Long-term survivors of glioblastoma: Tumor molecular, clinical, and imaging findings.

Background: Glioblastoma (GBM) is the most aggressive primary brain malignancy with <45% living a year beyond diagnosis. Previously published investigations of long-term survivors (LTS) provided clinical data but rarely incorporated a comprehensive clinical and molecular analysis. Herein, we identify clinical, imaging, molecular, and outcome features for 23 GBM-LTS patients and compare them with a matched cohort of short-term survivors (STS). Methods: Molecularly confirmed Isocitrate Dehydrogenase (IDH) wildtype GBM patients living ≥3 years post-diagnosis (NLTS = 23) or <3 years (NSTS = 75) were identified from our Natural History study. Clinical and demographic characteristics were compared. Tumor tissue was analyzed with targeted next generation sequencing (NGS) (NLTS = 23; NSTS = 74) and methylation analysis (NLTS = 18; NSTS = 28). Pre-surgical MRI scans for a subset of LTS (N = 14) and STS control (N = 28) matched on sex, age, and extent of resection were analyzed. Results: LTS tended to be younger. Diagnostic MRIs showed more LTS with T1 tumor hypointensity. LTS tumors were enriched for MGMTp methylation and tumor protein 53 (TP53) mutation. Three patients with classic GBM histology were reclassified based on NGS and methylation testing. Additionally, there were LTS with typical poor prognostic molecular markers. Conclusions: Our findings emphasize that generalized predictions of prognosis are inaccurate for individual patients and underscore the need for complete clinical evaluation including molecular work-up to confirm the diagnosis. Continued accrual of patients to LTS registries that containcomprehensive clinical, imaging, tumor molecular data, and outcomes measures may pro\vide important insights about individual patient prognosis.

Feasibility of a virtual reality intervention targeting distress and anxiety symptoms in patients with primary brain tumors: Interim analysis of a phase 2 clinical trial.

PURPOSE: Cancer patients experience distress and anxiety when undergoing imaging studies to monitor disease status, yet these symptoms are not always appropriately identified or well-managed. This interim analysis of a phase 2 clinical trial explored feasibility and acceptability of a virtual reality relaxation (VR) intervention for primary brain tumor (PBT) patients at the time of clinical evaluation. METHODS: English speaking, adult PBT patients with previous reports of distress and upcoming neuroimaging were recruited between March of 2021 and March 2022. A brief VR session was done within 2 weeks prior to neuroimaging with patient-reported outcomes (PROs) collected before and immediately post-intervention. Self-directed VR use over the next 1 month was encouraged with additional PROs assessments at 1 and 4 weeks. Feasibility metrics included enrollment, eligibility, attrition, and device-related adverse effects with satisfaction measured with qualitative phone interviews. RESULTS: Fifty-five patients were approached via email, 40 (73%) responded and 20 (50%) enrolled (9 declines, 11 screen fails). 65% of participants were ≤ 50 years, 50% were male, 90% were White/non-Hispanic, 85% had good KPS (≥ 90), and most were on active treatment. All patients completed the VR intervention, PROs questionnaires, weekly check-ins, and qualitative interview. Most (90%) reported frequent VR use and high satisfaction and only 7 mild AEs were recorded (headache, dizziness, nausea, neck pain). CONCLUSION: This interim analysis supports feasibility and acceptability of a novel VR intervention to target psychological symptoms for PBT patients. Trial enrollment will continue to assess for intervention efficacy. TRIAL REGISTRATION: NCT04301089 registered on 3/9/2020.

Heterogeneous clinicopathological findings and patient-reported outcomes in adults with MN1-altered CNS tumors: A case report and systematic literature review.

