Cerebrospinal fluid proteomics indicates immune dysregulation and neuronal dysfunction in antibody associated autoimmune encephalitis.

Autoimmune Encephalitis (AE) spans a group of non-infectious inflammatory conditions of the central nervous system due to an imbalanced immune response. Aiming to elucidate the pathophysiological mechanisms of AE, we applied an unsupervised proteomic approach to analyze the cerebrospinal fluid (CSF) protein profile of AE patients with autoantibodies against N-methyl-d-aspartate receptor (NMDAR) (n = 9), leucine-rich glioma-inactivated protein 1 (LGI1) (n = 9), or glutamate decarboxylase 65 (GAD65) (n = 8) compared to 9 patients with relapsing-remitting multiple sclerosis as inflammatory controls, and 10 patients with somatic symptom disorder as non-inflammatory controls. We found a dysregulation of the complement system, a disbalance between pro-inflammatory and anti-inflammatory proteins on the one hand, and dysregulation of proteins involved in synaptic transmission, synaptogenesis, brain connectivity, and neurodegeneration on the other hand to a different extent in all AE subtypes compared to non-inflammatory controls. Furthermore, elevated levels of several proteases and reduction in protease inhibitors could be detected in all AE subtypes compared to non-inflammatory controls. Moreover, the different AE subtypes showed distinct protein profiles compared to each other and inflammatory controls which may facilitate future identification of disease-specific biomarkers. Overall, CSF proteomics provides insights into the complex pathophysiological mechanisms of AE, including immune dysregulation, neuronal dysfunction, neurodegeneration, and altered protease function.

Cerebrospinal fluid flow cytometry distinguishes psychosis spectrum disorders from differential diagnoses.

Psychotic disorders are common and disabling mental conditions. The relative importance of immune-related mechanisms in psychotic disorders remains subject of debate. Here, we present a large-scale retrospective study of blood and cerebrospinal fluid (CSF) immune cell profiles of psychosis spectrum patients. We performed basic CSF analysis and multi-dimensional flow cytometry of CSF and blood cells from 59 patients with primary psychotic disorders (F20, F22, F23, and F25) in comparison to inflammatory (49 RRMS and 16 NMDARE patients) and non-inflammatory controls (52 IIH patients). We replicated the known expansion of monocytes in the blood of psychosis spectrum patients, that we identified to preferentially affect classical monocytes. In the CSF, we found a relative shift from lymphocytes to monocytes, increased protein levels, and evidence of blood-brain barrier disruption in psychosis. In fact, these CSF features confidently distinguished autoimmune encephalitis from psychosis despite similar (initial) clinical features. We then constructed machine learning models incorporating blood and CSF parameters and demonstrated their superior ability to differentiate psychosis from non-inflammatory controls compared to individual parameters. Multi-dimensional and multi-compartment immune cell signatures can thus support the diagnosis of psychosis spectrum disorders with the potential to accelerate diagnosis and initiation of therapy.

Exploring cellular markers of metabolic syndrome in peripheral blood mononuclear cells across the neuropsychiatric spectrum.

Recent evidence suggests that comorbidities between neuropsychiatric conditions and metabolic syndrome may precede and even exacerbate long-term side-effects of psychiatric medication, such as a higher risk of type 2 diabetes and cardiovascular disease, which result in increased mortality. In the present study we compare the expression of key metabolic proteins, including the insulin receptor (CD220), glucose transporter 1 (GLUT1) and fatty acid translocase (CD36), on peripheral blood mononuclear cell subtypes from patients across the neuropsychiatric spectrum, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions (n = 25/condition), relative to typical controls (n = 100). This revealed alterations in the expression of these proteins that were specific to schizophrenia. Further characterization of metabolic alterations in an extended cohort of first-onset antipsychotic drug-naïve schizophrenia patients (n = 58) and controls (n = 63) revealed that the relationship between insulin receptor expression in monocytes and physiological insulin sensitivity was disrupted in schizophrenia and that altered expression of the insulin receptor was associated with whole genome polygenic risk scores for schizophrenia. Finally, longitudinal follow-up of the schizophrenia patients over the course of antipsychotic drug treatment revealed that peripheral metabolic markers predicted changes in psychopathology and the principal side effect of weight gain at clinically relevant time points. These findings suggest that peripheral blood cells can provide an accessible surrogate model for metabolic alterations in schizophrenia and have the potential to stratify subgroups of patients with different clinical outcomes or a greater risk of developing metabolic complications following antipsychotic therapy.

