Rick F. Nelson

CHIP Suppresses Polyglutamine Aggregation and Toxicity In Vitro and In Vivo

Huntingtons disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As both a cochaperone and ubiquitin ligase, the C-terminal Hsp70 (heat shock protein 70)-interacting protein (CHIP) links the two major arms of protein quality control, molecular chaperones, and the ubiquitin-proteasome system. Here, we demonstrate that CHIP suppresses polyQ aggregation and toxicity in transfected cell lines, primary neurons, and a novel zebrafish model of disease. Suppression by CHIP requires its cochaperone function, suggesting that CHIP acts to facilitate the solubility of mutant polyQ proteins through its interactions with chaperones. Conversely, HD transgenic mice that are haploinsufficient for CHIP display a markedly accelerated disease phenotype. We conclude that CHIP is a critical mediator of the neuronal response to misfolded polyQ protein and represents a potential therapeutic target in this important class of neurodegenerative diseases.

Selective Cochlear Degeneration in Mice Lacking the F-Box Protein, Fbx2, a Glycoprotein-Specific Ubiquitin Ligase Subunit

Little is known about the role of protein quality control in the inner ear. We now report selective cochlear degeneration in mice deficient in Fbx2, a ubiquitin ligase F-box protein with specificity for high-mannose glycoproteins (Yoshida et al., 2002). Originally described as a brain-enriched protein (Erhardt et al., 1998), Fbx2 is also highly expressed in the organ of Corti, in which it has been called organ of Corti protein 1 (Thalmann et al., 1997). Mice with targeted deletion of Fbxo2 develop age-related hearing loss beginning at 2 months. Cellular degeneration begins in the epithelial support cells of the organ of Corti and is accompanied by changes in cellular membrane integrity and early increases in connexin 26, a cochlear gap junction protein previously shown to interact with Fbx2 (Henzl et al., 2004). Progressive degeneration includes hair cells and the spiral ganglion, but the brain itself is spared despite widespread CNS expression of Fbx2. Cochlear Fbx2 binds Skp1, the common binding partner for F-box proteins, and is an unusually abundant inner ear protein. Whereas cochlear Skp1 levels fall in parallel with the loss of Fbx2, other components of the canonical SCF (Skp1, Cullin1, F-box, Rbx1) ubiquitin ligase complex remain unchanged and show little if any complex formation with Fbx2/Skp1, suggesting that cochlear Fbx2 and Skp1 form a novel, heterodimeric complex. Our findings demonstrate that components of protein quality control are essential for inner ear homeostasis and implicate Fbx2 and Skp1 as potential genetic modifiers in age-related hearing loss.

Diversity in Tissue Expression, Substrate Binding, and SCF Complex Formation for a Lectin Family of Ubiquitin Ligases

Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.

A Novel Route for F-box Protein-mediated Ubiquitination Links CHIP to Glycoprotein Quality Control

In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.

Facial Nerve Outcome and Tumor Control Rate as a Function of Degree of Resection in Treatment of Large Acoustic Neuromas: Preliminary Report of the Acoustic Neuroma Subtotal Resection Study (ANSRS).

BACKGROUND Patients with large vestibular schwannomas are at high risk of poor facial nerve (cranial nerve VII [CNVII]) function after surgery. Subtotal resection potentially offers better outcome, but may lead to higher tumor regrowth. OBJECTIVE To assess long-term CNVII function and tumor regrowth in patients with large vestibular schwannomas. METHODS Prospective multicenter nonrandomized cohort study of patients with vestibular schwannoma ≥2.5 cm who received gross total resection, near total resection, or subtotal resection. Patients received radiation if tumor remnant showed signs of regrowth. RESULTS Seventy-three patients had adequate follow-up with mean tumor diameter of 3.33 cm. Twelve received gross total resection, 22 near total resection, and 39 subtotal resection. Fourteen (21%) remnant tumors continued to grow, of which 11 received radiation, 1 had repeat surgery, and 2 no treatment. Four of the postradiation remnants (36%) required surgical salvage. Tumor regrowth was related to non-cystic nature, larger residual tumor, and subtotal resection. Regrowth was 3 times as likely with subtotal resection compared to gross total resection and near total resection. Good CNVII function was achieved in 67% immediately and 81% at 1-year. Better immediate nerve function was associated with smaller preoperative tumor size and percentage of tumor left behind on magnetic resonance image. Degree of resection defined by surgeon and preoperative tumor size showed weak trend toward better late CNVII function. CONCLUSION Likelihood of tumor regrowth was 3 times higher in subtotal resection compared to gross total resection and near total resection groups. Rate of radiation control of growing remnants was suboptimal. Better immediate but not late CNVII outcome was associated with smaller tumors and larger tumor remnants. ABBREVIATIONS CNVII, cranial nerve VIIGTR, gross total resectionHB, House-BrackmannMRI, magnetic resonance imageNTR, near total resectionSTR, subtotal resection.

