Divergent Folding-Mediated Epistasis Among Unstable Membrane Protein Variants.

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Structure-Activity Relationship Study of Cannabidiol-Based Analogs as Negative Allosteric Modulators of the µ-Opioid Receptor.

The US faces an unprecedented surge in fatal drug overdoses. Naloxone, the only antidote for opiate overdose, competes at the mu opioid receptor (µOR) orthosteric site. Naloxone struggles against fentanyl-class synthetic opioids that now cause ∼80% of deaths. Negative allosteric modulators (NAMs) targeting secondary sites may noncompetitively downregulate µOR activation. (-)-Cannabidiol ((-)-CBD) is a candidate µOR NAM. To explore its therapeutic potential, we evaluated the structure-activity relationships among CBD analogs to identify NAMs with increased potency. Using a cyclic AMP assay, we characterize reversal of µOR activation by 15 CBD analogs, several of which proved more potent than (-)-CBD. Comparative docking investigations suggest that potent compounds interact with a putative allosteric pocket to stabilize the inactive µOR conformation. Finally, these compounds enhance naloxone displacement of fentanyl from the orthosteric site. Our results suggest that CBD analogs offer considerable potential for the development of next-generation antidotes for opioid overdose.

General trends in the effects of VX-661 and VX-445 on the plasma membrane expression of clinical CFTR variants.

Cystic fibrosis (CF) is caused by mutations that compromise the expression and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Most people with CF harbor a common misfolded variant (ΔF508) that can be partially rescued by therapeutic "correctors" that restore its expression. Nevertheless, many other CF variants are insensitive to correctors. Using deep mutational scanning, we quantitatively compare the effects of two correctors on the plasma membrane expression of 129 CF variants. Though structural calculations suggest corrector binding provides similar stabilization to most variants, it's those with intermediate expression and mutations near corrector binding pockets that exhibit the greatest response. Deviations in sensitivity appear to depend on the degree of variant destabilization and the timing of misassembly. Combining correctors appears to rescue more variants by doubling the binding energy and stabilizing distinct cotranslational folding transitions. These results provide an overview of rare CF variant expression and establish new tools for precision pharmacology.

Molecular basis for variations in the sensitivity of pathogenic rhodopsin variants to 9-cis-retinal.

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. Of these apparent class II variants, 67 exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts relative to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest that many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (ΔN73, S131P, and R135G) and show that two of these retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

Towards generalizable predictions for G protein-coupled receptor variant expression.

Missense mutations that compromise the plasma membrane expression (PME) of integral membrane proteins are the root cause of numerous genetic diseases. Differentiation of this class of mutations from those that specifically modify the activity of the folded protein has proven useful for the development and targeting of precision therapeutics. Nevertheless, it remains challenging to predict the effects of mutations on the stability and/ or expression of membrane proteins. In this work, we utilize deep mutational scanning data to train a series of artificial neural networks to predict the PME of transmembrane domain variants of G protein-coupled receptors from structural and/ or evolutionary features. We show that our best-performing network, which we term the PME predictor, can recapitulate mutagenic trends within rhodopsin and can differentiate pathogenic transmembrane domain variants that cause it to misfold from those that compromise its signaling. This network also generates statistically significant predictions for the relative PME of transmembrane domain variants for another class A G protein-coupled receptor (ß2 adrenergic receptor) but not for an unrelated voltage-gated potassium channel (KCNQ1). Notably, our analyses of these networks suggest structural features alone are generally sufficient to recapitulate the observed mutagenic trends. Moreover, our findings imply that networks trained in this manner may be generalizable to proteins that share a common fold. Implications of our findings for the design of mechanistically specific genetic predictors are discussed.

The development of ventricular fibrillation as a complication of pericardiectomy in 16 dogs.