Exploring the neuropsychiatric spectrum using high-content functional analysis of single-cell signaling networks.

Neuropsychiatric disorders overlap in symptoms and share genetic risk factors, challenging their current classification into distinct diagnostic categories. Novel cross-disorder approaches are needed to improve our understanding of the heterogeneous nature of neuropsychiatric diseases and overcome existing bottlenecks in their diagnosis and treatment. Here we employ high-content multi-parameter phospho-specific flow cytometry, fluorescent cell barcoding and automated sample preparation to characterize ex vivo signaling network responses (n = 1764) measured at the single-cell level in B and T lymphocytes across patients diagnosed with four major neuropsychiatric disorders: autism spectrum condition (ASC), bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ; n = 25 each), alongside matched healthy controls (n = 100). We identified 25 nodes (individual cell subtype-epitope-ligand combinations) significantly altered relative to the control group, with variable overlap between different neuropsychiatric diseases and heterogeneously expressed at the level of each individual patient. Reconstruction of the diagnostic categories from the altered nodes revealed an overlapping neuropsychiatric spectrum extending from MDD on one end, through BD and SCZ, to ASC on the other end. Network analysis showed that although the pathway structure of the epitopes was broadly preserved across the clinical groups, there were multiple discrete alterations in network connectivity, such as disconnections within the antigen/integrin receptor pathway and increased negative regulation within the Akt1 pathway in CD4+ T cells from ASC and SCZ patients, in addition to increased correlation of Stat1 (pY701) and Stat5 (pY694) responses in B cells from BD and MDD patients. Our results support the "dimensional" approach to neuropsychiatric disease classification and suggest potential novel drug targets along the neuropsychiatric spectrum.

Clinical characteristics of inflammation-associated depression: Monocyte gene expression is age-related in major depressive disorder.

Increased inflammatory activation might only be present in a subgroup of depressed individuals in which immune processes are especially relevant to disease development. We aimed to analyze demographic, depression, and trauma characteristics of major depressive disorder (MDD) patients with regard to inflammatory monocyte gene expression. Fifty-six naturalistically treated MDD patients (32 ± 12 years) and 57 healthy controls (HC; 31 ± 11 years) were analyzed by the Inventory of Depressive Symptomatology (IDS) and by the Childhood Trauma Questionnaire (CTQ). We determined the expression of 38 inflammatory and immune activation genes including the glucocorticoid receptor (GR)α and GRß genes in purified CD14(+) monocytes using quantitative-polymerase chain reaction (RT-qPCR). Monocyte gene expression was age-dependent, particularly in MDD patients. Increased monocyte gene expression and decreased GRα/ß ratio were only present in MDD patients aged ⩾ 28 years. Post hoc analyses of monocyte immune activation in patients <28 years showed two subgroups: a subgroup with a severe course of depression (recurrent type, onset <15 years) - additionally characterized by panic/arousal symptoms and childhood trauma - that had a monocyte gene expression similar to HC, and a second subgroup with a milder course of the disorder (73% first episode depression, onset ⩾15 years) - additionally characterized by the absence of panic symptoms - that exhibited a strongly reduced inflammatory monocyte activation compared to HC. In conclusion, monocyte immune activation was not uniformly raised in MDD patients but was increased only in patients of 28 years and older.

Diagnostic model development for schizophrenia based on peripheral blood mononuclear cell subtype-specific expression of metabolic markers.

A significant proportion of the personal and economic burden of schizophrenia can be attributed to the late diagnosis or misdiagnosis of the disorder. A novel, objective diagnostic approaches could facilitate the early detection and treatment of schizophrenia and improve patient outcomes. In the present study, we aimed to identify robust schizophrenia-specific blood biomarkers, with the goal of developing an accurate diagnostic model. The levels of selected serum and peripheral blood mononuclear cell (PBMC) markers relevant to metabolic and immune function were measured in healthy controls (n = 26) and recent-onset schizophrenia patients (n = 36) using multiplexed immunoassays and flow cytometry. Analysis of covariance revealed significant upregulation of insulin receptor (IR) and fatty acid translocase (CD36) levels in T helper cells (F = 10.75, P = 0.002, Q = 0.024 and F = 21.58, P = 2.8 × 10-5, Q = 0.0004, respectively), as well as downregulation of glucose transporter 1 (GLUT1) expression in monocytes (F = 21.46, P = 2.9 × 10-5, Q = 0.0004). The most robust predictors, monocyte GLUT1 and T helper cell CD36, were used to develop a diagnostic model, which showed a leave-one-out cross-validated area under the receiver operating characteristic curve (AUC) of 0.78 (95% CI: 0.66-0.92). The diagnostic model was validated in two independent datasets. The model was able to distinguish first-onset, drug-naïve schizophrenia patients (n = 34) from healthy controls (n = 39) with an AUC of 0.75 (95% CI: 0.64-0.86), and also differentiated schizophrenia patients (n = 22) from patients with other neuropsychiatric conditions, including bipolar disorder, major depressive disorder and autism spectrum disorder (n = 68), with an AUC of 0.83 (95% CI: 0.75-0.92). These findings indicate that PBMC-derived biomarkers have the potential to support an accurate and objective differential diagnosis of schizophrenia.