The uncommon MN1-altered primary central nervous system (CNS) tumors were recently added to the World Health Organization 2021 classification under the name Astroblastoma, MN1-altered. Another term used to describe them, "High-grade neuroepithelial tumor with MN1 alteration" (HGNET-MN1), makes reference to their distinct epigenetic profile but is currently not a recommended name. Thought to occur most commonly in children and predominantly in females, MN1-altered CNS tumors are associated with typical but not pathognomonic histological patterns and are characterized by a distinct DNA methylation profile and recurrent fusions implicating the MN1 (meningioma 1) gene. Diagnosis based on histological features alone is challenging: most cases with morphological features of astroblastoma (but not all) show these molecular features, whereas not all tumors with MN1 fusions show astroblastoma morphology. There is large variability in reported outcomes and detailed clinical and therapeutic information is frequently missing. Some patients experience multiple recurrences despite multimodality treatment, whereas others experience no recurrence after surgical resection alone, suggesting large clinical and biological heterogeneity despite unifying epigenetic features and recurrent fusions. In this report, we present the demographics, tumor characteristics, treatment, and outcome (including patient-reported outcomes) of three adults with MN1-altered primary CNS tumors diagnosed via genome-wide DNA methylation and RNA sequencing. All three patients were females and two of them were diagnosed as young adults. By reporting our neuropathological and clinical findings and comparing them with previously published cases we provide insight into the clinical heterogeneity of this tumor. Additionally, we propose a model for prospective, comprehensive, and systematic collection of clinical data in addition to neuropathological data, including standardized patient-reported outcomes.

Association of Employment Status With Symptom Burden and Health-Related Quality of Life in People Living With Primary CNS Tumors.

BACKGROUND AND OBJECTIVES: Financial toxicity significantly affects many patients, especially cancer survivors. We evaluated the association of unemployment as a major contributor to financial toxicity with patient-reported outcomes (PROs) assessing multiple illness experience domains in a primary CNS tumor (PCNST) cohort. METHODS: Patient and disease characteristics and PROs measuring symptom burden, interference, psychologic distress, functional impairment, and health-related quality of life (HRQOL) from participants enrolled in an institutional review board-approved observational study at the US NIH's Neuro-Oncology Branch were collected between September 2016 and December 2019. Descriptive statistics, tests of association, and comparison of group mean values were used to describe and evaluate PROs. RESULTS: Of the 277 participants diagnosed with a PCNST, 57% were male and 43% were female. Participants reported their race as White, non-Hispanic (78%); White, Hispanic/Latino (9%); Asian (7%); Black (4%); Native Hawaiian/Pacific Islander (1%); and other (2%) with 8% missing. The median age of the overall cohort was 45 years (range 18-74). Hispanic participants in the overall sample were 2.3 times more likely, and in the brain tumor group 3.2 times more likely, to report unemployment (p = 0.043, odds ratio [OR] 2.3, 95% CI 1.0-5.4 and p = 0.008, OR 3.2, 95% CI 1.3-7.9, respectively). 77 (28%) individuals unemployed due to tumor reported more functional impairment with walking, washing, dressing, and performing usual activities and reduced HRQOL (p < 0.001). More unemployed participants in the total sample reported moderate-to-severe depressive symptoms (25%) than those employed (8%) (χ2(1) = 13.9, p < 0.001, OR 3.7, 95% CI 1.8-7.8) and more moderate-to-severe anxiety symptoms (30%) than those employed (15%) (χ2(1) = 7.8, p = 0.005, OR 2.4, 95% CI 1.3-4.5). Unemployed participants with brain tumor reported on average 3 more symptoms as moderate-to-severe compared with those employed (t(83) = -4.0, 95% CI [Formula: see text] difference -5 to -2, p < 0.001, Hedge g = 0.70). DISCUSSION: Being unemployed due to a PCNST strongly correlated with high symptom burden, functional impairment, psychological distress, and reduced HRQOL, which may be impediments to returning to work that warrant intervention. Lack of employer-based health insurance and reduced earnings are financial sequelae of unemployment superimposed on the physical, social, and cognitive effects of living with a PCNST. Innovations to screen for and address financial toxicity and its contributing factors are needed.

Feasibility of a virtual reality intervention targeting distress and anxiety symptoms in patients with primary brain tumors: Interim analysis of a phase 2 clinical trial.

