Faculty Bibliography

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system (CNS). B lymphocytes in the cerebrospinal fluid (CSF) of MS patients contribute to inflammation and secrete oligoclonal immunoglobulins^1,2. Epstein-Barr virus (EBV) infection has been linked to MS epidemiologically, but its pathological role remains unclear³. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBNA1 and the CNS protein GlialCAM, and provide structural and in-vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements, and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment allowed for tracking the development of the naïve EBNA1-restricted antibody to a mature EBNA1/GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates the mouse model of MS and anti-EBNA1/GlialCAM antibodies are prevalent in MS patients. Our results provide a mechanistic link for the association between MS and EBV, and could guide the development of novel MS therapies.

OBJECTIVES/OBJECTIVE:The goals of this study were to 1) compare global protein expression in muscles of the larynx and hindlimb and 2) investigate differences in protein expression between aged and nonaged muscle using label-free global proteomic profiling methods. METHODS:Liquid chromatography-mass spectrometry (LC-MS/MS) analysis was performed on thyroarytenoid intrinsic laryngeal muscle and plantaris hindlimb muscle from 10 F344xBN F1 male rats (5 old and 5 young). Protein expression was compared and pathway enrichment analysis performed for each muscle type (larynx and limb) and age group (old and young muscle). RESULTS:Over 1,000 proteins were identified in common across both muscle types and age groups using LC-MS/MS analysis. Significant age-related differences were seen across 107 proteins in plantaris hindlimb and in 19 proteins in thyroarytenoid laryngeal muscle. Bioinformatic and enrichment analysis demonstrated protein differences between the hindlimb and larynx may relate to immune and stress redox responses and RNA repair. CONCLUSION/CONCLUSIONS:There are clear differences in protein expressions between the laryngeal and hindlimb skeletal muscles. Initial analysis suggests differences between the two muscle groups may relate to stress responses and repair mechanisms. Age-related changes in the thyroarytenoid appear to be less obvious than in the plantaris. Further in-depth study is needed to elucidate how aging affects protein expression in the laryngeal muscles. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2021.

BACKGROUND:Tuberous sclerosis complex (TSC) and some focal cortical dysplasias (FCDs) are associated with dysfunctional mTOR signaling, resulting in increased cell growth and ribosomal S6 protein phosphorylation (phospho-S6). mTOR inhibitors can reduce TSC tumor growth and seizure frequency, and preclinical FCD studies indicate seizure suppression. This pilot study evaluated safety of mTOR inhibitor everolimus in treatment resistant (failure of >2 anti-seizure medications) TSC and FCD patients undergoing surgical resection and to assess mTOR signaling and molecular pathways. METHODS AND FINDINGS/RESULTS:We evaluated everolimus in 14 treatment resistant epilepsy patients undergoing surgical resection (4.5 mg/m2 daily for 7 days; n = 4 Active, mean age 18.3 years, range 4-26; n = 10, Control, mean age 13.1, range 3-45). Everolimus was well tolerated. Mean plasma everolimus in Active participants were in target range (12.4 ng/ml). Brain phospho-S6 was similar in Active and Control participants with a lower trend in Active participants, with Ser235/236 1.19-fold (p = 0.67) and Ser240/244 1.15-fold lower (p = 0.66). Histologically, Ser235/236 was 1.56-fold (p = 0.37) and Ser240/244 was 5.55-fold lower (p = 0.22). Brain proteomics identified 11 proteins at <15% false discovery rate associated with coagulation system (p = 1.45x10-9) and acute phase response (p = 1.23x10-6) activation. A weighted gene correlation network analysis (WGCNA) of brain proteomics and phospho-S6 identified 5 significant modules. Higher phospho-S6 correlated negatively with cellular respiration and synaptic transmission and positively with organophosphate metabolic process, nuclear mRNA catabolic process, and neuron ensheathment. Brain metabolomics identified 14 increased features in Active participants, including N-acetylaspartylglutamic acid. Plasma proteomics and cytokine analyses revealed no differences. CONCLUSIONS:Short-term everolimus before epilepsy surgery in TSC and FCD resulted in no adverse events and trending lower mTOR signaling (phospho-S6). Future studies should evaluate implications of our findings, including coagulation system activation and everolimus efficacy in FCD, in larger studies with long-term treatment to better understand molecular and clinical effects. CLINICAL TRIALS REGISTRATION/BACKGROUND:ClinicalTrials.gov NCT02451696.

Brainstem nuclei dysfunction is implicated in sudden unexpected death in epilepsy. In animal models, deficient serotonergic activity is associated with seizure-induced respiratory arrest. In humans, glia are decreased in the ventrolateral medullary pre-Botzinger complex that modulate respiratory rhythm, as well as in the medial medullary raphe that modulate respiration and arousal. Finally, sudden unexpected death in epilepsy cases have decreased midbrain volume. To understand the potential role of brainstem nuclei in sudden unexpected death in epilepsy, we evaluated molecular signalling pathways using localized proteomics in microdissected midbrain dorsal raphe and medial medullary raphe serotonergic nuclei, as well as the ventrolateral medulla in brain tissue from epilepsy patients who died of sudden unexpected death in epilepsy and other causes in diverse epilepsy syndromes and non-epilepsy control cases (n = 15-16 cases per group/region). Compared with the dorsal raphe of non-epilepsy controls, we identified 89 proteins in non-sudden unexpected death in epilepsy and 219 proteins in sudden unexpected death in epilepsy that were differentially expressed. These proteins were associated with inhibition of EIF2 signalling (P-value of overlap = 1.29 × 10^-8, z = -2.00) in non-sudden unexpected death in epilepsy. In sudden unexpected death in epilepsy, there were 10 activated pathways (top pathway: gluconeogenesis I, P-value of overlap = 3.02 × 10^-6, z = 2.24) and 1 inhibited pathway (fatty acid beta-oxidation, P-value of overlap = 2.69 × 10^-4, z = -2.00). Comparing sudden unexpected death in epilepsy and non-sudden unexpected death in epilepsy, 10 proteins were differentially expressed, but there were no associated signalling pathways. In both medullary regions, few proteins showed significant differences in pairwise comparisons. We identified altered proteins in the raphe and ventrolateral medulla of epilepsy patients, including some differentially expressed in sudden unexpected death in epilepsy cases. Altered signalling pathways in the dorsal raphe of sudden unexpected death in epilepsy indicate a shift in cellular energy production and activation of G-protein signalling, inflammatory response, stress response and neuronal migration/outgrowth. Future studies should assess the brain proteome in relation to additional clinical variables (e.g. recent tonic-clonic seizures) and in more of the reciprocally connected cortical and subcortical regions to better understand the pathophysiology of epilepsy and sudden unexpected death in epilepsy.