Immune Cell Profiling of the Cerebrospinal Fluid Provides Pathogenetic Insights Into Inflammatory Neuropathies.

Objective: Utilize immune cell profiles in the cerebrospinal fluid (CSF) to advance the understanding and potentially support the diagnosis of inflammatory neuropathies. Methods: We analyzed CSF cell flow cytometry data of patients with definite Guillain-Barré syndrome (GBS, n = 26) and chronic inflammatory demyelinating polyneuropathy (CIDP, n = 32) based on established diagnostic criteria in comparison to controls with relapsing-remitting multiple sclerosis (RRMS, n = 49) and idiopathic intracranial hypertension (IIH, n = 63). Results: Flow cytometry revealed disease-specific changes of CSF cell composition with a significant increase of NKT cells and CD8+ T cells in CIDP, NK cells in GBS, and B cells and plasma cells in MS in comparison to IIH controls. Principal component analysis demonstrated distinct CSF immune cells pattern in inflammatory neuropathies vs. RRMS. Systematic receiver operator curve (ROC) analysis identified NKT cells as the best parameter to distinguish GBS from CIDP. Composite scores combing several of the CSF parameters differentiated inflammatory neuropathies from IIH and GBS from CIDP with high confidence. Applying a novel dimension reduction technique, we observed an intra-disease heterogeneity of inflammatory neuropathies. Conclusion: Inflammatory neuropathies display disease- and subtype-specific alterations of CSF cell composition. The increase of NKT cells and CD8+ T cells in CIDP and NK cells in GBS, suggests a central role of cytotoxic cell types in inflammatory neuropathies varying between acute and chronic subtypes. Composite scores constructed from multi-dimensional CSF parameters establish potential novel diagnostic tools. Intra-disease heterogeneity suggests distinct disease mechanisms in subgroups of inflammatory neuropathies.

Cerebrospinal Fluid Concentrations of Neuronal Proteins Are Reduced in Primary Angiitis of the Central Nervous System.

Primary angiitis of the central nervous system (PACNS) is a rare autoimmune vasculitis limited to the CNS often causing substantial disability. Understanding of this disease is impaired by the lack of available biomaterial. Here, we collected cerebrospinal fluid (CSF) from patients with PACNS and matched controls and performed unbiased proteomics profiling using ion mobility mass spectrometry to identify novel disease mechanisms and candidate biomarkers. We identified 14 candidate proteins, including amyloid-beta A4 protein (APP), with reduced abundance in the CSF of PACNS patients and validated APP by Enzyme-linked Immunosorbent Assay (ELISA) in an extended cohort of patients with PACNS. Subsequent functional annotation surprisingly suggested neuronal pathology rather than immune activation in PACNS. Our study is the first to employ mass spectrometry to local immune reactions in PACNS and it identifies candidates such as APP with pathogenic relevance in PACNS to improve patient care in the future.

Lithium reverses behavioral and axonal transport-related changes associated with ANK3 bipolar disorder gene disruption.

