotherapy in patients with PCNSL. On the other hand, hyperglutamylation of microtubules in neurons has been implicated in neurodegeneration via the accumulation of phosphorylated tau. This study aimed to elucidate the relationship of polyglutamylation with cognitive decline and neuropsychiatric changes at PCNSL onset. We retrospectively analyzed 145 patients with PCNSL treated at our institution between 2001 and 2022. Polyglutamylation and phosphorylated tau were evaluated using immunohistochemistry. In vitro studies were performed to evaluate the causal relationship between polyglutamylation and tau phosphorylation in PCNSL cell lines. Cognitive decline and neuropsychiatric changes at disease onset were observed in 48.3% of patients, with multivariate analysis identifying high polyglutamylation levels as an independent risk factor (odds ratio: 7.34, p < 0.001). Patient samples and cell lines consistently demonstrated a link between polyglutamylation and tau phosphorylation, implicating that polyglutamylation-induced tau phosphorylation may contribute to cognitive decline and neuropsychiatric changes. Our findings suggest that polyglutamylation contributes to neurocognitive dysfunction in PCNSL, and these results elucidate a novel aspect of PCNSL pathophysiology. Polyglutamylation Cognitive decline Primary central nervous system lymphoma Tau protein Figures Figure 1 Figure 2 Introduction 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. On the other hand, PCNSL is a potential cause of rapidly progressive dementias [ 5 ], and approximately 40% of patients experience nonspecific cognitive decline and neuropsychiatric changes [ 1 , 6 ], delaying diagnosis and treatment and resulting in poor prognosis [ 7 ]. However, 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 [ 8 ]. 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 [ 9 ]. Beyond cognitive deficits, tauopathies can manifest with a range of symptoms, including motor dysfunction and neuropsychiatric abnormalities [ 10 ]. Polyglutamylation, a reversible posttranslational modification that adds glutamate chains to proteins, plays a role in stabilizing proteins like microtubules [ 11 ]. However, hyperglutamylation of microtubules in neurons has been implicated in neurodegeneration [ 12 – 14 ]. Furthermore, a recent study has demonstrated that C-terminal-specific tubulin polyglutamylation is crucial for the aggregation of hyperphosphorylated tau, leading to neuronal dysfunction in tauopathy models [ 15 ]. We previously reported that polyglutamylation levels in tumor specimens were high in more than half of the patients with PCNSL and were associated with responsiveness to methotrexate (MTX)-based chemotherapy [ 16 , 17 ]. While these clinical benefits are evident, we hypothesized that polyglutamylation may play a role in the cognitive decline and neuropsychiatric changes observed in the early stages of PCNSL, potentially through the accumulation of phosphorylated tau. 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 their potential correlation with clinical symptoms such as cognitive decline and neuropsychiatric changes. 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. 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) [ 18 ]. 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 [ 19 ]. Cases in which tissue volume was not sufficient for immunohistological evaluation were excluded. Lesion locations were defined based on contrast enhancements 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 lesions was calculated by adding the number of locations. In cases of multiple lesions, histopathologic diagnoses were based on tissue samples from contrast-enhanced areas if a sufficient sample volume was safely available [ 17 ]. Patient evaluation of cognitive decline and neuropsychiatric changes We conducted a retrospective evaluation of cognitive decline and neuropsychiatric changes at symptom onset using detailed chart reviews and clinical episode documentation provided by family members and caregivers, following the criteria described below, and categorized the patients into groups with or without cognitive decline and neuropsychiatric changes. Cognitive decline at onset, defined according to the criteria for all-cause dementia [ 20 ], was identified as symptoms interfering with work or daily activities, representing a decline from previous levels of functioning or performance. Such symptoms included any of the following: impaired ability to acquire and remember new information, decreased ability to reason or handle complex tasks and judgments, impaired visuospatial abilities, or dysfunction in language skills (speaking, reading, or writing). Neuropsychiatric changes were assessed based on the presence of symptoms across the domains evaluated by the Neuropsychiatric Inventory (NPI) [ 21 ], which include delusions, hallucinations, agitation/aggression, depression/dysphoria, anxiety, euphoria/elation, apathy/indifference, disinhibition, irritability/lability, aberrant motor behavior, sleep and nighttime behavior disturbances, and appetite and eating changes. The presence of at least one symptom in any of these domains was considered indicative of neuropsychiatric changes. Survival analysis As previously reported [ 17 ], overall survival (OS) was defined as the time from initial diagnosis to death, whereas progression-free survival (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 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 [ 17 ]. 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-polyglutamylated group, and those with < 10% comprised the low-polyglutamylated group [ 17 ]. 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 [ 16 , 17 ], 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 (DMSO). Afterward, cells were incubated for 72 h and then used for various experiments. Western blotting The protocols for western blotting experiments were previously described [ 22 ]. 