Validation of a murine proteome-wide phage display library for identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

Autoantibodies to Perilipin-1 Define a Subset of Acquired Generalized Lipodystrophy.

Acquired lipodystrophy is often characterized as an idiopathic subtype of lipodystrophy. Despite suspicion of an immune-mediated pathology, biomarkers such as autoantibodies are generally lacking. Here, we used an unbiased proteome-wide screening approach to identify autoantibodies to the adipocyte-specific lipid droplet protein perilipin 1 (PLIN1) in a murine model of autoimmune polyendocrine syndrome type 1 (APS1). We then tested for PLIN1 autoantibodies in human subjects with acquired lipodystrophy with two independent severe breaks in immune tolerance (including APS1) along with control subjects using a specific radioligand binding assay and indirect immunofluorescence on fat tissue. We identified autoantibodies to PLIN1 in these two cases, including the first reported case of APS1 with acquired lipodystrophy and a second patient who acquired lipodystrophy as an immune-related adverse event following cancer immunotherapy. Lastly, we also found PLIN1 autoantibodies to be specifically enriched in a subset of patients with acquired generalized lipodystrophy (17 of 46 [37%]), particularly those with panniculitis and other features of autoimmunity. These data lend additional support to new literature that suggests that PLIN1 autoantibodies represent a marker of acquired autoimmune lipodystrophies and further link them to a break in immune tolerance.

Unveiling the autoreactome: Proteome-wide immunological fingerprints reveal the promise of plasma cell depleting therapy.

The prevalence and burden of autoimmune and autoantibody mediated disease is increasing worldwide, yet most disease etiologies remain unclear. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leverage advances in programmable-phage immunoprecipitation (PhIP-Seq) methodology to explore the modulation, or lack thereof, of proteome-wide autoantibody profiles in both health and disease. We demonstrate that each individual, regardless of disease state, possesses a distinct set of autoreactivities constituting a unique immunological fingerprint, or "autoreactome", that is remarkably stable over years. In addition to uncovering important new biology, the autoreactome can be used to better evaluate the relative effectiveness of various therapies in altering autoantibody repertoires. We find that therapies targeting B-Cell Maturation Antigen (BCMA) profoundly alter an individual's autoreactome, while anti-CD19 and CD-20 therapies have minimal effects, strongly suggesting a rationale for BCMA or other plasma cell targeted therapies in autoantibody mediated diseases.

Autoantibody discovery across monogenic, acquired, and COVID-19-associated autoimmunity with scalable PhIP-seq.

Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work (Vazquez et al., 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

ZSCAN1 Autoantibodies Are Associated with Pediatric Paraneoplastic ROHHAD.

OBJECTIVE: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation and Autonomic Dysregulation (ROHHAD), is a severe pediatric disorder of uncertain etiology resulting in hypothalamic dysfunction and frequent sudden death. Frequent co-occurrence of neuroblastic tumors have fueled suspicion of an autoimmune paraneoplastic neurological syndrome (PNS); however, specific anti-neural autoantibodies, a hallmark of PNS, have not been identified. Our objective is to determine if an autoimmune paraneoplastic etiology underlies ROHHAD. METHODS: Immunoglobulin G (IgG) from pediatric ROHHAD patients (n = 9), non-inflammatory individuals (n = 100) and relevant pediatric controls (n = 25) was screened using a programmable phage display of the human peptidome (PhIP-Seq). Putative ROHHAD-specific autoantibodies were orthogonally validated using radioactive ligand binding and cell-based assays. Expression of autoantibody targets in ROHHAD tumor and healthy brain tissue was assessed with immunohistochemistry and mass spectrometry, respectively. RESULTS: Autoantibodies to ZSCAN1 were detected in ROHHAD patients by PhIP-Seq and orthogonally validated in 7/9 ROHHAD patients and 0/125 controls using radioactive ligand binding and cell-based assays. Expression of ZSCAN1 in ROHHAD tumor and healthy human brain tissue was confirmed. INTERPRETATION: Our results support the notion that tumor-associated ROHHAD syndrome is a pediatric PNS, potentially initiated by an immune response to peripheral neuroblastic tumor. ZSCAN1 autoantibodies may aid in earlier, accurate diagnosis of ROHHAD syndrome, thus providing a means toward early detection and treatment. This work warrants follow-up studies to test sensitivity and specificity of a novel diagnostic test. Last, given the absence of the ZSCAN1 gene in rodents, our study highlights the value of human-based approaches for detecting novel PNS subtypes. ANN NEUROL 2022;92:279-291.

Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq.

Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.

Long-term MRI changes in a patient with Kelch-like protein 11-associated paraneoplastic neurological syndrome.

BACKGROUND AND PURPOSE: The aim of this study was to identify the long-term radiological changes, autoantibody specificities, and clinical course in a patient with kelch-like protein 11 (KLHL11)-associated paraneoplastic neurological syndrome (PNS). METHODS: Serial brain magnetic resonance images were retrospectively assessed. To test for KLHL11 autoantibodies, longitudinal cerebrospinal fluid (CSF) and serum samples were screened by Phage-display ImmunoPrecipitation and Sequencing (PhIP-Seq). Immunohistochemistry was also performed to assess for the presence of KLHL11 in the patient's seminoma tissue. RESULTS: A 42-year-old man presented with progressive ataxia and sensorineural hearing loss. Metastatic seminoma was detected 11 months after the onset of the neurological symptoms. Although immunotherapy was partially effective, his cerebellar ataxia gradually worsened over the next 8 years. Brain magnetic resonance imaging revealed progressive brainstem and cerebellar atrophy with a "hot-cross-bun sign", and low-signal intensity on susceptibility-weighted imaging (SWI) in the substantia nigra, red nucleus and dentate nuclei. PhIP-Seq enriched for KLHL11-derived peptides in all samples. Immunohistochemical staining of mouse brain with the patient CSF showed co-localization with a KLHL11 commercial antibody in the medulla and dentate nucleus. Immunohistochemical analysis of seminoma tissue showed anti-KLHL11 antibody-positive particles in cytoplasm. CONCLUSIONS: This study suggests that KLHL11-PNS should be included in the differential diagnosis for patients with brainstem and cerebellar atrophy and signal changes not only on T2-FLAIR but also on SWI, which might otherwise be interpreted as secondary to a neurodegenerative disease such as multiple system atrophy.

Phage display demonstrates durable differences in serological profile by route of inoculation in primary infections of non-human primates with Dengue Virus 1.

Natural dengue virus (DENV) infections occur by mosquito bite but how the inoculation route affects the humoral immune response is unknown. We serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection where animals were inoculated by mosquito (N = 10) or subcutaneous injection (N = 10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. Profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year with differences in sero-reactivity in the Envelope (E; residues 215-406; p < 0.08), and Nonstructural-3 (NS3; residues 549-615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339-384 in domain III accounted for > 99% of the observed sero-reactivity difference. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation.

High-resolution epitope mapping of anti-Hu and anti-Yo autoimmunity by programmable phage display.

Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

Expanded Clinical Phenotype, Oncological Associations, and Immunopathologic Insights of Paraneoplastic Kelch-like Protein-11 Encephalitis.