Ankyrin 3 (ANK3) has been implicated as a genetic risk factor for bipolar disorder (BD), however the resulting pathophysiological and treatment implications remain elusive. In a preclinical systems biological approach, we aimed to characterize the behavioral and proteomic effects of Ank3 haploinsufficiency and chronic mood-stabilizer treatment in mice. Psychiatric-related behavior was evaluated with the novelty-suppressed feeding (NSF) paradigm, elevated plus maze (EPM) and a passive avoidance task (PAT). Tandem mass spectrometry (MSE) was employed for hippocampal proteome profiling. A functional enrichment approach based on protein-protein interactions (PPIs) was performed to outline which biological processes in the hippocampus were affected by Ank3 haploinsufficiency and lithium treatment. Proteomic abundance changes as detected by MSE or highlighted by PPI network modelling were followed up by targeted selected reaction monitoring (SRM). Increased psychiatric-related behavior in Ank3+/- mice was ameliorated by lithium in all assessments (NSF, EPM, PAT). MSE followed by modular PPI clustering and functional annotation enrichment pointed towards kinesin-related axonal transport and glutamate signaling as mediators of Ank3+/- pathophysiology and lithium treatment. SRM validated this hypothesis and further confirmed abundance changes of ANK3 interaction partners. We propose that psychiatric-related behavior in Ank3+/- mice is connected to a disturbance of the kinesin cargo system, resulting in a dysfunction of neuronal ion channel and glutamate receptor transport. Lithium reverses this molecular signature, suggesting the promotion of anterograde kinesin transport as part of its mechanism of action in ameliorating Ank3-related psychiatric-related behavior.

Impact of electroconvulsive therapy on magnetoencephalographic correlates of dysfunctional emotional processing in major depression.

In major depressive disorder (MDD), electrophysiological and imaging studies provide evidence for a reduced neural activity in parietal and dorsolateral prefrontal regions. In the present study, neural correlates and temporal dynamics of visual affective perception have been investigated in patients with unipolar depression in a pre/post treatment design using magnetoencephalography (MEG). Nineteen in-patients and 19 balanced healthy controls passed MEG measurement while passively viewing pleasant, unpleasant and neutral pictures. After a 4-week treatment with electroconvulsive therapy or 4-week waiting period without intervention respectively, 16 of these patients and their 16 corresponding controls participated in a second MEG measurement. Before treatment neural source estimations of magnetic fields evoked by the emotional scenes revealed a general bilateral parietal hypoactivation in depressed patients compared to controls predominately at early and mid-latency time intervals. Successful ECT treatment, as reflected by a decline in clinical scores (Hamilton Depression Scale; HAMD) led to a normalization of this distinct parietal hypoactivation. Effective treatment was also accompanied by relatively increased neural activation at right temporo-parietal regions. The present study indicates dysfunctional parietal information processing and attention processes towards emotional stimuli in MDD patients which can be returned to normal by ECT treatment. Since convergent neural hypoactivations and treatment effects have recently been shown in MDD patients before and after pharmacological therapy, this electrophysiological correlate might serve as a biomarker for objective treatment evaluation and thereby potentially advance treatment options and support the prediction of individual treatment responses.

Molecular serum signature of treatment resistant depression.

RATIONALE: A substantial number of patients suffering from major depressive disorder (MDD) do not respond to multiple trials of anti-depressants, develop a chronic course of disease and become treatment resistant. Most of the studies investigating molecular changes in treatment-resistant depression (TRD) have only examined a limited number of molecules and genes. Consequently, biomarkers associated with TRD are still lacking. OBJECTIVES: This study aimed to use recently advanced high-throughput proteomic platforms to identify peripheral biomarkers of TRD defined by two staging models, the Thase and Rush staging model (TRM) and the Maudsley Staging Model (MSM). METHODS: Serum collected from an inpatient cohort of 65 individuals suffering from MDD was analysed using two different mass spectrometric-based platforms, label-free liquid chromatography mass spectrometry (LC-MS(E)) and selective reaction monitoring (SRM), as well as a multiplex bead based assay. RESULTS: In the LC-MS(E) analysis, proteins involved in the acute phase response and complement activation and coagulation were significantly different between the staging groups in both models. In the multiplex bead-based assay analysis TNF-α levels (log(odds) = -4.95, p = 0.045) were significantly different in the TRM comparison. Using SRM, significant changes of three apolipoproteins A-I (ß = 0.029, p = 0.035), M (ß = -0.017, p = 0.009) and F (ß = -0.031, p = 0.024) were associated with the TRM but not the MSM. CONCLUSION: Overall, our findings suggest that proteins, which are involved in immune and complement activation, may represent potential biomarkers that could be used by clinicians to identify high-risk patients. Nevertheless, given that the molecular changes between the staging groups were subtle, the results need to be interpreted cautiously.

Neuropeptide S receptor gene variation modulates anterior cingulate cortex Glx levels during CCK-4 induced panic.