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. Statistical analysis All uni- and multivariate analyses were performed as previously reported [ 16 , 17 ]. Between-group differences in numerical values were tested using Student’s t-test and 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. Two-tailed p -values < 0.05 were considered statistically significant. Results Clinical study Of 207 consecutive patients with PCNSL, 145 patients met the inclusion criteria of this study. The clinical characteristics of the 145 patients with PCNSL are presented in Table 1 . The median age was 68 (range: 41–92) years, the study population comprised 75 men and 70 women, and the median Karnofsky performance status (KPS) score was 50 (range: 20–100) at the time of diagnosis. Of the 145 patients with PCNSL, 143 were diagnosed with DLBCL, one with Burkitt lymphoma, and one with low-grade B-cell lymphoma. In 143 DLBCLs, the cell of origin could be assessed, and 100 (69.0%) were categorized as non-GCB type. Among the 145 patients with PCNSL, 87 (60.0%) showed high levels of polyglutamylation. Multiple lesions were observed in 92 patients (63.4%). Table 1 Characteristics of the enrolled patients with PCNSL Overall Variables n = 145 Age Median (range) 68 (41–92) Sex (# of pts) Male/female 75/70 KPS score (%) Median (range) 50 (20–100) Cell of origin GCB/non-GCB 43/100 LDH (# of pts) High/normal range 49/96 Polyglutamylation (# of pts) High/low 87/58 Number of lesions (# of pts) Single/multiple 53/92 Location of the lesions (Right/left, # of pts) Frontal lobe 37/39 Temporal lobe (including the hippocampus) 16/10 Temporoparietal lobe (excluding the hippocampus) 19/21 Occipital lobe 10/6 Basal ganglia and/or thalamus 42/34 Corpus callosum and/or cingulate gyrus 42 Ventricle 21 Brain stem and/or cerebellum 38 Abbreviations: GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; PCNSL, primary central nervous system lymphoma; pts, patients. Next, the clinical characteristics were compared regarding the presence or absence of cognitive decline and neuropsychiatric changes at symptom onset (Table 2 ). Cognitive decline and neuropsychiatric changes at the onset were observed in 70 patients (48.3%). The groups with and without cognitive decline and neuropsychiatric changes significantly differed in age ( p = 0.00049), KPS score ( p = 0.035), and polyglutamylation status ( p < 0.0001). Table 2 Comparison of the clinical characteristics between patients with and without cognitive decline and neuropsychiatric changes as the initial symptoms Cognitive decline and neuropsychiatric changes at disease onset Yes No Variables n = 70 n = 75 p Age Median (range) 71 (41–85) 66 (43–92) 0.00049 Sex (# of pts) Male/female 34/36 41/34 0.51 KPS score (%) Median (range) 60 (20–100) 50 (20–100) 0.035 Cell of origin GCB/non-GCB 19/49 24/51 0.39 LDH (# of pts) High/normal range 25/45 24/51 0.73 Polyglutamylation (# of pts) High/low 55/15 32/43 < 0.0001 Abbreviations: GCB, germinal center B-cell-like; KPS, Karnofsky performance status; LDH, lactate dehydrogenase; pts, patients. A comparison of each tumor localization is shown in Fig. 1 a and Supplementary Table 2. The group with cognitive decline and neuropsychiatric changes had significantly more cases with multiple lesions ( p = 0.026) and with corpus callosum and cingulate gyrus lesions ( p = 0.0018) than the group without such changes. Conversely, brain stem and cerebellar lesions were significantly more common ( p = 0.0023) in the group without cognitive decline and neuropsychiatric changes at disease onset (Fig. 1 b). Furthermore, multivariate analysis confirmed polyglutamylation as a significant independent risk factor for cognitive decline and neuropsychiatric changes as the initial symptoms (odds ratio [OR] 7.34, 95% confidence interval [CI] 2.99–18.0, p < 0.001, Table 3 ). These results suggest that in addition to aging and lower KPS score, polyglutamylation affects cognitive decline and neuropsychiatric status at symptom onset in PCNSL. Table 3 Multivariate logistic regression analysis of the predictors of cognitive decline and neuropsychiatric changes as the initial symptoms Variables OR 95% CI p Age (y) 1.05 1.01–1.09 0.014 KPS score (%) 0.977 0.958–0.998 0.029 Polyglutamylation 7.34 2.99–18.0 < 0.001 Number of lesions 1.54 0.619–3.82 0.35 Corpus callosum and/or cingulate gyrus 1.93 0.756–4.93 0.17 Brain stem and/or cerebellum 0.237 0.0869–0.644 0.0048 Abbreviations: CI, confidence interval; KPS, Karnofsky performance status; OR, odds ratio. Next, we compared survival times between the high- and low-polyglutamylation groups. Consistent with our previous reports, the high-polyglutamylation group showed significantly longer PFS ( p = 0.0055), whereas no difference in OS was observed ( p = 0.15, Fig. 1 c, d). 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 [ 23 ]. Surprisingly, the expression of total and phosphorylated tau coincided with that of polyglutamylation (Fig. 2 a). We statistically compared the expression of tau and phosphorylated tau between the low- and high-polyglutamylated 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-polyglutamylated group than in the low-polyglutamylated group (Fig. 2 b). Although phosphorylated tau was expressed, no β-amyloid deposits were observed in the samples of patients with PCNSL (Fig. 2 c). 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 [ 16 ]. We treated cells with the HDACI NaBu to increase the polyglutamylation level by upregulating FPGS (Fig. 2 d). First, we compared the levels of phosphorylated tau between cells with and without HDACI treatment. Immunoblots showed that NaBu treatment induced increased expression of both FPGS and phosphorylated tau in two different cell lines (Fig. 2 d, e). 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 [ 24 , 25 ]. In contrast, protein phosphatase 2A (PP2A) [ 26 ] and peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) [ 27 ] 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. Interestingly, 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. 2 f). 