Purpose: Cancer patients experience distress and anxiety when undergoing imaging studies to monitor disease status, yet these symptoms are not always appropriately identified or well-managed. This interim analysis of a phase 2 clinical trial explored feasibility and acceptability of a virtual reality relaxation (VR) intervention for primary brain tumor (PBT) patients at the time of clinical evaluation. Methods: English speaking, adult PBT patients with previous reports of distress and upcoming neuroimaging were recruited between March of 2021 and March 2022. A brief VR session was done within 2 weeks prior to neuroimaging with patient-reported outcomes (PROs) collected before and immediately post-intervention. Self-directed VR use over the next 1 month was encouraged with additional PROs assessments at 1 and 4 weeks. Feasibility metrics included enrollment, eligibility, attrition, and device-related adverse effects with satisfaction measured with qualitative phone interviews. Results: 55 patients were approached via email, 40 (73%) responded and 20 (50%) enrolled (9 declines, 11 screen fails). 65% of participants were ≤ 50 years, 50% were male, 90% were White/non-Hispanic, 85% had good KPS (≥ 90), and most were on active treatment. All patients completed the VR intervention, PROs questionnaires, weekly check-ins, and qualitative interview. Most (90%) reported frequent VR use and high satisfaction and only 7 mild AEs were recorded (headache, dizziness, nausea, neck pain). Conclusion: This interim analysis confirmed feasibility and acceptability of a novel VR intervention to target psychological symptoms for PBT patients. Trial enrollment will continue to assess for intervention efficacy. Trial Registration: NCT04301089 registered on 3/9/2020.

Relationship between RANO-PRO Working Group standardised priority constructs and disease progression among malignant glioma patients: A retrospective cohort study.

Background: Recognising the importance of clinical outcomes assessments (COAs), the Response Assessment in Neuro-Oncology-Patient Reported Outcome (RANO-PRO) Working Group recommended inclusion of core symptoms and functions in clinical care or research for malignant glioma patients. This study evaluated the association of the recommended symptoms (pain, perceived cognition, seizures, aphasia, symptomatic adverse events) and functions (weakness, walking, work, usual activities) with disease progression in these patients. Methods: In this retrospective cohort study, patients with malignant glioma were included from the US National Cancer Institute Neuro-Oncology Branch Natural History Study (NOB-NHS) which follows primary central nervous system tumour patients aged 18 years and older throughout their disease trajectory. The M.D. Anderson Symptom Inventory-Brain Tumor (MDASI-BT), EQ-5D-3L, Karnofsky Performance Status (KPS), and Neurologic Function scores (NFS) were evaluated in relation to disease progression by chi-square tests, independent- and paired-samples t-tests, adjusted for multiple comparisons at first assessment and over time to a second assessment. Radiographic disease progression was determined on the interpretation of the imaging study by a radiologist and neuro-oncologist using standard criteria as part of clinical trial participation or routine standard of care. The priority constructs were evaluated to provide initial evidence of their relevance, relationship to disease status over time, and sensitivity to change in a diverse group of patients with malignant glioma. Findings: Seven hundred and sixty-five patients had enrolled into the NOB-NHS between September 1, 2016 and January 31, 2020. Three hundred and thirty-six patients had a diagnosis of a malignant glioma (anaplastic astrocytoma, anaplastic oligodendroglioma, glioblastoma, and gliosarcoma) and were included in the current study. The sample was 64% male (n = 215), 36% female (n = 121), median age of 52 years (IQR = 18.75), 82% White (n = 276), and 65% had tumour recurrence (n = 219). One hundred and fifty-four (46%) had radiographic disease progression. Difficulty remembering, fatigue, and weakness were worse in the group whose imaging was interpreted as radiographic disease progression versus stable disease, as well as the functions of walking, work, activity, and self-care (1.1 < difference < 1.8). Patients with disease progression were four times more likely to have a poor KPS (≤80) and worse NFS. Among patients with disease progression at a second assessment (n = 112), all symptoms, except seizures, worsened between first assessment and disease progression and up to 22% of patients (n = 25) reported worsening mobility, self-care, and usual activity; 46% (n = 51) and 35% (n = 30) had worsened KPS and NFS, respectively. On average, 4 symptoms or functions (SD = 3) were reported as moderate-to-severe and 30% (n = 33) and 23% (n = 26) had a change to moderate-to-severe fatigue and walking, respectively, at time of disease progression. Over 7% of patients with worsening (n = 7 of 100) reported every symptom and function as having changed the most severely including seizures with fatigue and activity reported as the top symptom and function, respectively. Interpretation: The identified core symptoms and functions worsened at the time of progression, supporting the relevance and sensitivity of the priority constructs identified by the RANO-PRO Working Group for clinical care and clinical trials for malignant glioma patients. Funding: The Natural History Study is supported by Intramural Project 1ZIABC011786-03.