OBJECTIVE: To describe the clinical characteristics, perioperative protocols, and outcomes in dogs diagnosed with ventricular fibrillation (VF) while undergoing pericardiectomy. STUDY DESIGN: Retrospective, multi-institutional study. ANIMALS: Sixteen client-owned dogs. METHODS: Cases were accrued through a listserve request posted to 3 subspecialty veterinary societies. Dogs were included if they developed VF during a pericardiectomy performed through an open or thoracoscopic approach. Data collected included signalment, history and physical examination, surgical approach, histopathology, treatment, and outcome. RESULTS: Indications for pericardiectomy included idiopathic chylothorax (n = 7), neoplasia (4), idiopathic pericardial effusion (4), and foreign body granuloma (1). Surgical approaches included thoracoscopy (12), intercostal thoracotomy (3) and median sternotomy (1). Electrosurgical devices were used to complete at least part of the pericardiectomy in 15 of 16 dogs. Ventricular fibrillation appeared to be initiated during electrosurgical use in 8/15 dogs. However, in 5/15 dogs it was not obviously associated with electrosurgical use. In 3/16 dogs the timing of initiation of VF was unclear. In 7/16 dogs, cardiac arrhythmias were noted prior to the development of VF. Fourteen of 16 dogs died from intraoperative VF. CONCLUSION: In most dogs ventricular fibrillation was a fatal complication of pericardiectomy. Ventricular fibrillation might be associated with the use of electrosurgical devices and cardiac manipulation during pericardiectomy although a causal link could not be established from the data in this study. CLINICAL SIGNIFICANCE: Surgeons must be aware of the risk of VF during pericardial surgery. Electrosurgery might need to be used judiciously during pericardiectomy, particularly in dogs exhibiting cardiac arrythmias.

Coordination of -1 programmed ribosomal frameshifting by transcript and nascent chain features revealed by deep mutational scanning.

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of -1PRF is coordinated by a 'slippery' sequence in the transcript, an adjacent RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop reduce the efficiency of -1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of -1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

Systematic profiling of temperature- and retinal-sensitive rhodopsin variants by deep mutational scanning.

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of "temperature-sensitive" variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.

Molecular basis for the evolved instability of a human G-protein coupled receptor.

Membrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Although they function at the plasma membrane, mammalian GnRHRs accumulate within the secretory pathway. Their apparent instability is believed to have evolved through selection for attenuated GnRHR activity. Nevertheless, the molecular basis of this adaptation remains unclear. We show that adaptation coincides with a C-terminal truncation that compromises the translocon-mediated membrane integration of its seventh transmembrane domain (TM7). We also identify a series of polar residues in mammalian GnRHRs that compromise the membrane integration of TM2 and TM6. Reverting a lipid-exposed polar residue in TM6 to an ancestral hydrophobic residue restores expression with no impact on function. Evolutionary trends suggest variations in the polarity of this residue track with reproductive phenotypes. Our findings suggest that the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.

Transorbital craniectomy for treatment of frontal lobe and olfactory bulb neoplasms in two dogs.

CASE DESCRIPTION: An 8-year-old spayed female Shih Tzu crossbreed dog (dog 1) and a 13-year-old neutered male Miniature Fox Terrier (dog 2) were evaluated for removal of neoplasms involving both the frontal lobe and olfactory bulb. CLINICAL FINDINGS: Physical examination revealed decreased menace response and behavioral changes in both dogs. For dog 1, neuroanatomic localization of the lesion was the left forebrain region; for dog 2, neuroanatomic localization of the lesion was the right forebrain region. Both dogs underwent CT, and dog 1 also underwent MRI. Results of diagnostic imaging were consistent with frontal lobe and olfactory bulb neoplasia in both cases. Dog 1 had lysis of the frontal bone adjacent to the neoplasm. TREATMENT AND OUTCOME: Both dogs underwent a transorbital craniectomy to permit surgical tumor removal. Dog 1 was discharged from the hospital 48 hours after surgery, at which time its mentation and cranial nerve examination findings were considered normal. Dog 2 developed neurologic deterioration after surgery but was ultimately discharged from the hospital after 72 hours, at which time its mentation appeared normal. CLINICAL RELEVANCE: The transorbital approach to the cranium provided excellent access to facilitate removal of frontal lobe and olfactory bulb neoplasms in these 2 dogs.