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 considerably involved in the association of polyglutamylation with cognitive decline and neuropsychiatric changes in patients with PCNSL. Discussion In the present study, we found for the first time that in patients with PCNSL, highly polyglutamylated tumors are involved in cognitive decline and neuropsychiatric changes at symptom onset. Furthermore, our data suggest that the cause of these symptoms may be the accumulation of phosphorylated tau due to high levels of polyglutamylation. Additionally, analysis of PCNSL tumor samples and PCNSL cell lines revealed a positive correlation between polyglutamylation levels and phosphorylated tau expression, suggesting that polyglutamylation may regulate the expression of phosphorylated tau. Few studies have investigated the relationship between polyglutamylation and tau expression. Hausrat et al. recently demonstrated in a mouse model that polyglutamylation plays a key role in tauopathy caused by the accumulation of phosphorylated tau [ 15 ]. Polyglutamylation is deeply involved in the regulation of tau phosphorylation and accumulation, but the details of the regulation of polyglutamylation itself are unknown. As we previously reported, polyglutamylation occurs at a high rate in the presence of HDACIs, suggesting that epigenetics may be involved [ 16 ]. Tau, one of the microtubule-associate proteins, regulates microtubule assembly and stabilization [ 9 ]. Tau phosphorylation reduces the affinity of tau and causes its detachment from microtubules, resulting in the formation of hyperphosphorylated tau aggregation, which can potentially activate microglia and induce gliosis and neurodegeneration [ 28 ]. The question is how tau, which is present in highly polyglutamylated tumor cells in PCNSL, reaches neurons and affects cognitive function in patients. 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 [ 28 – 32 ]. A similar pathological process can occur in PCNSL, where tau is shed from tumor cells and affects neurons. Tau positron emission tomography (PET) may be useful in assessing the phosphorylated tau status in the whole brain [ 33 ]. Regarding the localization of lesions, a report demonstrated that neuropsychological changes in PCNSL correlate with diffuse involvement of the periventricular white matter or corpus callosum [ 34 ]. However, our results showed that lesions in the corpus callosum and cingulate gyrus were significant risk factors for cognitive decline and neuropsychiatric changes only in univariate analysis, but they were not significant independent risk factors in multivariate analysis. Brain stem and cerebellar tumor lesions were significant positive factors for cognitive function. The correlation of the KPS at diagnosis with cognitive decline and neuropsychiatric changes at onset is likely due to these changes being initially unrecognized, with the patient seeking medical attention only after other neurological symptoms appeared, leading to a further decline in KPS at the time of diagnosis. While the present study demonstrated a significant extension of PFS in the high-polyglutamylation group, OS was not significantly different between the low- and high-polyglutamylation groups. It is possible that the high-polyglutamylation group often presents with nonspecific cognitive changes, resulting in delayed diagnosis and therapeutic intervention. Actually, 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. If early therapeutic intervention is achievable in patients with highly polyglutamylated PCNSL, further improvement in OS can be anticipated. This study has several limitations. PCNSL progresses more quickly than dementia such as Alzheimer’s dementia. Therefore, even if cognitive decline and neuropsychiatric changes exist at the time of onset, symptoms such as impaired consciousness, aphasia, or paralysis may already be apparent at the time of the first medical examination, often making it difficult to conduct objective cognitive function tests. Thus, it is necessary to adopt a retrospective approach to understanding the symptoms at the time of onset based on information from family members and caregivers. As wearable AI and other technologies become more widespread in daily life, it might become possible to evaluate cognitive function over time in older adults and to detect cognitive decline and neuropsychiatric changes at an early stage. Moreover, as the tissue samples were obtained from the main tumor mass, the extent of tau spread into surrounding tissues could not be determined. Future studies utilizing tau PET or other advanced methods may offer additional insights. In conclusion, polyglutamylation-induced tau phosphorylation may contribute to cognitive decline and neuropsychiatric changes at the onset of PCNSL. Cases with these changes often show high polyglutamylation levels, which are associated with a favorable response to MTX. In cases of rapidly progressive cognitive decline, it is highly likely that the condition may be MTX-responsive PCNSL, and early therapeutic intervention is expected to improve prognosis. Therefore, it is critically important to consider PCNSL as a differential diagnosis in patients presenting with these symptoms. Declarations 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. Funding This work was supported by a Grant-in-Aid for Scientific Research [19K09485] from the Japanese Society for the Promotion of Science. Data availability All original data from this manuscript will be made available from the authors upon reasonable request. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Ethics approval 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). References Grommes C, Rubenstein JL, DeAngelis LM et al (2019) Comprehensive approach to diagnosis and treatment of newly diagnosed primary CNS lymphoma. Neuro Oncol 21:296–305 Makino K, Nakamura H, Kino T et al (2006) Rising incidence of primary central nervous system lymphoma in Kumamoto, Japan. 