A cross-disease resource of living human microglia identifies disease-enriched subsets and tool compounds recapitulating microglial states.

Human microglia play a pivotal role in neurological diseases, but we still have an incomplete understanding of microglial heterogeneity, which limits the development of targeted therapies directly modulating their state or function. Here, we use single-cell RNA sequencing to profile 215,680 live human microglia from 74 donors across diverse neurological diseases and CNS regions. We observe a central divide between oxidative and heterocyclic metabolism and identify microglial subsets associated with antigen presentation, motility and proliferation. Specific subsets are enriched in susceptibility genes for neurodegenerative diseases or the disease-associated microglial signature. We validate subtypes in situ with an RNAscope-immunofluorescence pipeline and high-dimensional MERFISH. We also leverage our dataset as a classification resource, finding that induced pluripotent stem cell model systems capture substantial in vivo heterogeneity. Finally, we identify and validate compounds that recapitulate certain subtypes in vitro, including camptothecin, which downregulates the signature of disease-enriched subtypes and upregulates a signature previously associated with Alzheimer's disease.

A structured evaluation of cryopreservation in generating single-cell transcriptomes from cerebrospinal fluid.

Single-cell transcriptomics allows characterization of cerebrospinal fluid (CSF) cells at an unprecedented level. Here, we report a robust cryopreservation protocol adapted for the characterization of fragile CSF cells by single-cell RNA sequencing (RNA-seq) in moderate- to large-scale studies. Fresh CSF was collected from twenty-one participants at two independent sites. Each CSF sample was split into two fractions: one was processed fresh, while the second was cryopreserved for months and profiled after thawing. B and T cell receptor sequencing was also performed. Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. We find no significant difference in cell type proportions and cellular transcriptomes between fresh and cryopreserved cells. Results were comparable at both sites and with different single-cell sequencing chemistries. Cryopreservation did not affect recovery of T and B cell clonotype diversity. Our CSF cell cryopreservation protocol provides an important alternative to fresh processing of fragile CSF cells.

A multi-step genomic approach prioritized TBKBP1 gene as relevant for multiple sclerosis susceptibility.

BACKGROUND: Over 200 genetic loci have been associated with multiple sclerosis (MS) explaining ~ 50% of its heritability, suggesting that additional mechanisms may account for the "missing heritability" phenomenon. OBJECTIVE: To analyze a large cohort of Italian individuals to identify markers associated with MS with potential functional impact in the disease. METHODS: We studied 2571 MS and 3234 healthy controls (HC) of continental Italian origin. Discovery phase included a genome wide association study (1727 MS, 2258 HC), with SNPs selected according to their association in the Italian cohort only or in a meta-analysis of signals with a cohort of European ancestry (4088 MS, 7144 HC). Top associated loci were then tested in two Italian cohorts through array-based genotyping (903 MS, 884 HC) and pool-based target sequencing (588 MS, 408 HC). Finally, functional prioritization through conditional eQTL and mQTL has been performed. RESULTS: Top associated signals overlap with already known MS loci on chromosomes 3 and 17. Three SNPs (rs4267364, rs8070463, rs67919208), all involved in the regulation of TBKBP1, were prioritized to be functionally relevant. CONCLUSIONS: No evidence of novel signal of association with MS specific for the Italian continental population has been found; nevertheless, two MS loci seems to play a relevant role, raising the interest to further investigations for TBKBP1 gene.

Proximal and distal effects of genetic susceptibility to multiple sclerosis on the T cell epigenome.

Identifying the effects of genetic variation on the epigenome in disease-relevant cell types can help advance our understanding of the first molecular contributions of genetic susceptibility to disease onset. Here, we establish a genome-wide map of DNA methylation quantitative trait loci in CD4+ T-cells isolated from multiple sclerosis patients. Utilizing this map in a colocalization analysis, we identify 19 loci where the same haplotype drives both multiple sclerosis susceptibility and local DNA methylation. We also identify two distant methylation effects of multiple sclerosis susceptibility loci: a chromosome 16 locus affects PRDM8 methylation (a chromosome 4 region not previously associated with multiple sclerosis), and the aggregate effect of multiple sclerosis-associated variants in the major histocompatibility complex influences DNA methylation near PRKCA (chromosome 17). Overall, we present a new resource for a key cell type in inflammatory disease research and uncover new gene targets for the study of predisposition to multiple sclerosis.

A pharmacogenetic study implicates NINJ2 in the response to Interferon-ß in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a disease in which biomarker identification is fundamental to predict response to treatments and to deliver the optimal drug to patients. We previously found an association between rs7298096, a polymorphism upstream to the NINJ2 gene, and the 4-year response to interferon-ß (IFNß) treatment in MS patients. OBJECTIVES: To analyse the association between rs7298096 and time to first relapse (TTFR) during IFNß therapy in MS patients and to better investigate its functional role. METHODS: Survival analysis was applied in three MS cohorts from different countries (n = 1004). We also studied the role of the polymorphism on gene expression using GTEx portal and a luciferase assay. We interrogated GEO datasets to explore the relationship between NINJ2 expression, IFNß and TTFR. RESULTS: Rs7298096AA patients show a shorter TTFR than rs7298096G-carriers (Pmeta-analysis = 3 × 10-4, hazard ratio = 1.41). Moreover, rs7298096AA is associated with a higher NINJ2 expression in blood (p = 7.0 × 10-6), which was confirmed in vitro (p = 0.009). Finally, NINJ2 expression is downregulated by IFNß treatment and related to TTFR. CONCLUSIONS: Rs7298096 could influence MS disease activity during IFNß treatment by modulating NINJ2 expression in blood. The gene encodes for an adhesion molecule involved in inflammation and endothelial cells activation, supporting its role in MS.