Importance: Recognizing the presenting and immunopathological features of Kelch-like protein-11 immunoglobulin G seropositive (KLHL11 IgG+) patients may aid in early diagnosis and management. Objective: To describe expanding neurologic phenotype, cancer associations, outcomes, and immunopathologic features of KLHL11 encephalitis. Design, Setting, and Participants: This retrospective tertiary care center study, conducted from October 15, 1998, to November 1, 2019, prospectively identified 31 KLHL11 IgG+ cases in the neuroimmunology laboratory. Eight were identified by retrospective testing of patients with rhomboencephalitis (confirmed by tissue-based-immunofluorescence and transfected-cell-based assays). Main Outcomes and Measures: Outcome variables included modified Rankin score and gait aid use. Results: All 39 KLHL11 IgG+ patients were men (median age, 46 years; range, 28-73 years). Initial clinical presentations were ataxia (n = 32; 82%), diplopia (n = 22; 56%), vertigo (n = 21; 54%), hearing loss (n = 15; 39%), tinnitus (n = 14; 36%), dysarthria (n = 11; 28%), and seizures (n = 9; 23%). Atypical neurologic presentations included neuropsychiatric dysfunction, myeloneuropathy, and cervical amyotrophy. Hearing loss or tinnitus preceded other neurologic deficits by 1 to 8 months in 10 patients (26%). Among patients screened for malignancy (n = 36), testicular germ-cell tumors (n = 23; 64%) or testicular microlithiasis and fibrosis concerning for regressed germ cell tumor (n = 7; 19%) were found in 83% of the patients (n = 30). In 2 patients, lymph node biopsy diagnosed metastatic lung adenocarcinoma in one and chronic lymphocytic leukemia in the other. Initial brain magnetic resonance imaging revealed T2 hyperintensities in the temporal lobe (n = 12), cerebellum (n = 9), brainstem (n = 3), or diencephalon (n = 3). Among KLHL11 IgG+ patients who underwent HLA class I and class II genotyping (n = 10), most were found to have HLA-DQB1*02:01 (n = 7; 70%) and HLA-DRB1*03:01 (n = 6; 60%) associations. A biopsied gadolinium-enhancing temporal lobe lesion demonstrated T cell-predominant inflammation and nonnecrotizing granulomas. Cerebellar biopsy (patient with chronic ataxia) and 2 autopsied brains demonstrated Purkinje neuronal loss and Bergmann gliosis, supporting early active inflammation and later extensive neuronal loss. Compared with nonautoimmune control peripheral blood mononuclear cells, cluster of differentiation (CD) 8+ and CD4+ T cells were significantly activated when patient peripheral blood mononuclear cells were cultured with KLHL11 protein. Most patients (58%) benefitted from immunotherapy and/or cancer treatment (neurological disability stabilized [n = 10] or improved [n = 9]). Kaplan-Meier curve demonstrated significantly higher probability of wheelchair dependence among patients without detectable testicular cancer. Long-term outcomes in KLHL11-IgG+ patients were similar to Ma2 encephalitis. Conclusions and Relevance: Kelch-like protein-11 IgG is a biomarker of testicular germ-cell tumor and paraneoplastic neurologic syndrome, often refractory to treatment. Described expanded neurologic phenotype and paraclinical findings may aid in its early diagnosis and treatment.

Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by proteome-wide PhIP-Seq.

The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to more common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from a cohort of people with APS1 and discovered multiple common antibody targets. These novel APS1 autoantigens exhibit tissue-restricted expression, including expression in enteroendocrine cells, pineal gland, and dental enamel. Using detailed clinical phenotyping, we find novel associations between autoantibodies and organ-restricted autoimmunity, including a link between anti-KHDC3L autoantibodies and premature ovarian insufficiency, and between anti-RFX6 autoantibodies and diarrheal-type intestinal dysfunction. Our study highlights the utility of PhIP-Seq for extensively interrogating antigenic repertoires in human autoimmunity and the importance of antigen discovery for improved understanding of disease mechanisms.