Exploring the prevalence and burden of sleep disturbance in primary brain tumor patients.

Background: Sleep disturbance (SD) is common in patients with cancer and has been associated with worse clinical outcomes. This cross-sectional study explored the prevalence of SD in a primary brain tumor (PBT) population, identified associated demographic and clinical characteristics, and investigated co-occurrence of SD with other symptoms and mood disturbance. Methods: Demographic, clinical characteristics, MD Anderson Symptom Inventory-Brain Tumor, and Patient Reported Outcome Measurement Information System Depression and Anxiety Short-Forms were collected from PBT patients at study entry. Descriptive statistics, Chi-square tests, and independent t-tests were used to report results. Results: The sample included 424 patients (58% male, 81% Caucasian) with a mean age of 49 years (range 18-81) and 58% with high-grade gliomas. Moderate-severe SD was reported in 19% of patients and was associated with younger age, poor Karnofsky Performance Status, tumor progression on MRI, and active corticosteroid use. Those with moderate-severe SD had higher overall symptom burden and reported more moderate-severe symptoms. These individuals also reported higher severity in affective and mood disturbance domains, with 3 to 4 times higher prevalence of depressive and anxiety symptoms, respectively. The most frequently co-occurring symptoms with SD were, drowsiness, and distress, though other symptoms typically associated with tumor progression also frequently co-occurred. Conclusions: PBT patients with moderate-severe SD are more symptomatic, have worse mood disturbance, and have several co-occurring symptoms. Targeting interventions for sleep could potentially alleviate other co-occurring symptoms, which may improve life quality for PBT patients. Future longitudinal work examining objective and detailed subjective sleep reports, as well as underlying genetic risk factors, will be important.

The Montreal Cognitive Assessment (MoCA) in neuro-oncology: A pilot study of feasibility and utility in telehealth and in-person clinical assessments.

Background: Cognitive impairments are a common burden for patients with primary CNS tumors. Neuropsychological assessment batteries can be too lengthy, which limits their use as an objective measure of cognition during routine care. The purpose of this study was to evaluate the feasibility and utility of the brief Montreal Cognitive Assessment (MoCA) in routine in-person and telehealth visits (as a result of the global COVID-19 pandemic) with neuro-oncology patients. Methods: Seventy-one adults with primary CNS tumors completed MoCA testing in person (n = 47) and via telehealth (n = 24). Correlation analysis and patient-reported outcomes (PROs), including symptom burden and interference, perceived cognition, general health status, and anxiety and depression, were included in this study. Feasibility was assessed through a provider satisfaction questionnaire. Results: Patients were primarily White (83%), college-educated (71%) males (54%) with high-grade tumors (66%). The average total score on the MoCA administered in person was 25 (range: 6-30), with 34% classified as abnormal, and the average total score via telehealth was 26 (range: 12-30), with 29% classified as abnormal. Providers reported satisfaction in using the MoCA during routine clinical care, both in person and via telehealth. Lower MoCA scores correlated with worse symptom severity, KPS, age, education, and previous treatment. Conclusions: The MoCA was feasible in clinical and telehealth settings, and its relationship to clinical characteristics and PROs highlights the need for both objective and patient-reported measures of cognition to understand the overall cognitive profile of a patient with a CNS tumor.

Evaluation of the key geriatric assessment constructs in primary brain tumor population - a descriptive study.