Tumor size as a predictor of lymphatic invasion in oral melanomas of dogs.

OBJECTIVE: To investigate putative associations between oral melanoma size and variables of histologic grade such as mitotic index, nuclear atypia, junctional activity, ulceration, lymphatic invasion, and degree of pigmentation. SAMPLE: 59 samples of oral melanomas from dogs sourced from 6 diagnostic laboratories within Australia. PROCEDURES: The size of each melanoma was microscopically measured, and each sample was evaluated for variables of histologic grade including mitotic index, nuclear atypia, junctional activity, ulceration, lymphatic invasion, and degree of pigmentation by a veterinary pathologist. The association between tumor size and histologic outcomes was then statistically evaluated. RESULTS: A significant relationship was identified between the size of oral melanomas and a single variable of histologic grade, lymphatic invasion, with larger tumors more likely to show lymphatic invasion. Further analysis revealed 2 applicable size thresholds for different clinical scenarios. Results indicated lymphatic invasion can confidently be ruled out for tumors < 6.5 mm in diameter (100% sensitivity) and ruled in for tumors ≥ 24.5 mm in diameter (100% specificity). CONCLUSIONS AND CLINICAL RELEVANCE: An association was found for oral melanomas of dogs between tumor size and lymphatic invasion.

Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning.

Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin's expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin's plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

Classification of the Molecular Defects Associated with Pathogenic Variants of the SLC6A8 Creatine Transporter.

More than 80 loss-of-function (LOF) mutations in the SLC6A8 creatine transporter (hCRT1) are responsible for cerebral creatine deficiency syndrome (CCDS), which gives rise to a spectrum of neurological defects, including intellectual disability, epilepsy, and autism spectrum disorder. To gain insight into the nature of the molecular defects caused by these mutations, we quantitatively profiled the cellular processing, trafficking, expression, and function of eight pathogenic CCDS variants in relation to the wild type (WT) and one neutral isoform. All eight CCDS variants exhibit measurable proteostatic deficiencies that likely contribute to the observed LOF. However, the magnitudes of their specific effects on the expression and trafficking of hCRT1 vary considerably, and we find that the LOF associated with two of these variants primarily arises from the disruption of the substrate-binding pocket. In conjunction with an analysis of structural models of the transporter, we use these data to suggest mechanistic classifications for these variants. To evaluate potential avenues for therapeutic intervention, we assessed the sensitivity of these variants to temperature and measured their response to the proteostasis regulator 4-phenylbutyrate (4-PBA). Only one of the tested variants (G132V) is sensitive to temperature, though its response to 4-PBA is negligible. Nevertheless, 4-PBA significantly enhances the activity of WT hCRT1 in HEK293T cells, which suggests it may be worth evaluating as a therapeutic for female intellectual disability patients carrying a single CCDS mutation. Together, these findings reveal that pathogenic SLC6A8 mutations cause a spectrum of molecular defects that should be taken into consideration in future efforts to develop CCDS therapeutics.

Parathyroid hormone receptor 1 (PTHR1) is a prognostic indicator in canine osteosarcoma.

Osteosarcoma (OS) is the most common malignant primary bone tumour in humans and dogs. Several studies have established the vital role of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation and remodeling. In addition, these molecules play a role in the progression and metastasis of many human tumour types. This study investigated the expression of PTHR1 and PTHrP in canine OS tissues and assessed their prognostic value. Formalin-fixed, paraffin-embedded tissue samples from 50 dogs diagnosed with primary OS were immunolabeled with antibodies specific for PTHR1 and PTHrP. The immunostaining intensity of tumours from patients with OS was correlated with survival time. Both PTHR1 and PTHrP were detected in all OS samples (n = 50). Dogs with OS tumours showing high immunostaining intensity for PTHR1 (n = 36) had significantly shorter survival times (p = 0.028, Log Rank; p = 0.04, Cox regression) when compared with OS that had low immunostaining intensity for PTHR1 (n = 14).PTHrP immunostaining intensity did not correlate with survival time (p > 0.05). The results of this study indicate that increased expression of PTHR1 antigen in canine OS is associated with poor prognosis. This suggests that PTHR1 may be useful as a prognostic indicator in canine OS.