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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-5685172","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":400019197,"identity":"4e1b59ba-02e9-4a28-abe6-227db4d09b41","order_by":0,"name":"Yuki Takeshima","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Takeshima","suffix":""},{"id":400019198,"identity":"12b41c4a-99d5-4fb1-82a7-4c19448fd8df","order_by":1,"name":"Naoki Shinojima","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-3352-7936","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":true,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Shinojima","suffix":""},{"id":400019199,"identity":"fd1cab3d-98b8-4014-b80f-a0c05a808ec9","order_by":2,"name":"Kenji Fujimoto","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Fujimoto","suffix":""},{"id":400019200,"identity":"fa5cffc0-f979-4484-bcf0-9ec3436d2c2e","order_by":3,"name":"Daiki Yoshii","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Yoshii","suffix":""},{"id":400019201,"identity":"0f549a45-4778-4b7e-8f21-25d0102bdd8e","order_by":4,"name":"Yasushi Hayakata","email":"","orcid":"","institution":"Kumamoto University: Kumamoto Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Hayakata","suffix":""},{"id":400019202,"identity":"c3dd9450-0952-4d30-a35b-3657b1d2c016","order_by":5,"name":"Masafumi Oya","email":"","orcid":"","institution":"Kumamoto University: Kumamoto Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Masafumi","middleName":"","lastName":"Oya","suffix":""},{"id":400019203,"identity":"fa919918-955b-48c5-b840-4b3fe7b5ab68","order_by":6,"name":"Masayoshi Tasaki","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Masayoshi","middleName":"","lastName":"Tasaki","suffix":""},{"id":400019204,"identity":"8829cd2a-242a-41fe-9d81-9068424bcc57","order_by":7,"name":"Yoshiki Mikami","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Yoshiki","middleName":"","lastName":"Mikami","suffix":""},{"id":400019205,"identity":"c2d08967-b89a-476a-b886-73ee56763f7d","order_by":8,"name":"Hiroyuki Uetani","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Uetani","suffix":""},{"id":400019206,"identity":"efd09f5a-a75a-47fb-8941-51cfaf322afe","order_by":9,"name":"Toshinori Hirai","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Toshinori","middleName":"","lastName":"Hirai","suffix":""},{"id":400019207,"identity":"24d4c891-7994-4d53-83fe-0ecceb33a3d9","order_by":10,"name":"Akitake Mukasa","email":"","orcid":"","institution":"Kumamoto University Hospital: Kumamoto Daigaku Byoin","correspondingAuthor":false,"prefix":"","firstName":"Akitake","middleName":"","lastName":"Mukasa","suffix":""}],"badges":[],"createdAt":"2024-12-20 15:18:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5685172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5685172/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73703005,"identity":"bdb716c8-44f0-4b35-b714-24cdb1661bad","added_by":"auto","created_at":"2025-01-13 17:36:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5078636,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of tumor localizations between patients with and without cognitive decline and neuropsychiatric changes. \u003cstrong\u003ea\u003c/strong\u003e Black and gray bars represent the percentages of each localization in patients with and without cognitive decline and neuropsychiatric changes, respectively. \u003cstrong\u003eb\u003c/strong\u003e Representative contrast-enhanced magnetic resonance images of lesions in the corpus callosum or cingulate gyrus (upper) and lesions in the brain stem or cerebellum (lower). \u003cstrong\u003ec, d\u003c/strong\u003e Kaplan–Meier curves for (\u003cstrong\u003ec\u003c/strong\u003e) progression-free survival and (\u003cstrong\u003ed\u003c/strong\u003e) overall survival based on the polyglutamylation status in 145 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":"Fig.2241220.png","url":"https://assets-eu.researchsquare.com/files/rs-5685172/v1/e8f5bd16298aa2889054afca.png"},{"id":73703006,"identity":"5df424c7-905f-445c-9561-c406ffe4405a","added_by":"auto","created_at":"2025-01-13 17:36:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1190873,"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% DMSO (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. \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":"Fig1241220.png","url":"https://assets-eu.researchsquare.com/files/rs-5685172/v1/97cbe6ae06dd3886cef1c8a9.png"},{"id":77882876,"identity":"b25393ca-f3fc-4c78-b8f2-dad4bde08946","added_by":"auto","created_at":"2025-03-06 12:32:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6871234,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5685172/v1/4e83eba6-8be9-4755-847d-f513eae9b2b3.pdf"},{"id":73703016,"identity":"24d89d39-4130-41c2-926a-4931634f1b9a","added_by":"auto","created_at":"2025-01-13 17:36:55","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":24881,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable12241220.docx","url":"https://assets-eu.researchsquare.com/files/rs-5685172/v1/2209400e2c51c0e2772638fb.docx"}],"financialInterests":"","formattedTitle":"Elevated polyglutamylation and tau phosphorylation levels in patients with primary central nervous system lymphoma are related to cognitive decline and neuropsychiatric changes at disease onset","fulltext":[{"header":"Introduction","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. On the other hand, PCNSL is a potential cause of rapidly progressive dementias [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and approximately 40% of patients experience nonspecific cognitive decline and neuropsychiatric changes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], delaying diagnosis and treatment and resulting in poor prognosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, 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=\"CR8\" class=\"CitationRef\"\u003e8\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=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Beyond cognitive deficits, tauopathies can manifest with a range of symptoms, including motor dysfunction and neuropsychiatric abnormalities [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\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=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, hyperglutamylation of microtubules in neurons has been implicated in neurodegeneration [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, a recent study has demonstrated that C-terminal-specific tubulin polyglutamylation is crucial for the aggregation of hyperphosphorylated tau, leading to neuronal dysfunction in tauopathy models [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe previously reported that polyglutamylation levels in tumor specimens were high in more than half of the patients with PCNSL and were associated with responsiveness to methotrexate (MTX)-based chemotherapy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While these clinical benefits are evident, we hypothesized that polyglutamylation may play a role in the cognitive decline and neuropsychiatric changes observed in the early stages of PCNSL, potentially through the accumulation of phosphorylated tau. 