Neuropathological correlates and genetic architecture of microglial activation in elderly human brain.

Microglia, the resident immune cells of the brain, have important roles in brain health. However, little is known about the regulation and consequences of microglial activation in the aging human brain. Here we report that the proportion of morphologically activated microglia (PAM) in postmortem cortical tissue is strongly associated with ß-amyloid, tau-related neuropathology, and the rate of cognitive decline. Effect sizes for PAM measures are substantial, comparable to that of APOE ε4, the strongest genetic risk factor for Alzheimer's disease, and mediation models support an upstream role for microglial activation in Alzheimer's disease via accumulation of tau. Further, we identify a common variant (rs2997325) influencing PAM that also affects in vivo microglial activation measured by [11C]-PBR28 PET in an independent cohort. Thus, our analyses begin to uncover pathways regulating resident neuroinflammation and identify overlaps of PAM's genetic architecture with those of Alzheimer's disease and several other traits.

Convergent effects of a functional C3 variant on brain atrophy, demyelination, and cognitive impairment in multiple sclerosis.

BACKGROUND: Complement system activation products are present in areas of neuroinflammation, demyelination, and neurodegeneration in brains of patients with multiple sclerosis (MS). C3 is a central element in the activation of complement cascades. A common coding variant in the C3 gene (rs2230199, C3R102G) affects C3 activity. OBJECTIVES: To assess the effects of rs2230199 on MS severity using clinical, cognitive, and imaging measures. METHODS: In total, 161 relapse-onset MS patients (Expanded Disability Status Scale (EDSS) ≤ 6) underwent physical assessments, cognitive tests (Paced Auditory Serial Addition Test (PASAT), Symbol Digit Modalities Test (SDMT), and California Verbal Learning Test (CVLT)), and magnetic resonance imaging (MRI). Lesion volumes were quantified semi-automatically. Voxel-wise analyses were performed to assess the effects of rs2230199 genotype on gray matter (GM) atrophy ( n = 155), white matter (WM) fractional anisotropy (FA; n = 105), and WM magnetization transfer ratio (MTR; n = 90). RESULTS: While rs2230199 minor-allele dosage (C3-102G) showed no significant effect on EDSS and Multiple Sclerosis Functional Composite (MSFC), it was associated with worse cognitive performance ( p = 0.02), lower brain parenchymal fraction ( p = 0.003), and higher lesion burden ( p = 0.02). Moreover, voxel-wise analyses showed lower GM volume in subcortical structures and insula, and lower FA and MTR in several WM areas with higher copies of rs2230199 minor allele. CONCLUSION: C3-rs2230199 affects white and GM damage as well as cognitive impairment in MS patients. Our findings support a causal role for complement system activity in the pathophysiology of MS.

Genetic influence of plasma homocysteine on Alzheimer's disease.

Observational studies have consistently reported elevated plasma homocysteine as a risk factor for Alzheimer's disease (AD). However, results from clinical trials of homocysteine-lowering treatments are inconsistent. This discrepancy may be explained by a lack of causal association between homocysteine and AD. Mendelian randomization studies have the potential to provide insight into the causality of this association through studying the effect of genetic predisposition to high homocysteine on AD. Our analyses using summarized (n = 54,162) and individual participant (n = 6987) data from Caucasian participants did not show an effect of plasma homocysteine genetic risk on susceptibility to AD. Although with smaller sample sizes, further subanalyses also did not support an effect of genetically determined plasma homocysteine on cognitive impairment and decline, beta-amyloid and tau pathology and gray matter atrophy in AD. However, we found associations with tau tangle burden (n = 251) and gray matter atrophy (n = 605) in cognitively normal elderly. Our results do not support a causal association between elevated homocysteine and risk, severity, and progression of AD. However, the relationship between genetically determined homocysteine and brain pathology in cognitively normal elderly requires further exploration.

Gray Matter Neuritic Microstructure Deficits in Schizophrenia and Bipolar Disorder.