The immune system uses antibodies to fight microbes that cause disease. White blood cells pump antibodies into the bloodstream, and these antibodies latch onto bacteria and viruses, targeting them for destruction. But sometimes, the immune system gets it wrong. In autoimmune diseases, white blood cells mistakenly make antibodies that target the body's own tissues. Detecting these 'autoantibodies' in the blood can help doctors to diagnose autoimmune diseases. But the identities and targets of many autoantibodies remain unknown. In one rare disease, called autoimmune polyendocrine syndrome type 1 (APS-1), a faulty gene makes the immune system much more likely to make autoantibodies. People with this disease can develop an autoimmune response against many different healthy organs. Although APS-1 is rare, some of the autoantibodies made by individuals with the disease are the same as the ones in more common autoimmune diseases, like type 1 diabetes. Therefore, investigating the other autoantibodies produced by individuals with APS-1 could reveal the autoantibodies driving other autoimmune diseases. Autoantibodies bind to specific regions of healthy proteins, and one way to identify them is to use hundreds of thousands of tiny viruses in a technique called proteome-wide programmable phage-display, or PhIP-Seq. Each phage carries one type of protein segment. When mixed with blood serum from a patient, the autoantibodies stick to the phages that carry the target proteins for that autoantibody. These complexes can be isolated using biochemical techniques. Sequencing the genes of these phages then reveals the identity of the autoantibodies' targets. Using this technique, Vazquez et al successfully pulled 23 known autoantibodies from the serum of patients with APS-1. Then, experiments to search for new targets began. This revealed many new autoantibodies, targeting proteins found only in specific tissues. They included one that targets a protein found on cells in the gut, and another that targets a protein found on egg cells in the ovaries. Matching the PhIP-Seq data to patient symptoms confirmed that these new antibodies correlate with the features of specific autoimmune diseases. For example, patients with antibodies that targeted the gut protein were more likely to have gut symptoms, while patients with antibodies that targeted the egg cell protein were more likely to have problems with their ovaries. Further investigations using PhIP-Seq could reveal the identities of even more autoantibodies. This might pave the way for new antibody tests to diagnose autoimmune diseases and identify tissues at risk of damage. This could be useful not only for people with APS-1, but also for more common autoimmune diseases that target the same organs.

Kelch-like Protein 11 Antibodies in Seminoma-Associated Paraneoplastic Encephalitis.

A 37-year-old man with a history of seminoma presented with vertigo, ataxia, and diplopia. An autoantibody specific for kelch-like protein 11 (KLHL11) was identified with the use of programmable phage display. Immunoassays were used to identify KLHL11 IgG in 12 other men with similar neurologic features and testicular disease. Immunostaining of the patient's IgG on mouse brain tissue showed sparse but distinctive points of staining in multiple brain regions, with enrichment in perivascular and perimeningeal tissues. The onset of the neurologic syndrome preceded the diagnosis of seminoma in 9 of the 13 patients. An age-adjusted estimate of the prevalence of autoimmune KLHL11 encephalitis in Olmsted County, Minnesota, was 2.79 cases per 100,000 men. (Funded by the Rochester Epidemiology Project and others.).

Exploratory proteomic analysis implicates the alternative complement cascade in primary CNS vasculitis.

OBJECTIVE: To identify molecular correlates of primary angiitis of the CNS (PACNS) through proteomic analysis of CSF from a biopsy-proven patient cohort. METHODS: Using mass spectrometry, we quantitatively compared the CSF proteome of patients with biopsy-proven PACNS (n = 8) to CSF from individuals with noninflammatory conditions (n = 11). Significantly enriched molecular pathways were identified with a gene ontology workflow, and high confidence hits within enriched pathways (fold change >1.5 and concordant Benjamini-Hochberg-adjusted p < 0.05 on DeSeq and t test) were identified as differentially regulated proteins. RESULTS: Compared to noninflammatory controls, 283 proteins were differentially expressed in the CSF of patients with PACNS, with significant enrichment of the complement cascade pathway (C4-binding protein, CD55, CD59, properdin, complement C5, complement C8, and complement C9) and neural cell adhesion molecules. A subset of clinically relevant findings were validated by Western blot and commercial ELISA. CONCLUSIONS: In this exploratory study, we found evidence of deregulation of the alternative complement cascade in CSF from biopsy-proven PACNS compared to noninflammatory controls. More specifically, several regulators of the C3 and C5 convertases and components of the terminal cascade were significantly altered. These preliminary findings shed light on a previously unappreciated similarity between PACNS and systemic vasculitides, especially anti-neutrophil cytoplasmic antibody-associated vasculitis. The therapeutic implications of this common biology and the diagnostic and therapeutic utility of individual proteomic findings warrant validation in larger cohorts.