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 their potential correlation with clinical symptoms such as cognitive decline and neuropsychiatric changes. 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. 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=\"CR18\" class=\"CitationRef\"\u003e18\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=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Cases in which tissue volume was not sufficient for immunohistological evaluation were excluded. Lesion locations were defined based on contrast enhancements 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 lesions was calculated by adding the number of locations. In cases of multiple lesions, histopathologic diagnoses were based on tissue samples from contrast-enhanced areas if a sufficient sample volume was safely available [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePatient evaluation of cognitive decline and neuropsychiatric changes\u003c/p\u003e \u003cp\u003e We conducted a retrospective evaluation of cognitive decline and neuropsychiatric changes at symptom onset using detailed chart reviews and clinical episode documentation provided by family members and caregivers, following the criteria described below, and categorized the patients into groups with or without cognitive decline and neuropsychiatric changes.\u003c/p\u003e \u003cp\u003eCognitive decline at onset, defined according to the criteria for all-cause dementia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], was identified as symptoms interfering with work or daily activities, representing a decline from previous levels of functioning or performance. Such symptoms included any of the following: impaired ability to acquire and remember new information, decreased ability to reason or handle complex tasks and judgments, impaired visuospatial abilities, or dysfunction in language skills (speaking, reading, or writing).\u003c/p\u003e \u003cp\u003eNeuropsychiatric changes were assessed based on the presence of symptoms across the domains evaluated by the Neuropsychiatric Inventory (NPI) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which include delusions, hallucinations, agitation/aggression, depression/dysphoria, anxiety, euphoria/elation, apathy/indifference, disinhibition, irritability/lability, aberrant motor behavior, sleep and nighttime behavior disturbances, and appetite and eating changes. The presence of at least one symptom in any of these domains was considered indicative of neuropsychiatric changes.\u003c/p\u003e \u003cp\u003eSurvival analysis\u003c/p\u003e \u003cp\u003eAs previously reported [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], overall survival (OS) was defined as the time from initial diagnosis to death, whereas progression-free survival (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 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=\"CR17\" class=\"CitationRef\"\u003e17\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-polyglutamylated group, and those with \u0026lt;\u0026thinsp;10% comprised the low-polyglutamylated group [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\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=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\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 (DMSO). 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=\"CR22\" class=\"CitationRef\"\u003e22\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.\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=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Between-group differences in numerical values were tested using Student\u0026rsquo;s t-test and 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. 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":"\u003cp\u003eClinical study\u003c/p\u003e\n\u003cp\u003eOf 207 consecutive patients with PCNSL, 145 patients met the inclusion criteria of this study. The clinical characteristics of the 145 patients with PCNSL are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age was 68 (range: 41\u0026ndash;92) years, the study population comprised 75 men and 70 women, and the median Karnofsky performance status (KPS) score was 50 (range: 20\u0026ndash;100) at the time of diagnosis. Of the 145 patients with PCNSL, 143 were diagnosed with DLBCL, one with Burkitt lymphoma, and one with low-grade B-cell lymphoma. In 143 DLBCLs, the cell of origin could be assessed, and 100 (69.0%) were categorized as non-GCB type. Among the 145 patients with PCNSL, 87 (60.0%) showed high levels of polyglutamylation. Multiple lesions were observed in 92 patients (63.4%).