An excitatory-inhibitory neurotransmitter dysbalance has been suggested in pathogenesis of panic disorder. The neuropeptide S (NPS) system has been implicated in modulating GABA and glutamate neurotransmission in animal models and to genetically drive altered fear circuit function and an increased risk of panic disorder in humans. Probing a multi-level imaging genetic risk model of panic, in the present magnetic resonance spectroscopy (MRS) study brain glutamate+glutamine (Glx) levels in the bilateral anterior cingulate cortex (ACC) during a pharmacological cholecystokinin tetrapeptide (CCK-4) panic challenge were assessed depending on the functional neuropeptide S receptor gene (NPSR1) rs324981 A/T variant in a final sample of 35 healthy male subjects. The subjective panic response (Panic Symptom Scale; PSS) as well as cortisol and ACTH levels were ascertained throughout the experiment. CCK-4 injection was followed by a strong panic response. A significant time×genotype interaction was detected (p=.008), with significantly lower ACC Glx/Cr levels in T allele carriers as compared to AA homozygotes 5min after injection (p=.003). CCK-4 induced significant HPA axis stimulation, but no effect of genotype was discerned. The present pilot data suggests NPSR1 gene variation to modulate Glx levels in the ACC during acute states of stress and anxiety, with blunted, i.e. possibly maladaptive ACC glutamatergic reactivity in T risk allele carriers. Our results underline the notion of a genetically driven rapid and dynamic response mechanism in the neural regulation of human anxiety and further strengthen the emerging role of the NPS system in anxiety.

Applications of blood-based protein biomarker strategies in the study of psychiatric disorders.

Major psychiatric disorders such as schizophrenia, major depressive and bipolar disorders are severe, chronic and debilitating, and are associated with high disease burden and healthcare costs. Currently, diagnoses of these disorders rely on interview-based assessments of subjective self-reported symptoms. Early diagnosis is difficult, misdiagnosis is a frequent occurrence and there are no objective tests that aid in the prediction of individual responses to treatment. Consequently, validated biomarkers are urgently needed to help address these unmet clinical needs. Historically, psychiatric disorders are viewed as brain disorders and consequently only a few researchers have as yet evaluated systemic changes in psychiatric patients. However, promising research has begun to challenge this concept and there is an increasing awareness that disease-related changes can be traced in the peripheral system which may even be involved in the precipitation of disease onset and course. Converging evidence from molecular profiling analysis of blood serum/plasma have revealed robust molecular changes in psychiatric patients, suggesting that these disorders may be detectable in other systems of the body such as the circulating blood. In this review, we discuss the current clinical needs in psychiatry, highlight the importance of biomarkers in the field, and review a representative selection of biomarker studies to highlight opportunities for the implementation of personalized medicine approaches in the field of psychiatry. It is anticipated that the implementation of validated biomarker tests will not only improve the diagnosis and more effective treatment of psychiatric patients, but also improve prognosis and disease outcome.

Acute shift in glutamate concentrations following experimentally induced panic with cholecystokinin tetrapeptide--a 3T-MRS study in healthy subjects.

According to preclinical studies, glutamate has been implicated in the pathogenesis of anxiety. In order to elucidate the role of glutamate in anxiety and panic in humans, brain glutamate+glutamine (Glx) levels were measured during cholecystokinin-tetrapeptide (CCK-4)-induced panic using magnetic resonance spectroscopy (MRS). Eighteen healthy subjects underwent a CCK-4 challenge. MR spectra were obtained from the anterior cingulate cortex (ACC) using a single voxel point-resolved spectroscopy method and analyzed using LCModel. A combined fitting of Glx was performed. Panic was assessed using the Acute Panic Inventory (API) and Panic Symptom Scale (PSS) scores. Moreover, hypothalamic-pituitary-adrenal axis stimulation was monitored throughout the challenge. There was a significant panic response following CCK-4 as revealed by a marked increase in both the panic scores (API: F(1,17)=149.41; p<0.0001; PSS: F(1,17)=88.03; p<0.0001) and heart rate (HR: F(1,17)=72.79; p<0.0001). MRS measures showed a significant increase of brain Glx/creatine (Glx/Cr) levels peaking at 2-10 min after challenge (F(1,17)=15.94; p=0.001). There was also a significant increase in CCK-4-related cortisol release (F(6,11)=8.68; p=0.002). Finally, significant positive correlations were found between baseline Glx/Cr and both APImax (r=0.598; p=0.009) and maximum heart rate (HR(max)) during challenge (r=0.519; p=0.027). Our results suggest that CCK-4-induced panic is accompanied by a significant glutamate increase in the bilateral ACC. The results add to the hypothesis of a disturbance of the inhibitory-excitatory equilibrium and suggest that apart from static alterations rapid and dynamic neurochemical changes might also be relevant for the neural control of panic attacks.