Zika virus cell tropism in the developing human brain and inhibition by azithromycin.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Seizure-like activity in a juvenile Angelman syndrome mouse model is attenuated by reducing Arc expression.

Angelman syndrome (AS) is a neurodevelopmental disorder arising from loss-of-function mutations in the maternally inherited copy of the UBE3A gene, and is characterized by an absence of speech, excessive laughter, cognitive delay, motor deficits, and seizures. Despite the fact that the symptoms of AS occur in early childhood, behavioral characterization of AS mouse models has focused primarily on adult phenotypes. In this report we describe juvenile behaviors in AS mice that are strain-independent and clinically relevant. We find that young AS mice, compared with their wild-type littermates, produce an increased number of ultrasonic vocalizations. In addition, young AS mice have defects in motor coordination, as well as abnormal brain activity that results in an enhanced seizure-like response to an audiogenic challenge. The enhanced seizure-like activity, but not the increased ultrasonic vocalizations or motor deficits, is rescued in juvenile AS mice by genetically reducing the expression level of the activity-regulated cytoskeleton-associated protein, Arc. These findings suggest that therapeutic interventions that reduce the level of Arc expression have the potential to reverse the seizures associated with AS. In addition, the identification of aberrant behaviors in young AS mice may provide clues regarding the neural circuit defects that occur in AS and ultimately allow new approaches for treating this disorder.

The nogo receptor family restricts synapse number in the developing hippocampus.

Neuronal development is characterized by a period of exuberant synaptic growth that is well studied. However, the mechanisms that restrict this process are less clear. Here we demonstrate that glycosylphosphatidylinositol-anchored cell-surface receptors of the Nogo Receptor family (NgR1, NgR2, and NgR3) restrict excitatory synapse formation. Loss of any one of the NgRs results in an increase in synapse number in vitro, whereas loss of all three is necessary for abnormally elevated synaptogenesis in vivo. We show that NgR1 inhibits the formation of new synapses in the postsynaptic neuron by signaling through the coreceptor TROY and RhoA. The NgR family is downregulated by neuronal activity, a response that may limit NgR function and facilitate activity-dependent synapse development. These findings suggest that NgR1, a receptor previously shown to restrict axon growth in the adult, also functions in the dendrite as a barrier that limits excitatory synapse number during brain development.

Crh and Oprm1 mediate anxiety-related behavior and social approach in a mouse model of MECP2 duplication syndrome.

Genomic duplications spanning Xq28 are associated with a spectrum of phenotypes, including anxiety and autism. The minimal region shared among affected individuals includes MECP2 and IRAK1, although it is unclear which gene when overexpressed causes anxiety and social behavior deficits. We report that doubling MECP2 levels causes heightened anxiety and autism-like features in mice and alters the expression of genes that influence anxiety and social behavior, such as Crh and Oprm1. To test the hypothesis that alterations in these two genes contribute to heightened anxiety and social behavior deficits, we analyzed MECP2 duplication mice (MECP2-TG1) that have reduced Crh and Oprm1 expression. In MECP2-TG1 animals, reducing the levels of Crh or its receptor, Crhr1, suppressed anxiety-like behavior; in contrast, reducing Oprm1 expression improved abnormal social behavior. These data indicate that increased MeCP2 levels affect molecular pathways underlying anxiety and social behavior and provide new insight into potential therapies for MECP2-related disorders.

Exercise and genetic rescue of SCA1 via the transcriptional repressor Capicua.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). To determine the long-term effects of exercise, we implemented a mild exercise regimen in a mouse model of SCA1 and found a considerable improvement in survival accompanied by up-regulation of epidermal growth factor and consequential down-regulation of Capicua, which is an ATXN1 interactor. Offspring of Capicua mutant mice bred to SCA1 mice showed significant improvement of all disease phenotypes. Although polyglutamine-expanded Atxn1 caused some loss of Capicua function, further reduction of Capicua levels--either genetically or by exercise--mitigated the disease phenotypes by dampening the toxic gain of function. Thus, exercise might have long-term beneficial effects in other ataxias and neurodegenerative diseases.