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics of the enrolled patients with PCNSL\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOverall\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;145\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68 (41\u0026ndash;92)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75/70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKPS score (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCell of origin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGCB/non-GCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43/100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDH (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh/normal range\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49/96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolyglutamylation (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh/low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87/58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of lesions (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSingle/multiple\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53/92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLocation of the lesions (Right/left, # of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFrontal lobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37/39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTemporal lobe (including the hippocampus)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16/10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTemporoparietal lobe (excluding the hippocampus)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOccipital lobe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBasal ganglia and/or thalamus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42/34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCorpus callosum and/or cingulate gyrus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVentricle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrain stem and/or cerebellum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38\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\u003c/div\u003e\n\u003cp\u003eNext, the clinical characteristics were compared regarding the presence or absence of cognitive decline and neuropsychiatric changes at symptom onset (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Cognitive decline and neuropsychiatric changes at the onset were observed in 70 patients (48.3%). The groups with and without cognitive decline and neuropsychiatric changes significantly differed in age (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00049), KPS score (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035), and polyglutamylation status (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of the clinical characteristics between patients with and without cognitive decline and neuropsychiatric changes as the initial symptoms\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eCognitive decline and neuropsychiatric changes at disease onset\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71 (41\u0026ndash;85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66 (43\u0026ndash;92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00049\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34/36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41/34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKPS score (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMedian (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 (20\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCell of origin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGCB/non-GCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24/51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDH (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh/normal range\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25/45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24/51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolyglutamylation (# of pts)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh/low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55/15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32/43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\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\u003c/div\u003e\n\u003cp\u003eA comparison of each tumor localization is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea and Supplementary Table\u0026nbsp;2. The group with cognitive decline and neuropsychiatric changes had significantly more cases with multiple lesions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026) and with corpus callosum and cingulate gyrus lesions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0018) than the group without such changes. Conversely, brain stem and cerebellar lesions were significantly more common (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0023) in the group without cognitive decline and neuropsychiatric changes at disease onset (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Furthermore, multivariate analysis confirmed polyglutamylation as a significant independent risk factor for cognitive decline and neuropsychiatric changes as the initial symptoms (odds ratio [OR] 7.34, 95% confidence interval [CI] 2.99\u0026ndash;18.0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that in addition to aging and lower KPS score, polyglutamylation affects cognitive decline and neuropsychiatric status at symptom onset in PCNSL.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMultivariate logistic regression analysis of the predictors of cognitive decline and neuropsychiatric changes as the initial symptoms\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.01\u0026ndash;1.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKPS score (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.977\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.958\u0026ndash;0.998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolyglutamylation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.99\u0026ndash;18.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of lesions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.619\u0026ndash;3.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCorpus callosum and/or cingulate gyrus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.756\u0026ndash;4.