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.

Staphylococcus aureus bi-component pore-forming leukocidins are secreted toxins that directly target and lyse immune cells. Intriguingly, one of the leukocidins, Leukocidin AB (LukAB), is found associated with the bacterial cell envelope in addition to secreted into the extracellular milieu. Here, we report that retention of LukAB on the bacterial cells provides S. aureus with a pre-synthesized active toxin that kills immune cells. On the bacteria, LukAB is distributed as discrete foci in two distinct compartments: membrane-proximal and surface-exposed. Through genetic screens, we show that a membrane lipid, lysyl-phosphatidylglycerol (LPG), and lipoteichoic acid (LTA) contribute to LukAB deposition and release. Furthermore, by studying non-covalently surface-bound proteins we discovered that the sorting of additional exoproteins, such as IsaB, Hel, ScaH, and Geh, are also controlled by LPG and LTA. Collectively, our study reveals a multistep secretion system that controls exoprotein storage and protein translocation across the S. aureus cell wall.

PD-1 is a critical therapeutic target in cancer immunotherapy and antibodies blocking PD-1 are approved for multiple types of malignancies. The phosphatase SHP2 is the main effector mediating PD-1 downstream signaling and accordingly attempts have been made to target this enzyme as an alternative approach to treat immunogenic tumors. Unfortunately, small molecule inhibitors of SHP2 do not work as expected, suggesting that the role of SHP2 in T cells is more complex than initially hypothesized. To better understand the perplexing role of SHP2 in T cells, we performed interactome mapping of SAP, an adapter protein that is associated with SHP2 downstream signaling. Using genetic and pharmacological approaches, we discovered that SHP2 dephosphorylates ITK specifically downstream of PD-1 and that this event was associated with PD-1 inhibitory cellular functions. This study suggests that ITK is a unique target in this pathway, and since ITK is a SHP2-dependent specific mediator of PD-1 signaling, the combination of ITK inhibitors with PD-1 blockade may improve upon PD-1 monotherapy in the treatment of cancer.

OBJECTIVE:To identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to epilepsy control patients. METHODS:For proteomics analyses, we evaluated the hippocampus and frontal cortex from microdissected post-mortem brain tissue of 12 SUDEP and 14 non-SUDEP epilepsy patients. For transcriptomics analyses, we evaluated hippocampus and temporal cortex surgical brain tissue from mesial temporal lobe epilepsy (MTLE) patients: 6 low-risk and 8 high-risk SUDEP as determined by a short (< 50 seconds) or prolonged (≥ 50 seconds) postictal generalized EEG suppression (PGES) that may indicate severely depressed brain activity impairing respiration, arousal, and protective reflexes. RESULTS:In autopsy hippocampus and cortex, we observed no proteomic differences between SUDEP and non-SUDEP epilepsy patients, contrasting with our previously reported robust differences between epilepsy and non-epilepsy control patients. Transcriptomics in hippocampus and cortex from surgical epilepsy patients segregated by PGES identified 55 differentially expressed genes (37 protein-coding, 15 lncRNAs, three pending) in hippocampus. CONCLUSION/CONCLUSIONS:The SUDEP proteome and high-risk SUDEP transcriptome were similar to other epilepsy patients in hippocampus and frontal cortex, consistent with diverse epilepsy syndromes and comorbidities associated with SUDEP. Studies with larger cohorts and different epilepsy syndromes, as well as additional anatomic regions may identify molecular mechanisms of SUDEP.

Proteogenomics is an increasingly common method for species identification as it allows for rapid and inexpensive interrogation of an unknown organism's proteome-even when the proteome is partially degraded. The proteomic method typically uses tandem mass spectrometry to survey all peptides detectable in a sample that frequently contains hundreds or thousands of proteins. Species identification is based on detection of a small numbers of species-specific peptides. Genetic analysis of proteins by mass spectrometry, however, is a developing field, and the bone proteome, typically consisting of only two proteins, pushes the limits of this technology. Nearly 20% of highly confident spectra from modern human bone samples identify non-human species when searched against a vertebrate database-as would be necessary with a fragment of unknown bone. These non-human peptides are often the result of current limitations in mass spectrometry or algorithm interpretation errors. Consequently, it is difficult to know if a "species-specific" peptide used to identify a sample is actually present in that sample. Here we evaluate the causes of peptide sequence errors and propose an unbiased, probabilistic approach to determine the likelihood that a species is correctly identified from bone without relying on species-specific peptides.