Adverse events and outcomes in dogs with appendicular osteosarcoma treated with limb amputation and a single subcutaneous infusion of carboplatin.

OBJECTIVE: To evaluate adverse events and outcomes in dogs with appendicular osteosarcoma treated with limb amputation followed by a single SC infusion of carboplatin. ANIMALS: 45 client-owned dogs with appendicular osteosarcoma treated with limb amputation and SC infusion of carboplatin between January 1, 2006, and January 15, 2017. PROCEDURES: Medical records were reviewed, and data collected included signalment, tumor location, treatment, results of clinicopathologic analyses and diagnostic imaging, adverse effects of chemotherapy, metastasis-free interval, survival time, and communications with owners and referring veterinarians. Findings were evaluated with the Kaplan-Meier survival analysis and Mantel-Haenszel log-rank test. RESULTS: 45 dogs were identified that met the inclusion criteria (12 of the 45 dogs had been reported in a previous case series). No dogs had pulmonary metastases detectable by CT or radiography before treatment. All dogs completed the protocol as planned. Median survival time (MST) was 196 days; metastasis-free interval was 197 days. Three of the 45 (7%) dogs required hospitalization for gastrointestinal signs related to chemotherapy. There were no chemotherapy-related deaths. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that although treatment with SC infusion of carboplatin was well tolerated, the MST for dogs in the present study was similar to reported MSTs in dogs with appendicular osteosarcoma treated with limb amputation alone and was in the lower range of historically reported survival times for dogs receiving IV adjunctive chemotherapy. Therefore, we could not recommend this protocol of SC infusion of carboplatin but recommended that protocols with IV administration of carboplatin be used instead.

Apocrine gland anal sac adenocarcinoma in cats: 30 cases (1994-2015).

OBJECTIVE To describe the signalment, clinical signs, biological behavior, and outcome for cats with apocrine gland anal sac adenocarcinoma (AGASACA) that underwent surgical excision. DESIGN Retrospective case series. ANIMALS 30 client-owned cats. PROCEDURES Databases of 13 Veterinary Society of Surgical Oncology member-affiliated institutions were searched for records of cats with a histologic diagnosis of AGASACA that underwent tumor excision. For each cat, information regarding signalment, clinical signs, diagnostic test results, treatment, and outcome was extracted from the medical record. The Kaplan-Meier method was used to determine median time to local recurrence (TLR), disease-free interval (DFI), and survival time. Cox regression was used to identify factors associated with TLR, DFI, and survival time. RESULTS Perineal ulceration or discharge was the most common clinical sign in affected cats. Eleven cats developed local recurrence at a median of 96 days after AGASACA excision. Incomplete tumor margins and a high nuclear pleomorphic score were risk factors for local recurrence. Nuclear pleomorphic score was negatively associated with DFI. Local recurrence and a high nuclear pleomorphic score were risk factors for death. Median DFI and survival time were 234 and 260 days, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, in cats, perineal ulceration or discharge should raise suspicion of AGASACA and prompt rectal and anal sac examinations. Local recurrence was the most common life-limiting event in cats that underwent surgery for treatment of AGASACA, suggesting that wide margins should be obtained whenever possible during AGASACA excision. Efficacy of chemotherapy and radiation therapy for treatment of cats with AGASACA requires further investigation. (J Am Vet Med Assoc 2019;254:716-722).

The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux.

The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser.

Pia Mater Significantly Contributes to Spinal Cord Intraparenchymal Pressure in a Simulated Model of Edema.