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBrain stem and/or cerebellum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0869\u0026ndash;0.644\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0048\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\u003c/div\u003e\n\u003cp\u003eNext, we compared survival times between the high- and low-polyglutamylation groups. Consistent with our previous reports, the high-polyglutamylation group showed significantly longer PFS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0055), whereas no difference in OS was observed (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.15, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, d).\u003c/p\u003e\n\u003cp\u003eHistopathology and immunohistochemistry\u003c/p\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 [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Surprisingly, the expression of total and phosphorylated tau coincided with that of polyglutamylation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). We statistically compared the expression of tau and phosphorylated tau between the low- and high-polyglutamylated 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-polyglutamylated group than in the low-polyglutamylated group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Although phosphorylated tau was expressed, no \u0026beta;-amyloid deposits were observed in the samples of patients with PCNSL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eElevated polyglutamylation levels increase the levels of phosphorylated tau in PCNSL cells\u003c/p\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 [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. We treated cells with the HDACI NaBu to increase the polyglutamylation level by upregulating FPGS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed).\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 induced increased expression of both FPGS and phosphorylated tau in two different cell lines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, e).\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 [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. In contrast, protein phosphatase 2A (PP2A) [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] and peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] 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. Interestingly, 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.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef). 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 considerably involved in the association of polyglutamylation with cognitive decline and neuropsychiatric changes in patients with PCNSL.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we found for the first time that in patients with PCNSL, highly polyglutamylated tumors are involved in cognitive decline and neuropsychiatric changes at symptom onset. Furthermore, our data suggest that the cause of these symptoms may be the accumulation of phosphorylated tau due to high levels of polyglutamylation. Additionally, analysis of PCNSL tumor samples and PCNSL cell lines revealed a positive correlation between polyglutamylation levels and phosphorylated tau expression, suggesting that polyglutamylation may regulate the expression of phosphorylated tau. Few studies have investigated the relationship between polyglutamylation and tau expression. Hausrat et al. recently demonstrated in a mouse model that polyglutamylation plays a key role in tauopathy caused by the accumulation of phosphorylated tau [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Polyglutamylation is deeply involved in the regulation of tau phosphorylation and accumulation, but the details of the regulation of polyglutamylation itself are unknown. As we previously reported, polyglutamylation occurs at a high rate in the presence of HDACIs, suggesting that epigenetics may be involved [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTau, one of the microtubule-associate proteins, regulates microtubule assembly and stabilization [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Tau phosphorylation reduces the affinity of tau and causes its detachment from microtubules, resulting in the formation of hyperphosphorylated tau aggregation, which can potentially activate microglia and induce gliosis and neurodegeneration [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The question is how tau, which is present in highly polyglutamylated tumor cells in PCNSL, reaches neurons and affects cognitive function in patients. 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 [\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A similar pathological process can occur in PCNSL, where tau is shed from tumor cells and affects neurons. Tau positron emission tomography (PET) may be useful in assessing the phosphorylated tau status in the whole brain [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the localization of lesions, a report demonstrated that neuropsychological changes in PCNSL correlate with diffuse involvement of the periventricular white matter or corpus callosum [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, our results showed that lesions in the corpus callosum and cingulate gyrus were significant risk factors for cognitive decline and neuropsychiatric changes only in univariate analysis, but they were not significant independent risk factors in multivariate analysis. Brain stem and cerebellar tumor lesions were significant positive factors for cognitive function.\u003c/p\u003e \u003cp\u003eThe correlation of the KPS at diagnosis with cognitive decline and neuropsychiatric changes at onset is likely due to these changes being initially unrecognized, with the patient seeking medical attention only after other neurological symptoms appeared, leading to a further decline in KPS at the time of diagnosis.\u003c/p\u003e \u003cp\u003eWhile the present study demonstrated a significant extension of PFS in the high-polyglutamylation group, OS was not significantly different between the low- and high-polyglutamylation groups. It is possible that the high-polyglutamylation group often presents with nonspecific cognitive changes, resulting in delayed diagnosis and therapeutic intervention. Actually, 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. If early therapeutic intervention is achievable in patients with highly polyglutamylated PCNSL, further improvement in OS can be anticipated.