The rising incidence of spontaneous cerebrospinal fluid leaks in the United States and the association with obesity and obstructive sleep apnea.

Objective To determine the national rates of spontaneous CSF leaks and to determine the association with risk factors. Study Design Retrospective review from 2002 to 2012. Setting University HealthSystem Consortium (UHC) database of 127 of the leading academic medical centers in the United States (81 centers participated all years of the study). Patients Those who underwent craniotomy for CSF leak repair in the UHC database and those who have undergone repair of spontaneous CSF leaks at one UHC center. Intervention Assessment of procedure code rates and patient demographics from 2002 to 2012. Main Outcome Measure National rates of craniotomy for spontaneous CSF leak repair each year, the relation to U.S. regional obesity rates, and the proportion of patients with coincident obstructive sleep apnea. Spontaneous CSF leak patient characteristics (age, sex, BMI, hypertension, and OSA) were calculated. Results The rate of craniotomy for spontaneous CSF leak repair has risen 2 fold from 2002 (218 cases per year) to 2012 (488 cases per year). There was no change in the rate of nonspontaneous CSF leaks over the same period. The rate of spontaneous CSF leak repair is twice as high (2.54 versus 1.07 per million people per year) in regions of the United States with the highest obesity rate (Midwest) compared with the lowest obesity rate (West). All patients with spontaneous CSF leaks were overweight (BMI, >25 kg/m2) with an average BMI of 37.8 kg/m2. The average age was 57.03 years, and 72% were female. Patients with spontaneous CSF leaks presented with high rate of OSA (14.8% nationally and 37.1% at the University of Iowa) and hypertension (85.7%). Conclusion The national rate of craniotomy for spontaneous CSF leak repair is rising. This condition is yet another public health problem related to the rising obesity epidemic. All patients with spontaneous CSF leaks should be evaluated for OSA.

Calvarium thinning in patients with spontaneous cerebrospinal fluid leak.

Objective To determine the thickness of the calvarium in patients with spontaneous cerebrospinal fluid (CSF) leaks. Study Design Case control study. Setting University of Iowa Hospitals and Clinics. Patients Those with a confirmed spontaneous CSF leak compared to non-obese (body mass index, BMI < 30) and obese (BMI ≥ 30) cochlear implant (CI) control groups. All patients had to have temporal bone CT scans that fit specified criteria. Intervention Bilateral volumetric analysis of the squamous temporal bone and the zygoma in all patients. Assessment of patient age, sex, BMI, and medical comorbidities. Main Outcome Measure Assessment of the average thickness of the squamous temporal bone and zygoma compared to control groups. Results The average BMI of patients with spontaneous CSF leaks was significantly higher than non-obese CI controls (43.73 ± 9.19 vs. 24.60 ± 3.10; P < 0.0001). The calvarium in patients with spontaneous CSF leaks was 23% thinner than both non-obese CI controls (3.29 ± 0.68 vs. 4.25 ± 0.58; P < 0.0001) and obese CI controls (3.29 ± 0.68 vs. 4.27 ± 0.68; P < 0.0001). In addition, the skull thickness of obese CI patients (body mass index, BMI = 37.34 ± 6.1) was not significantly different from non-obese CI controls (4.27 ± 0.68 vs. 4.25 ± 0.58; P = 0.92). The extracranial zygoma was not significantly different among the three groups (ANOVA = 0.9). In our study groups, 5.8% of both CI control groups had the diagnosis of obstructive sleep apnea (OSA), whereas 46.2% of the spontaneous CSF leak patients presented with the diagnosis of OSA. Conclusion Patients with spontaneous CSF leak are more likely to be obese, have the diagnosis of OSA, and show thinning of their entire calvarium that is independent of BMI. These data suggest an additional obesity-associated intracranial process contributes to skull thinning.

