Localized proteomic differences in the choroid plexus of Alzheimer's disease and epilepsy patients.

Introduction: Alzheimer's disease (AD) and epilepsy are reciprocally related. Among sporadic AD patients, clinical seizures occur in 10-22% and subclinical epileptiform abnormalities occur in 22-54%. Cognitive deficits, especially short-term memory impairments, occur in most epilepsy patients. Common neurophysiological and molecular mechanisms occur in AD and epilepsy. The choroid plexus undergoes pathological changes in aging, AD, and epilepsy, including decreased CSF turnover, amyloid beta (Aß), and tau accumulation due to impaired clearance and disrupted CSF amino acid homeostasis. This pathology may contribute to synaptic dysfunction in AD and epilepsy. Methods: We evaluated control (n = 8), severe AD (n = 8; A3, B3, C3 neuropathology), and epilepsy autopsy cases (n = 12) using laser capture microdissection (LCM) followed by label-free quantitative mass spectrometry on the choroid plexus adjacent to the hippocampus at the lateral geniculate nucleus level. Results: Proteomics identified 2,459 proteins in the choroid plexus. At a 5% false discovery rate (FDR), 616 proteins were differentially expressed in AD vs. control, 1 protein in epilepsy vs. control, and 438 proteins in AD vs. epilepsy. There was more variability in the epilepsy group across syndromes. The top 20 signaling pathways associated with differentially expressed proteins in AD vs. control included cell metabolism pathways; activated fatty acid beta-oxidation (p = 2.00 x 10-7, z = 3.00), and inhibited glycolysis (p = 1.00 x 10-12, z = -3.46). For AD vs. epilepsy, the altered pathways included cell metabolism pathways, activated complement system (p = 5.62 x 10-5, z = 2.00), and pathogen-induced cytokine storm (p = 2.19 x 10-2, z = 3.61). Of the 617 altered proteins in AD and epilepsy vs. controls, 497 (81%) were positively correlated (p < 0.0001, R2 = 0.27). Discussion: We found altered signaling pathways in the choroid plexus of severe AD cases and many correlated changes in the protein expression of cell metabolism pathways in AD and epilepsy cases. The shared molecular mechanisms should be investigated further to distinguish primary pathogenic changes from the secondary ones. These mechanisms could inform novel therapeutic strategies to prevent disease progression or restore normal function. A focus on dual-diagnosed AD/epilepsy cases, specific epilepsy syndromes, such as temporal lobe epilepsy, and changes across different severity levels in AD and epilepsy would add to our understanding.

Metabolomic, proteomic, and transcriptomic changes in adults with epilepsy on modified Atkins diet.

OBJECTIVE: High-fat and low-carbohydrate diets can reduce seizure frequency in some treatment-resistant epilepsy patients, including the more flexible modified Atkins diet (MAD), which is more palatable, mimicking fasting and inducing high ketone body levels. Low-carbohydrate diets may shift brain energy production, particularly impacting neuron- and astrocyte-linked metabolism. METHODS: We evaluated the effect of short-term MAD on molecular mechanisms in adult epilepsy patients from surgical brain tissue and plasma compared to control participants consuming a nonmodified higher carbohydrate diet (n = 6 MAD, mean age = 43.7 years, range = 21-53, diet for average 10 days; n = 10 control, mean age = 41.9 years, range = 28-64). RESULTS: By metabolomics, there were 13 increased metabolites in plasma (n = 15 participants with available specimens), which included 4.10-fold increased ketone body 3-hydroxybutyric acid, decreased palmitic acid in cortex (n = 16), and 11 decreased metabolites in hippocampus (n = 6), which had top associations with mitochondrial functions. Cortex and plasma 3-hydroxybutyric acid levels had a positive correlation (p = .0088, R2  = .48). Brain proteomics and RNAseq identified few differences, including 2.75-fold increased hippocampal MT-ND3 and trends (p < .01, false discovery rate > 5%) in hippocampal nicotinamide adenine dinucleotide (NADH)-related signaling pathways (activated oxidative phosphorylation and inhibited sirtuin signaling). SIGNIFICANCE: Short-term MAD was associated with metabolic differences in plasma and resected epilepsy brain tissue when compared to control participants, in combination with trending expression changes observed in hippocampal NADH-related signaling pathways. Future studies should evaluate how brain molecular mechanisms are altered with long-term MAD in a larger cohort of epilepsy patients, with correlations to seizure frequency, epilepsy syndrome, and other clinical variables. [Clinicaltrials.gov NCT02565966.].

Brain molecular mechanisms in Rasmussen encephalitis.