INTRODUCTION: Despite an increasing aging population, older adults (≥ 65 years) with primary brain tumors (PBTs) are not routinely assessed for geriatric vulnerabilities. Recent reports of geriatric assessment (GA) in patients with glioblastomas demonstrated that GA may serve as a sensitive prognosticator of overall survival. Yet, current practice does not include routine evaluation of geriatric vulnerabilities and the relevance of GA has not been previously evaluated in broader cohorts of PBT patients. The objective of this descriptive study was to assess key GA constructs in adults with PBT dichotomized into older versus younger groups. MATERIALS AND METHODS: A cross-sectional analysis of data collected from 579 participants with PBT recruited between 2016 and 2020, dichotomized into older (≥ 65 years, n = 92) and younger (≤ 64 years, n = 487) from an ongoing observational trial. GA constructs were evaluated using socio-demographic characteristics, Charlson Comorbidity Index (CCI), polypharmacy (>5 daily medications), Karnofsky Performance Status (KPS), Neurologic Function Score (NFS), and patient-reported outcome assessments including general health, functional status, symptom burden and interference, and mood. Descriptive statistics, t-tests, chi-square tests, and Pearson correlations were used to evaluate differences between age groups. RESULTS: Older participants were more likely to have problems with mobility (58% vs. 44%), usual activities (64% vs 50%) and self-care (38% vs 26%) compared to the younger participants (odds ratios [ORs] = 1.3-1.4, ps < 0.05), while older participants were less likely to report feeling distressed (OR = 0.4, p < 0.05). Older participants also had higher CCI and were more likely to have polypharmacy (OR = 1.7, ps < 0.05). Increasing age strongly correlated with worse KPS score (r = -0.232, OR = 1.4, p < 0.001) and worse NFS (r = 0.210, OR = 1.5, p < 0.001). No differences were observed in overall symptom burden, symptom interference, and anxiety/depression scores. DISCUSSION: While commonly used GA tools were not available, the study employed patient- and clinician-reported outcomes to identify potential future research directions for the use of GA in the broader neuro-oncology population. Findings illustrate missed opportunities in neuro-oncology practice and underscore the need for incorporation of GA into routine care of this population. Future studies are warranted to further evaluate the prognostic utility of GA and to better understand functional aging outcomes in this patient population.

Living with a central nervous system (CNS) tumor: findings on long-term survivorship from the NIH Natural History Study.

BACKGROUND: Primary central nervous system (CNS) tumors are often associated with high symptom burden and a poor prognosis from the time of diagnosis. The purpose of this study is to describe patient-reported outcomes (PRO) data from long-term survivors (LTS; ≥5-year survival post-diagnosis). METHODS: Clinical/treatment/molecular characteristics and PROs (symptom burden/interference (MDASI-BT/SP), perceived cognition (Neuro-QoL), anxiety/depression (PROMIS), and general health status (EQ-5D-3L)) were collected on 248 adult LTS between 9/2016 and 8/2019. Descriptive statistics and regression analysis were used to report results. RESULTS: Participants had a median age of 47 years (19-82) and were primarily White (83%) males (51%) with high-grade tumors (59%) and few mutations. Forty-two percent of the 222 brain tumor LTS reported no moderate-to-severe symptoms, whereas 45% reported three or more; most common symptoms were fatigue (40%), difficulty remembering (29%), and drowsiness (28%). Among spine tumor LTS (n = 42), nearly half reported moderate-to-severe weakness, pain, fatigue, and numbness/tingling, with 72% experiencing activity-related interference. Severe anxiety, depression, and cognitive symptoms were reported in up to 23% of the sample. Brain tumor LTS at higher risk for severe symptoms were more likely to be young, unemployed, and have poor KPS (Karnofsky Performance Status), whereas high symptom-risk spinal cord tumor LTS had poor KPS and received any tumor treatment. CONCLUSIONS: Findings indicate LTS fall into distinct cohorts with no significant symptoms or very high symptom burden, regardless of tumor grade or mutational profile. These LTS data demonstrate the need for survivorship care programs and future studies to explore the symptom trajectory of all CNS tumor patients for prevention and early interventions.

Phase I Study of Zotiraciclib in Combination with Temozolomide for Patients with Recurrent High-grade Astrocytomas.