Loss of F-box Only Protein 2 (Fbxo2) Disrupts Levels and Localization of Select NMDA Receptor Subunits, and Promotes Aberrant Synaptic Connectivity

NMDA receptors (NMDARs) play an essential role in some forms of synaptic plasticity, learning, and memory. Therefore, these receptors are highly regulated with respect to their localization, activation, and abundance both within and on the surface of mammalian neurons. Fundamental questions remain, however, regarding how this complex regulation is achieved. Using cell-based models and F-box Only Protein 2 (Fbxo2) knock-out mice, we found that the ubiquitin ligase substrate adaptor protein Fbxo2, previously reported to facilitate the degradation of the NMDAR subunit GluN1 in vitro, also functions to regulate GluN1 and GluN2A subunit levels in the adult mouse brain. In contrast, GluN2B subunit levels are not affected by the loss of Fbxo2. The loss of Fbxo2 results in greater surface localization of GluN1 and GluN2A, together with increases in the synaptic markers PSD-95 and Vglut1. These synaptic changes do not manifest as neurophysiological differences or alterations in dendritic spine density in Fbxo2 knock-out mice, but result instead in increased axo-dendritic shaft synapses. Together, these findings suggest that Fbxo2 controls the abundance and localization of specific NMDAR subunits in the brain and may influence synapse formation and maintenance.

Middle Cranial Fossa (MCF) approach without the use of lumbar drain for the management of spontaneous cerebral spinal fluid (CSF) Leaks

Objective: To determine the efficacy and morbidity of repairing spontaneous cerebrospinal fluid (CSF) leaks with the middle cranial fossa (MCF) approach without the use of a lumbar drain (LD), as perioperative use of LD remains controversial. Study Design: Retrospective review from 2003 to 2015. Setting: University of Iowa Hospitals and Clinics and Indiana University Health Center. Patients: Those with a confirmed lateral skull base spontaneous CSF leaks and/or encephaloceles. Intervention: MCF approach for repair of spontaneous CSF leak and/or encephalocele without the use of lumbar drain. Assessment of patient age, sex, body mass index (BMI), and medical comorbidities. Main Outcome Measure: Spontaneous CSF leak patient characteristics (age, sex, BMI, obstructive sleep apnea) were collected. Length of stay (LOS), hospital costs, postoperative complications, CSF leak rate, and need for LD were calculated. Results: Sixty-five operative MCF repairs were performed for spontaneous CSF leaks on 60 patients (five had bilateral CSF leaks). CSF diversion with LD was used in 15 of 60 patients, mostly before 2010. After 2010, only three of 44 patients (6.7%) had postoperative otorrhea requiring LD. The use of LD resulted in significantly longer LOS (3.6 ± 1.6 versus 8.7 ± 2.9 d) and hospital costs (

Tension pneumocephalus related to spontaneous skull base dehiscence in a patient on BiPAP

29,621). There were no postoperative complications in 77% (50 of 65) of cases. Three cases required return to the operating room for complications including frontal subdural hematoma (1), subdural CSF collection (1), and tension pneumocephalus (1). No patients experienced long-term neurologic sequelae or long-term CSF leak recurrence with an average length of follow-up of 19.5 months (range 3–137 mo). The average patient BMI was 37.5 ± 8.6 kg/m2. The average age was 57.5 ± 11.4 years and 68% were female. Obstructive sleep apnea was present in 43.3% (26 of 60) of patients. Conclusion: The morbidity of the MCF craniotomy for repair of spontaneous CSF leaks is low and the long-term efficacy of repair is high. Universal use of perioperative lumbar drain is not indicated and significantly increases length of stay and hospital costs. Obesity and obstructive sleep apnea are highly associated with spontaneous CSF leaks.