OBJECTIVE: This study was undertaken to identify molecular mechanisms in brain tissue of Rasmussen encephalitis (RE) when compared to people with non-RE epilepsy (PWE) and control cases using whole exome sequencing (WES), RNAseq, and proteomics. METHODS: Frozen brain tissue (ages = 2-19 years) was obtained from control autopsy (n = 14), surgical PWE (n = 10), and surgical RE cases (n = 27). We evaluated WES variants in RE associated with epilepsy, seizures, RE, and human leukocyte antigens (HLAs). Differential expression was evaluated by RNAseq (adjusted p < .05) and label-free quantitative mass spectrometry (false discovery rate < 5%) in the three groups. RESULTS: WES revealed no common pathogenic variants in RE, but several rare and likely deleterious variants of unknown significance (VUS; ANGPTL7/MTOR, SCN1A, FCGR3B, MTOR) and more common HLA VUS in >25% of RE cases (HLA-DRB1, HLA-DQA2), all with allele frequency < 5% in the general population. RNAseq in RE versus PWE (1516 altered transcripts) revealed significant activation of crosstalk between dendritic and natural killer cells (p = 7.94 × 10-6 , z = 2.65), in RE versus control (7466 transcripts) neuroinflammation signaling activation (p = 6.31 × 10-13 , z = 5.07), and in PWE versus control (945 transcripts) phagosome formation activation (p = 2.00 × 10-13 , z = 5.61). Proteomics detected fewer altered targets. SIGNIFICANCE: In RE, we identified activated immune signaling pathways and immune cell type annotation enrichment that suggest roles of the innate and adaptive immune responses, as well as HLA variants that may increase vulnerability to RE. Follow-up studies could evaluate cell type density and subregional localization associated with top targets, clinical history (neuropathology, disease duration), and whether modulating crosstalk between dendritic and natural killer cells may limit disease progression.

Raphe and ventrolateral medulla proteomics in epilepsy and sudden unexpected death in epilepsy.

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.

Pilot study evaluating everolimus molecular mechanisms in tuberous sclerosis complex and focal cortical dysplasia.

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: 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: ClinicalTrials.gov NCT02451696.

Proteomic differences in hippocampus and cortex of sudden unexplained death in childhood.

Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and complete autopsy with ancillary testing. Multiple etiologies may cause SUDC. SUDC and sudden unexpected death in epilepsy (SUDEP) share clinical and pathological features, suggesting some similarities in mechanism of death and possible abnormalities in hippocampus and cortex. To identify molecular signaling pathways, we performed label-free quantitative mass spectrometry on microdissected frontal cortex, hippocampal dentate gyrus (DG), and cornu ammonis (CA1-3) in SUDC (n = 19) and pediatric control cases (n = 19) with an explained cause of death. At a 5% false discovery rate (FDR), we found differential expression of 660 proteins in frontal cortex, 170 in DG, and 57 in CA1-3. Pathway analysis of altered proteins identified top signaling pathways associated with activated oxidative phosphorylation (p = 6.3 × 10-15, z = 4.08) and inhibited EIF2 signaling (p = 2.0 × 10-21, z = - 2.56) in frontal cortex, and activated acute phase response in DG (p = 8.5 × 10-6, z = 2.65) and CA1-3 (p = 4.7 × 10-6, z = 2.00). Weighted gene correlation network analysis (WGCNA) of clinical history indicated that SUDC-positive post-mortem virology (n = 4/17) had the most significant module in each brain region, with the top most significant associated with decreased mRNA metabolic processes (p = 2.8 × 10-5) in frontal cortex. Additional modules were associated with clinical history, including fever within 24 h of death (top: increased mitochondrial fission in DG, p = 1.8 × 10-3) and febrile seizure history (top: decreased small molecule metabolic processes in frontal cortex, p = 8.8 × 10-5) in all brain regions, neuropathological hippocampal findings in the DG (top: decreased focal adhesion, p = 1.9 × 10-3). Overall, cortical and hippocampal protein changes were present in SUDC cases and some correlated with clinical features. Our studies support that proteomic studies of SUDC cohorts can advance our understanding of the pathogenesis of these tragedies and may inform the development of preventive strategies.

Blinded review of hippocampal neuropathology in sudden unexplained death in childhood reveals inconsistent observations and similarities to explained paediatric deaths.

