Christian Iorio-Morin

Novel, Gel-free Proteomics Approach Identifies RNF5 and JAMP as Modulators of GPCR Stability

The maturation and folding of G protein-coupled receptors are governed by mechanisms that remain poorly understood. In an effort to characterize these biological events, we optimized a novel, gel-free proteomic approach to identify partners of the β2-adrenergic receptor (β2AR). In addition to a number of known interacting proteins such as heterotrimeric G protein subunits, this allowed us to identify proteins involved in endoplasmic reticulum (ER) QC of the receptor. Among β2AR-associated proteins is Ring finger protein 5 (RNF5), an E3 ubiquitin ligase anchored to the outer membrane of the ER. Coimmunoprecipitation assays confirmed, in a cellular context, the interaction between RNF5 and the β2AR as well as the prostaglandin D2 receptor (DP). Confocal microscopy revealed that DP colocalized with RNF5 at the ER. Coexpression of RNF5 with either receptor increased levels of their expression, whereas small interfering RNA-mediated knockdown of endogenous RNF5 promoted the opposite. RNF5 did not modulate the ubiquitination state of β2AR or DP. Instead, RNF5 ubiquitinated JNK-associated membrane protein (JAMP), a protein that recruits the proteasome to the ER membrane and that is negatively regulated by RNF5-mediated ubiquitination. JAMP coimmunoprecipitated with both β2AR and DP and decreased total receptor protein levels through proteasomal degradation. Expression of DP, a receptor largely retained in the ER, promoted proteasome recruitment by JAMP. Degradation of both receptors via JAMP was increased when RNF5 was depleted. Our data suggest that RNF5 regulates the turnover of specific G protein-coupled receptors by ubiquitinating JAMP and preventing proteasome recruitment.

Inverse agonist and pharmacochaperone properties of MK-0524 on the prostanoid DP1 receptor.

Prostaglandin D2 (PGD2) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gαs-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gαi/o proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD2 induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.

ANKRD13C Acts as a Molecular Chaperone for G Protein-coupled Receptors

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D2. In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A2 (TPα), and β2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.

Safety and Efficacy of Gamma Knife Radiosurgery for the Management of Koos Grade 4 Vestibular Schwannomas.

BACKGROUND Gamma Knife radiosurgery (GKRS) is commonly used in treating small vestibular schwannomas; however, its use for larger vestibular schwannomas is still controversial. OBJECTIVE To assess the long-term safety and efficacy of treating eligible Koos grade 4 vestibular schwannomas with GKRS. METHODS We conducted a single-center, retrospective evaluation of patient undergoing GKRS for Koos grade 4 vestibular schwannomas. We evaluated clinical, imaging, and treatment characteristics and assessed treatment outcome. Inclusion criteria were tumor size of ≥4 cm and follow-up of at least 6 months. Patients with neurofibromatosis type 2 were excluded. Primary outcomes measured were tumor control rate, hearing and facial function preservation rate, and complications. All possible factors were analyzed to assess clinical significance. RESULTS Sixty-eight patients met inclusion criteria. Median follow-up was 47 months (range, 6-125 months). Baseline hearing was serviceable in 60%. Median tumor volume at radiosurgery was 7.4 cm (range, 4-19 cm). The median marginal dose used was 12 Gy at the 50% isodose line. Actuarial tumor control rates were 95% and 92% at 2 and 10 years, respectively. Actuarial serviceable hearing preservation rates were 89% and 49% at 2 and 5 years, respectively. Facial nerve preservation was 100%. Clinical complications included balance disturbance (11%), facial pain (10%), facial numbness (5%), and tinnitus (10%). Most complications were mild and transient. Hydrocephalus occurred in 3 patients, requiring ventriculoperitoneal shunt insertion. Larger tumor size was significantly associated with persisting symptoms post-treatment. CONCLUSION Patients with Koos grade 4 vestibular schwannomas and minimal symptoms can be treated safely and effectively with GKRS.