PURPOSE: To investigate the toxicity profile and establish an optimal dosing schedule of zotiraciclib with temozolomide in patients with recurrent high-grade astrocytoma. PATIENTS AND METHODS: This two-stage phase I trial determined the MTD of zotiraciclib combined with either dose-dense (Arm1) or metronomic (Arm2) temozolomide using a Bayesian Optimal Interval design; then a randomized cohort expansion compared the progression-free survival rate at 4 months (PFS4) of the two arms for an efficient determination of a temozolomide schedule to combine with zotiraciclib at MTD. Pharmacokinetic and pharmacogenomic profiling were included. Patient-reported outcome was evaluated by longitudinal symptom burden. RESULTS: Fifty-three patients were enrolled. Dose-limiting toxicities were neutropenia, diarrhea, elevated liver enzymes, and fatigue. MTD of zotiraciclib was 250 mg in both arms and thus selected for the cohort expansion. Dose-dense temozolomide plus zotiraciclib (PSF4 40%) compared favorably with metronomic temozolomide (PFS4 25%). Symptom burden worsened at cycle 2 but stabilized by cycle 4 in both arms. A significant decrease in absolute neutrophil count and neutrophil reactive oxygen species production occurred 12-24 hours after an oral dose of zotiraciclib but both recovered by 72 hours. Pharmacokinetic/pharmacogenomic analyses revealed that the CYP1A2_5347T>C (rs2470890) polymorphism was associated with higher AUCinf value. CONCLUSIONS: Zotiraciclib combined with temozolomide is safe in patients with recurrent high-grade astrocytomas. Zotiraciclib-induced neutropenia can be profound but mostly transient, warranting close monitoring rather than treatment discontinuation. Once validated, polymorphisms predicting drug metabolism may allow personalized dosing of zotiraciclib.

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism.

The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, ß-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function.

A real-time method for measuring cAMP production modulated by Gαi/o-coupled metabotropic glutamate receptors.

Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.

Ligand bias at metabotropic glutamate 1a receptors: molecular determinants that distinguish ß-arrestin-mediated from G protein-mediated signaling.

The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ß-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the ß-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for ß-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward ß-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate-induced PI hydrolysis. Taken together, these data indicate that ligand bias at mGlu1 receptors is attributable to different modes of receptor-glutamate interactions, which are differentially coupled to PI hydrolysis and ß-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.

Heat shock enhances CMV-IE promoter-driven metabotropic glutamate receptor expression and toxicity in transfected cells.

In CHO-K1 cells, heat shock strongly activated reporter-gene expression driven by the cytomegalovirus immediate-early (CMV-IE) promoter from adenoviral and plasmid vectors. Heat shock treatment (2h at 42.5 °C) significantly enhanced the promoter DNA-binding activity in nuclear extracts. In CHO cells expressing mGluR1a and mGluR5a receptors under the control of the CMV promoter, heat shock increased receptor protein expression, mRNA levels and receptor function estimated by measurement of PI hydrolysis, intracellular Ca²+ and cAMP. Hyperthermia increased average amplitudes of Ca²+ responses, the number of responding cells, and revealed the toxic properties of mGluR1a receptor. Heat shock also effectively increased the expression of EGFP. Hence, heat shock effects on mGluR expression and function in CHO cells may be attributed to the activation of the CMV promoter. Moreover, this effect was not limited to CHO cells as heat shock also increased EGFP expression in PC-12 and HEK293 cells. Heat shock treatment may be a useful tool to study the function of proteins expressed in heterologous systems under control of the CMV promoter. It may be especially valuable for increasing protein expression in transient transfections, for enhancing receptor expression in drug screening applications and to control the expression of proteins endowed with toxic properties. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Dual neurotoxic and neuroprotective role of metabotropic glutamate receptor 1 in conditions of trophic deprivation - possible role as a dependence receptor.

Group I metabotropic glutamate receptors have been often implicated in various models of neuronal toxicity, however, the role played by the individual receptors and their putative mechanisms of action contributing to neurotoxicity or neuroprotection remain unclear. Here, using primary cultures of rat cerebellar granule cells and mouse cortical neurons, we show that conditions of trophic deprivation increased mGlu1 expression which correlated with the developing cell death. The inhibition of mGlu1 expression by specific siRNA attenuated toxicity, while adenovirus-mediated overexpression of mGlu1 resulted in increased cell death, indicating a causal relationship between the level of receptor expression and neuronal survival. In pharmacological experiments selective mGlu1 antagonists failed to protect from mGlu1-induced cell death, instead, neuronal survival was promoted by glutamate acting at mGlu1 receptors. Such properties are characteristics of a novel heterogeneous family of dependence receptors which control neuronal apoptosis. Our findings indicate that increased expression of mGlu1 in neurons creates a state of cellular dependence on the presence of its endogenous agonist glutamate. We propose a new role and a new mechanism for mGlu1 action. This receptor may play a crucial role in determining the fate of individual neurons during the development of the nervous system.