STUDY DESIGN: Intraparenchymal pressure (IPP) measurements in an in vitro cadaveric model of CNS edema. OBJECTIVE: To assess the contribution of pia mater to IPP and the effect of piotomy. SUMMARY OF BACKGROUND DATA: Multicenter randomized control trials have shown that decompression with durotomy/duroplasty significantly decreases intracranial pressure (ICP). There is a paucity of evidence regarding the effectiveness of decompression of the spinal cord by piotomy. METHODS: The supratentorial brain and spinal cord were removed from six fresh cadavers. Dura and arachnoid mater were removed. ICP monitors were placed bilaterally in the frontal and parietal lobes, and centrally in the cervical and thoracic spinal cord. To simulate edema, specimens were submerged in hypotonic solution. IPP was recorded for 5 days. A complete dorsal midline piotomy was performed on the spinal cord and resulting IPP was recorded. RESULTS: Brain and spinal cord both increased in weight. IPP significantly increased in both brain and spinal cord. The IPP increase within the spinal cord was substantially greater (averages: all four lobes = 4.0 mm Hg; cervical = 73.7 mm Hg; thoracic = 49.3 mm Hg). After piotomy, cervical and thoracic spinal cord IPP decreased immediately (avg. postpiotomy IPP = 9.7 and 10.3, respectively). CONCLUSION: There were differential effects on brain and spinal cord IPP. Brain IPP increased only slightly, possibly because of the absence of the cranium and dura mater. In contrast, spinal cord IPP increased substantially even in the absence of the laminae, dura, and arachnoid mater. Piotomy immediately and dramatically reduced spinal cord IPP. These data are consistent with the hypothesis that spinal cord IPP is primarily dependent on constraints imposed by the pia mater. Conversely, in the absence of the cranium and dura mater, the sulci may permit the pia-invested brain to better accommodate edema without significant increases in IPP. LEVEL OF EVIDENCE: N/A.

Spinal cord intramedullary pressure in thoracic scoliotic deformity: a cadaveric study.

STUDY DESIGN: In vitro cadaveric study of thoracic spinal cord intramedullary pressure (IMP) in scoliotic deformity. OBJECTIVE: To define the relationship between thoracic scoliotic deformity and spinal cord IMP. SUMMARY OF BACKGROUND DATA: Clinical studies of patients with thoracic scoliosis without other spinal pathology (spinal stenosis, etc.) have rarely reported an associated thoracic myelopathy. Previous clinical and cadaveric studies of kyphosis have reported associated myelopathy and increased spinal cord IMP. We sought to determine if IMP changes in response to main thoracic scoliotic deformity. METHODS: In 6 fresh-frozen cadavers, a progressive main thoracic scoliotic deformity was created. Cadavers were positioned sitting with physiological spinal alignment, head stabilized using a skull clamp and spine segmentally instrumented from occiput to L3. The T3-T4 ligamentum flavum was removed, dura opened, and 3 pressure sensors were advanced caudally to T4-T5, T7-T8, and T10-T11 within the cord parenchyma. A step-wise main thoracic scoliotic deformity was then induced by sequentially releasing and retightening the skull clamp while coronally bending, concavity compressing, and convexity distracting posterior segmental instrumentation, allowing closure of lateral segmental osteotomies. After each step, fluoroscopic images and pressure measurements were obtained; the T4-T11 coronal Cobb angle was measured. RESULTS: Induction of main thoracic scoliosis did not significantly increase IMP. The mean main thoracic maximal scoliotic deformity created was 77° ± 2° (range: 71°-84°). At maximal deformity, the mean ΔIMP at T4-T5, T7-T8, T10-T11 was 2.2 ± 1.9 mm Hg, 1.0 ± 0.7 mm Hg, and 1.0 ± 0.8 mm Hg, respectively. CONCLUSION: In this cadaveric study, main thoracic scoliotic deformity did not significantly increase thoracic IMP. This correlates with clinical presentation such that clinical studies of patients with thoracic scoliosis without other spinal pathology have rarely reported an associated thoracic myelopathy with the thoracic scoliosis. This study helps explain the relative absence of myelopathy in isolated main thoracic coronal plane deformity. LEVEL OF EVIDENCE: 5.