AIMS: Hippocampal findings are implicated in the pathogenesis of sudden unexplained death in childhood (SUDC), although some studies have identified similar findings in sudden explained death in childhood (SEDC) cases. We blindly reviewed hippocampal histology in SUDC and SEDC controls. METHODS: Hippocampal haematoxylin and eosin (H&E) slides (n = 67; 36 SUDC, 31 controls) from clinical and forensic collaborators were evaluated by nine blinded reviewers: three board-certified forensic pathologists, three neuropathologists and three dual-certified neuropathologists/forensic pathologists. RESULTS: Among nine reviewers, about 50% of hippocampal sections were rated as abnormal (52.5% SUDC, 53.0% controls), with no difference by cause of death (COD) (p = 0.16) or febrile seizure history (p = 0.90). There was little agreement among nine reviewers on whether a slide was within normal range (Fleiss' κ = 0.014, p = 0.47). Within reviewer groups, there were no findings more frequent in SUDC compared with controls, with variability in pyramidal neuron and dentate gyrus findings. Across reviewer groups, there was concordance for bilamination and granule cell loss. Neither SUDC (51.2%) nor control (55.9%) slides were considered contributory to determining COD (p = 0.41). CONCLUSIONS: The lack of an association of hippocampal findings in SUDC and controls, as well as inconsistency of observations by multiple blinded reviewers, indicates discrepancy with previous studies and an inability to reliably identify hippocampal maldevelopment associated with sudden death (HMASD). These findings underscore a need for larger studies to standardise evaluation of hippocampal findings, identifying the range of normal variation and changes unrelated to SUDC or febrile seizures. Molecular studies may help identify novel immunohistological markers that inform on COD.

Neuropathology in the North American sudden unexpected death in epilepsy registry.

Sudden unexpected death in epilepsy is the leading category of epilepsy-related death and the underlying mechanisms are incompletely understood. Risk factors can include a recent history and high frequency of generalized tonic-clonic seizures, which can depress brain activity postictally, impairing respiration, arousal and protective reflexes. Neuropathological findings in sudden unexpected death in epilepsy cases parallel those in other epilepsy patients, with no implication of novel structures or mechanisms in seizure-related deaths. Few large studies have comprehensively reviewed whole brain examination of such patients. We evaluated 92 North American Sudden unexpected death in epilepsy Registry cases with whole brain neuropathological examination by board-certified neuropathologists blinded to the adjudicated cause of death, with an average of 16 brain regions examined per case. The 92 cases included 61 sudden unexpected death in epilepsy (40 definite, 9 definite plus, 6 probable, 6 possible) and 31 people with epilepsy controls who died from other causes. The mean age at death was 34.4 years and 65.2% (60/92) were male. The average age of death was younger for sudden unexpected death in epilepsy cases than for epilepsy controls (30.0 versus 39.6 years; P = 0.006), and there was no difference in sex distribution respectively (67.3% male versus 64.5%, P = 0.8). Among sudden unexpected death in epilepsy cases, earlier age of epilepsy onset positively correlated with a younger age at death (P = 0.0005) and negatively correlated with epilepsy duration (P = 0.001). Neuropathological findings were identified in 83.7% of the cases in our cohort. The most common findings were dentate gyrus dysgenesis (sudden unexpected death in epilepsy 50.9%, epilepsy controls 54.8%) and focal cortical dysplasia (FCD) (sudden unexpected death in epilepsy 41.8%, epilepsy controls 29.0%). The neuropathological findings in sudden unexpected death in epilepsy paralleled those in epilepsy controls, including the frequency of total neuropathological findings as well as the specific findings in the dentate gyrus, findings pertaining to neurodevelopment (e.g. FCD, heterotopias) and findings in the brainstem (e.g. medullary arcuate or olivary dysgenesis). Thus, like prior studies, we found no neuropathological findings that were more common in sudden unexpected death in epilepsy cases. Future neuropathological studies evaluating larger sudden unexpected death in epilepsy and control cohorts would benefit from inclusion of different epilepsy syndromes with detailed phenotypic information, consensus among pathologists particularly for more subjective findings where observations can be inconsistent, and molecular approaches to identify markers of sudden unexpected death in epilepsy risk or pathogenesis.

Proteomics and Transcriptomics of the Hippocampus and Cortex in SUDEP and High-Risk SUDEP Patients.

OBJECTIVE: To identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to control patients with epilepsy. METHODS: For proteomics analyses, we evaluated the hippocampus and frontal cortex from microdissected postmortem brain tissue of 12 patients with SUDEP and 14 with non-SUDEP epilepsy. For transcriptomics analyses, we evaluated hippocampus and temporal cortex surgical brain tissue from patients with mesial temporal lobe epilepsy: 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 patients with SUDEP and those with non-SUDEP epilepsy, contrasting with our previously reported robust differences between epilepsy and controls without epilepsy. Transcriptomics in hippocampus and cortex from patients with surgical epilepsy segregated by PGES identified 55 differentially expressed genes (37 protein-coding, 15 long noncoding RNAs, 3 pending) in hippocampus. CONCLUSION: The SUDEP proteome and high-risk SUDEP transcriptome were similar to those in other patients with epilepsy in hippocampus and cortex, consistent with diverse epilepsy syndromes and comorbid conditions associated with SUDEP. Studies with larger cohorts and different epilepsy syndromes, as well as additional anatomic regions, may identify molecular mechanisms of SUDEP.