Cyclothiazide selectively inhibits mGluR1 receptors interacting with a common allosteric site for non-competitive antagonists.

Metabotropic glutamate receptors mGluR1 and mGluR5 stimulate phospholipase C, leading to an increased inositol trisphosphate level and to Ca(2+) release from intracellular stores. Cyclothiazide (CTZ), known as a blocker of AMPA receptor desensitization, produced a non-competitive inhibition of [Ca(2+)](i) increases induced by mGluR agonists in HEK 293 cells transfected with rat mGluR1a but had no effect on the [Ca(2+)](i) signals in cells expressing rat mGluR5a. In cells expressing mGluR1, CTZ also inhibited phosphoinositide hydrolysis, as well as cAMP accumulation and arachidonic acid release induced by mGluR1 agonists, indicating a direct inhibition of the receptor and not of a particular signal transduction system. However, CTZ failed to antagonize cAMP inhibition stimulated by rat mGluR2, -3, -4, -6, -7 and -8 receptors confirming its selectivity for mGluR1. The use of chimeric receptors with substituted N-terminal domains showed that CTZ did not interact with the N-terminal mGluR1a domain. Instead, mutation analysis revealed that CTZ interacts with the Thr-815 and Ala-818 residues, located at the 7th transmembrane domain, similarly as the mGluR1-selective antagonist CPCCOEt. In primary cultures of cerebellar granule neurons, expressing native metabotropic and ionotropic glutamate receptors, the final outcome of CTZ effects depended on its combined ability to potentiate AMPA receptors and inhibit mGluR1 receptors.

Synthesis and receptor assay of aromatic-ethynyl-aromatic derivatives with potent mGluR5 antagonist activity.

Noncompetitive antagonists of the human metabotropic glutamate receptor subtype 5 (mGluR5) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and drug addiction. To discover novel noncompetitive antagonists to the mGluR5, we initiated an SAR study around the known lead compounds MPEP and M-MPEP. Our results pointed out the critical role of the para position of the two aromatic rings, which leads to inactive products and permitted the discovery of potent mGluR5 antagonists (e.g., 16, 25, 28, 34 IC(50) = 13.5, 11.9, 21, 15 nM, respectively).

Antinociceptive effects of N-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in the rat formalin test and in the rat neuropathic pain model.

The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) acts as an agonist at group II metabotropic glutamate receptors (mGluRs). NAAG is inactivated by extracellular peptidase activity yielding glutamate and N-acetylaspartate. We recently developed a series of potent NAAG peptidase inhibitors, including ZJ-11, ZJ-17 and ZJ-43. In the present study, we examined the effects of intrathecally administered ZJ-11 and ZJ-17 and intravenously administered ZJ-11 and ZJ-43 in the rat formalin test (an inflammatory pain model) and in the rat partial sciatic nerve ligation model (a neuropathic pain model). Intrathecal injection of ZJ-11 or ZJ-17 or intravenous injection of ZJ-11 or ZJ-43 suppressed both phases of the agitation behaviour induced by paw formalin injection. Intrathecal and intravenous injection of ZJ-11 suppressed the expression of Fos-like immunoreactivity, induced by paw formalin injection, in laminae I-II in segments L4-L5 of the spinal cord, suggesting an action on sensory spinal transmission. Partial sciatic nerve ligation induced significant mechanical allodynia 7 days after the nerve injury. Intrathecal injection of ZJ-11 or ZJ-17 or intravenous administration of ZJ-11 or ZJ-43 attenuated the level of mechanical allodynia induced by this nerve ligation. These effects of intrathecally or intravenously administered ZJ compounds in both the formalin test and the partial sciatic nerve ligation model were completely antagonized by pretreatment with LY-341495, a highly selective group II mGluR antagonist. Thus, elevation of extracellular NAAG, induced by the inhibition of NAAG peptidase, activates group II mGluRs and produces an analgesic effect in neuropathic and inflammatory and pain models. In contrast, peptidase inhibition did not affect the threshold for withdrawal from a noxious mechanical stimulus or from an acute thermal stimulus in the hotplate test.