\u003c/p\u003e \u003cp\u003eThis study has several limitations. PCNSL progresses more quickly than dementia such as Alzheimer\u0026rsquo;s dementia. Therefore, even if cognitive decline and neuropsychiatric changes exist at the time of onset, symptoms such as impaired consciousness, aphasia, or paralysis may already be apparent at the time of the first medical examination, often making it difficult to conduct objective cognitive function tests. Thus, it is necessary to adopt a retrospective approach to understanding the symptoms at the time of onset based on information from family members and caregivers. As wearable AI and other technologies become more widespread in daily life, it might become possible to evaluate cognitive function over time in older adults and to detect cognitive decline and neuropsychiatric changes at an early stage. Moreover, as the tissue samples were obtained from the main tumor mass, the extent of tau spread into surrounding tissues could not be determined. Future studies utilizing tau PET or other advanced methods may offer additional insights.\u003c/p\u003e \u003cp\u003eIn conclusion, polyglutamylation-induced tau phosphorylation may contribute to cognitive decline and neuropsychiatric changes at the onset of PCNSL. Cases with these changes often show high polyglutamylation levels, which are associated with a favorable response to MTX. In cases of rapidly progressive cognitive decline, it is highly likely that the condition may be MTX-responsive PCNSL, and early therapeutic intervention is expected to improve prognosis. Therefore, it is critically important to consider PCNSL as a differential diagnosis in patients presenting with these symptoms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor contributions\u003c/h2\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\u003ch2\u003eAcknowledgements\u003c/h2\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\n\u003ch2\u003eFunding\u003c/h2\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\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eAll original data from this manuscript will be made available from the authors upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\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"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGrommes C, Rubenstein JL, DeAngelis LM et al (2019) Comprehensive approach to diagnosis and treatment of newly diagnosed primary CNS lymphoma. Neuro Oncol 21:296\u0026ndash;305\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMakino K, Nakamura H, Kino T et al (2006) Rising incidence of primary central nervous system lymphoma in Kumamoto, Japan. Surg Neurol 66:503\u0026ndash;506\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura H, Makino K, Yano S et al (2011) Epidemiological study of primary intracranial tumors: a regional survey in Kumamoto prefecture in southern Japan\u0026mdash;20-year study. 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Nat Med 28:2381\u0026ndash;2387\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK\u0026uuml;ker W, N\u0026auml;gele T, Korfel A et al (2005) Primary central nervous system lymphomas (PCNSL): MRI features at presentation in 100 patients. J Neurooncol 72:169\u0026ndash;177\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polyglutamylation, Cognitive decline, Primary central nervous system lymphoma, Tau protein","lastPublishedDoi":"10.21203/rs.3.rs-5685172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5685172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrimary central nervous system lymphoma (PCNSL) often presents with nonspecific cognitive decline and neuropsychiatric changes at onset, yet the underlying mechanisms remain poorly understood. Polyglutamylation, a posttranslational modification, is associated with better responses to methotrexate-based chemotherapy in patients with PCNSL. On the other hand, hyperglutamylation of microtubules in neurons has been implicated in neurodegeneration via the accumulation of phosphorylated tau. This study aimed to elucidate the relationship of polyglutamylation with cognitive decline and neuropsychiatric changes at PCNSL onset. We retrospectively analyzed 145 patients with PCNSL treated at our institution between 2001 and 2022. Polyglutamylation and phosphorylated tau were evaluated using immunohistochemistry. In vitro studies were performed to evaluate the causal relationship between polyglutamylation and tau phosphorylation in PCNSL cell lines. Cognitive decline and neuropsychiatric changes at disease onset were observed in 48.3% of patients, with multivariate analysis identifying high polyglutamylation levels as an independent risk factor (odds ratio: 7.34, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Patient samples and cell lines consistently demonstrated a link between polyglutamylation and tau phosphorylation, implicating that polyglutamylation-induced tau phosphorylation may contribute to cognitive decline and neuropsychiatric changes. Our findings suggest that polyglutamylation contributes to neurocognitive dysfunction in PCNSL, and these results elucidate a novel aspect of PCNSL pathophysiology.\u003c/p\u003e","manuscriptTitle":"Elevated polyglutamylation and tau phosphorylation levels in patients with primary central nervous system lymphoma are related to cognitive decline and neuropsychiatric changes at disease onset","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 17:36:49","doi":"10.21203/rs.3.rs-5685172/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fe0edd32-6907-4173-9023-b4284fc65fce","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-06T12:23:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-13 17:36:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5685172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5685172","identity":"rs-5685172","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}