An Interaction between L-prostaglandin D Synthase and Arrestin Increases PGD2 Production

L-type prostaglandin synthase (L-PGDS) produces PGD2, a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD2-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2−/−-arr-3−/−. Arrestin-3 promotes PGD2 production by L-PGDS in vitro. IL-1β-induced PGD2 production is significantly lower in MEFs-arr-2−/−-arr-3−/− than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86–100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD2 production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD2 synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.

Demographics, Interests, and Quality of Life of Canadian Neurosurgery Residents

BACKGROUND Neurosurgical residents face a unique combination of challenges, including long duty hours, technically challenging cases, and uncertain employment prospects. We sought to assess the demographics, interests, career goals, self-rated happiness, and overall well-being of Canadian neurosurgery residents. METHODS A cross-sectional survey was developed and sent through the Canadian Neurosurgery Research Collaborative to every resident enrolled in a Canadian neurosurgery program as of April 1, 2016. RESULTS We analyzed 76 completed surveys of 146 eligible residents (52% response rate). The median age was 29 years, with 76% of respondents being males. The most popular subspecialties of interest for fellowship were spine, oncology, and open vascular neurosurgery. The most frequent self-reported number of worked hours per week was the 80- to 89-hour range. The majority of respondents reported a high level of happiness as well as stress. Sense of accomplishment and fatigue were reported as average to high and overall quality of life was low for 19%, average for 49%, and high for 32%. Satisfaction with work-life balance was average for 44% of respondents and was the only tested domain in which significant dissatisfaction was identified (18%). Overall, respondents were highly satisfied with their choice of specialty, choice of program, surgical exposure, and work environment; however, intimidation was reported in 36% of respondents and depression by 17%. CONCLUSIONS Despite a challenging residency and high workload, the majority of Canadian neurosurgery residents are happy and satisfied with their choice of specialty and program. However, work-life balance, employability, resident intimidation, and depression were identified as areas of active concern.

Histology-Stratified Tumor Control and Patient Survival After Stereotactic Radiosurgery for Pineal Region Tumors: A Report From the International Gamma Knife Research Foundation

BACKGROUND Pineal region tumors represent a rare and histologically diverse group of lesions. Few studies are available to guide management and the outcomes after stereotactic radiosurgery (SRS). METHODS Patients who underwent SRS for a pineal region tumor and for whom at least 6 months of imaging follow-up was available were retrospectively assessed in 5 centers. Data were collected from the medical record and histology level analyses were performed, including actuarial tumor control and survival analyses. RESULTS A total of 70 patients were treated between 1989 and 2014 with a median follow-up of 47 months. Diagnoses were pineocytoma (37%), pineoblastoma (19%), pineal parenchymal tumor of intermediate differentiation (10%), papillary tumor of the pineal region (9%), germinoma (7%), teratoma (3%), embryonal carcinoma (1%), and unknown (14%). Median prescription dose was 15 Gy at the 50% isodose line. Actuarial local control and survival rates were 81% and 76% at 20 years for pineocytoma, 50% and 56% at 5 years for pineal parenchymal tumor of intermediate differentiation, 27% and 48% at 5 years for pineoblastoma, 33% and 100% at 5 years for papillary tumor of the pineal region, 80% and 80% at 20 years for germinoma, and 61% and 67% at 5 years for tumors of unknown histology. New focal neurological deficit, Parinaud syndrome, and hydrocephalus occurred in 9%, 7%, and 3% of cases, respectively. CONCLUSIONS SRS is a safe modality for the management of pineal region tumors. Its specific role is highly dependent on tumor histology. As such, all efforts should be made to obtain a reliable histologic diagnosis.