The HFE genotype and a formulated diet controlling for iron status attenuate experimental cerebral malaria in mice.

Plasmodium falciparum infects approximately 500million individuals each year. A small but significant number of infections lead to complications such as cerebral malaria. Cerebral malaria is associated with myelin damage and neurological deficits in survivors, and iron status is thought to impact the outcome of infection. We evaluated whether a mouse model of experimental cerebral malaria with Plasmodium berghei ANKA strain was altered by dietary iron deficiency or genetic iron overload (H67D HFE). We found that H67D mice had increased survival over H67H (wild type) mice. Moreover, a specifically designed formulation diet increased survival regardless of whether the diet was iron deficient or iron adequate. To determine potential mechanisms underlying demyelination in experimental cerebral malaria, we measured Semaphorin4A (Sema4A) protein levels in the brain because we found it is cytotoxic to oligodendrocytes. Sema4A was increased in wild type mice that developed experimental cerebral malaria while consuming standard rodent chow, consistent with a decrease in myelin basic protein, an indicator of myelin integrity. The brains of iron deficient and H67D mice had lower levels of Sema4A. Myelin basic protein was decreased in brains of mice fed the iron deficient diet as has been previously reported. We also examined erythropoietin, which is under consideration for treatment of cerebral malaria, and IL-6, which is known to increase during infection. We found that plasma erythropoietin was elevated and IL-6 was low in H67D mice and in the mice fed the formulation diets. These data reveal a paradigm-shifting concept that maintaining iron status may not increase the mortality associated with malaria and provide a dietary strategy for further examination. Moreover, the data provide clues for exploring the mechanism to limit the co-morbidity associated with experimental cerebral malaria that appears to include decreased Sema4A in brain as well as elevated erythropoietin and lower IL-6 in plasma.

Semaphorin4A Is Cytotoxic to Oligodendrocytes and Is Elevated in Microglia and Multiple Sclerosis.

We have previously established that T cell immunoglobulin and mucin domain containing 2 (Tim2) is an H-ferritin receptor on oligodendrocytes (OLs). Tim2 also binds Semaphorin4A (Sema4A). Sema4A is expressed by lymphocytes, and its role in immune activation is known; however, its relationship to diseases that are known to have myelin damage has not been studied. In this study, we demonstrate that Sema4A is cytotoxic to OLs in culture: an effect accompanied by process collapse, membrane blebbing, and phosphatidylserine inversion. We further demonstrate that Sema4A preferentially binds to primary OLs but not astrocytes: an observation consistent with the lack of expression of Tim2 on astrocytes. We found that Sema4A protein levels are increased within multiple sclerosis plaques compared with normal-appearing white matter and that Sema4A induces lactate dehydrogenase release in a human OL cell line. The chief cellular source of Sema4A within the multiple sclerosis plaques appears to be infiltrating lymphocytes and microglia. Macrophages are known to express Sema4A, so we interrogated microglia as a potential source of Sema4A in the brain. We found that rat primary microglia express Sema4A which increased after lipopolysaccharide activation. Because activated microglia accumulate iron, we determined whether iron status influenced Sema4A and found that iron chelation decreased Sema4A and iron loading increased Sema4A in activated microglia. Overall, our data implicate Sema4A in the destruction of OLs and reveal that its expression is sensitive to iron levels.

Functional roles of transferrin in the brain.

BACKGROUND: Transferrin is synthesized in the brain by choroid plexus and oligodendrocytes, but only that in the choroid plexus is secreted. Transferrin is a major iron delivery protein to the brain, but the amount transcytosed across the brain microvasculature is minimal. Transferrin is the major source of iron delivery to neurons. It may deliver iron to immature oligodendrocytes but this trophic effect declines over time while iron requirements for maintaining myelination continue. Finally, transferrin may play an important role in neurodegenerative diseases through its ability to mobilize iron. SCOPE OF REVIEW: The role of transferrin in maintaining brain iron homeostasis and the mechanism by which it enters the brain and delivers iron will be discussed. Its relevance to neurological disorders will also be addressed. MAJOR CONCLUSIONS: Transferrin is the major iron delivery protein for neurons and the microvasculature, but has a limited role for glial cells. The main source of transferrin in the brain is likely from the choroid plexus although the concentration of transferrin at any given time in the brain includes that synthesized in oligodendrocytes. Little is known about brain iron egress or the role of transferrin in this process. GENERAL SIGNIFICANCE: Neuron survival requires iron, which is predominantly delivered by transferrin. The concentration of transferrin in the cerebrospinal fluid is reflective of brain iron availability and can function as a biomarker in disease. Accumulation of iron in the brain contributes to neurodegenerative processes, thus an understanding of the role that transferrin plays in regulating brain iron homeostasis is essential. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.