Operative Landscape at Canadian Neurosurgery Residency Programs

Background Currently, the literature lacks reliable data regarding operative case volumes at Canadian neurosurgery residency programs. Our objective was to provide a snapshot of the operative landscape in Canadian neurosurgical training using the trainee-led Canadian Neurosurgery Research Collaborative. METHODS Anonymized administrative operative data were gathered from each neurosurgery residency program from January 1, 2014, to December 31, 2014. Procedures were broadly classified into cranial, spine, peripheral nerve, and miscellaneous procedures. A number of prespecified subspecialty procedures were recorded. We defined the resident case index as the ratio of the total number of operations to the total number of neurosurgery residents in that program. Resident number included both Canadian medical and international medical graduates, and included residents on the neurosurgery service, off-service, or on leave for research or other personal reasons. RESULTS Overall, there was an average of 1845 operative cases per neurosurgery residency program. The mean numbers of cranial, spine, peripheral nerve, and miscellaneous procedures were 725, 466, 48, and 193, respectively. The nationwide mean resident case indices for cranial, spine, peripheral nerve, and total procedures were 90, 58, 5, and 196, respectively. There was some variation in the resident case indices for specific subspecialty procedures, with some training programs not performing carotid endarterectomy or endoscopic transsphenoidal procedures. CONCLUSIONS This study presents the breadth of neurosurgical training within Canadian neurosurgery residency programs. These results may help inform the implementation of neurosurgery training as the Royal College of Physicians and Surgeons residency training transitions to a competence-by-design curriculum.

Repeat Stereotactic Radiosurgery for Progressive or Recurrent Vestibular Schwannomas

BACKGROUND Stereotactic radiosurgery (SRS) is a highly effective management approach for patients with vestibular schwannomas (VS), with 10-yr control rates up 98%. When it fails, however, few data are available to guide management. OBJECTIVE To perform a retrospective analysis of patients who underwent 2 SRS procedures on the same VS to assess the safety and efficacy of this practice. METHODS This study was opened to centers of the International Gamma Knife Research Foundation (IGKRF). Data collected included patient characteristics, clinical symptoms at the time of SRS, radiosurgery dosimetric data, imaging response, clinical evolution, and survival. Actuarial analyses of tumor responses were performed. RESULTS Seventy-six patients from 8 IGKRF centers were identified. Median follow-up from the second SRS was 51.7 mo. Progression after the first SRS occurred at a median of 43 mo. Repeat SRS was performed using a median dose of 12 Gy. Actuarial tumor control rates at 2, 5, and 10 yr following the second SRS were 98.6%, 92.2%, and 92.2%, respectively. Useful hearing was present in 30%, 8%, and 5% of patients at first SRS, second SRS, and last follow-up, respectively. Seventy-five percent of patients reported stable or improved symptoms following the second SRS. Worsening of facial nerve function attributable to SRS occurred in 7% of cases. There were no reports of radionecrosis, radiation-associated edema requiring corticosteroids, radiation-related neoplasia, or death attributable to the repeat SRS procedure. CONCLUSION Patients with progressing VS after radiosurgery can be safely and effectively managed using a second SRS procedure.

Chronic Subdural Hematoma: Toward a New Management Paradigm for an Increasingly Complex Population

Chronic subdural hematoma (cSDH) is a frequent yet poorly studied entity. Patients with cSDH are increasingly using antithrombotic medication, are now older, and present with a variety of clinical symptoms, including incidental discoveries. Despite this increasing complexity, management has remained roughly unchanged since the late 1990s. We review here the state of cSDH research under way at Université de Sherbrooke and around the world with a focus on studies addressing specific gaps in the current evidence base. We show that evidence is lacking at many decision points in the typical cSDH patient treatment algorithm. No definition of cSDH is universally accepted, and a formal definition project, along with suggested common data elements to be reported in future trials (CODE-CSDH: formal cSDH definition project) is ongoing. An amendment to International Statistical Classification of Diseases and Related Health Problems (ICD-11) has also been proposed to improve classification and registry research. Within the cSDH clinical assessment, evidence for the occurrence of nonepileptic, stereotypical, and intermittent symptoms (NESIS) is emerging. The GENESIS study (Generation Evidence on the etiology and management of NESIS) will test etiological and therapeutic hypotheses for this patient subpopulation. For patients at high risk of recurrence, the TRACS (TXA for cSDH) and EMMACS studies (Embolization of the Middle Meningeal Artery in Chronic Subdural Hematoma study) are, respectively, assessing the use of tranexamic acid and meningeal artery embolization. The overarching vision is that patients with cSDH might be stratified for operative versus conservative treatment based on the need for mass effect removal, then be offered adjuvant therapies based on their risk of recurrence and thrombotic complications. We believe that such tailoring of therapy to each individual should help improve outcomes.