BACKGROUND: Postmortem studies have demonstrated considerable dendritic pathologies among persons with schizophrenia and to some extent among those with bipolar I disorder. Modeling gray matter (GM) microstructural properties is now possible with a recently proposed diffusion-weighted magnetic resonance imaging modeling technique: neurite orientation dispersion and density imaging. This technique may bridge the gap between neuroimaging and histopathological findings. METHODS: We performed an extended series of multishell diffusion-weighted imaging and other structural imaging series using 3T magnetic resonance imaging. Participants scanned included individuals with schizophrenia (n = 36), bipolar I disorder (n = 29), and healthy controls (n = 35). GM-based spatial statistics was used to compare neurite orientation dispersion and density imaging-driven microstructural measures (orientation dispersion index and neurite density index [NDI]) among groups and to assess their relationship with neurocognitive performance. We also investigated the accuracy of these measures in the prediction of group membership, and whether combining them with cortical thickness and white matter fractional anisotropy further improved accuracy. RESULTS: The GM-NDI was significantly lower in temporal pole, anterior parahippocampal gyrus, and hippocampus of the schizophrenia patients than the healthy controls. The GM-NDI of patients with bipolar I disorder did not differ significantly from either schizophrenia patients or healthy controls, and it was intermediate between the two groups in the post hoc analysis. Regardless of diagnosis, higher performance in spatial working memory was significantly associated with higher GM-NDI mainly in the frontotemporal areas. The addition of GM-NDI to cortical thickness resulted in higher accuracy to predict group membership. CONCLUSIONS: GM-NDI captures brain differences in the major psychoses that are not accessible with other structural magnetic resonance imaging methods. Given the strong association of GM-NDI with disease state and neurocognitive performance, its potential utility for biological subtyping should be further explored.

Effect of Vitamin A Supplementation on fatigue and depression in Multiple Sclerosis patients: A Double-Blind Placebo-Controlled Clinical Trial.

Decreasing the population and activation of inflammatory T helper cells in multiple sclerosis (MS) patients using vitamin A derivatives (retinoic acids) has been well documented. The present study determined the effect of vitamin A supplementation on psychiatric signs in MS patients. The subjects were 101 relapsing-remitting MS patients enrolled in a placebo-controlled randomized clinical trial. The treatment group was administered 25000 IU/d retinyl palmitate (RP) for 6 months followed by 10000 IU/d RP for another 6 months. The results for baseline characteristics, modified fatigue impact scale and Beck Depression Inventory-II were recorded at the beginning and end of the one-year study. The non-normal distribution data was compared between groups using a nonparametric test and normal distribution data was analyzed using a parametric test. (ClinicalTrials.gov Identifiers: NCT01417273). The results showed significant improvement in the treatment group for fatigue (p=0.004) and depression (p=0.01). Vitamin A supplementation helped during interferon therapy in the treatment process and improved psychiatric outcomes for anti-inflammatory mechanisms.

Channelopathy-related SCN10A gene variants predict cerebellar dysfunction in multiple sclerosis.

OBJECTIVE: To determine the motor-behavioral and neural correlates of putative functional common variants in the sodium-channel NaV1.8 encoding gene (SCN10A) in vivo in patients with multiple sclerosis (MS). METHODS: We recruited 161 patients with relapsing-onset MS and 94 demographically comparable healthy participants. All patients with MS underwent structural MRI and clinical examinations (Expanded Disability Status Scale [EDSS] and Multiple Sclerosis Functional Composite [MSFC]). Whole-brain voxel-wise and cerebellar volumetry were performed to assess differences in regional brain volumes between genotype groups. Resting-state fMRI was acquired from 62 patients with MS to evaluate differences in cerebellar functional connectivity. All participants were genotyped for 4 potentially functional SCN10A polymorphisms. RESULTS: Two SCN10A polymorphisms in high linkage disequilibrium (r(2) = 0.95) showed significant association with MSFC performance in patients with MS (rs6795970: p = 6.2 × 10(-4); rs6801957: p = 0.0025). Patients with MS with rs6795970(AA) genotype performed significantly worse than rs6795970(G) carriers in MSFC (p = 1.8 × 10(-4)) and all of its subscores. This association was independent of EDSS and cerebellar atrophy. Although the genotype groups showed no difference in regional brain volumes, rs6795970(AA) carriers demonstrated significantly diminished cerebellar functional connectivity with the thalami and midbrain. No significant SCN10A-genotype effect was observed on MSFC performance in healthy participants. CONCLUSIONS: Our data suggest that SCN10A variation substantially influences functional status, including prominent effects on motor coordination in patients with MS. These findings were supported by the effects of this variant on a neural system important for motor coordination, namely cerebello-thalamic circuitry. Overall, our findings add to the emerging evidence that suggests that sodium channel NaV1.8 could serve as a target for future drug-based interventions to treat cerebellar dysfunction in MS.

Genome-wide variant by serum urate interaction in Parkinson's disease.

OBJECTIVE: Serum urate levels have been associated with risk for and progression of Parkinson's disease (PD). Urate-related compounds are therapeutic candidates in neuroprotective efforts to slow PD progression. A urate-elevating agent is currently under investigation as a potential disease-modifying strategy in people with PD. However, PD is a heterogeneous disorder, and genetic variation may explain divergence in disease severity and progression. METHODS: We conducted a genome-wide association study to identify gene variant × serum urate interaction effects on the striatal (123) I-ioflupane (DaTscan) binding ratio measured using single photon emission computed tomography in patients with possible PD from the Parkinson's Progression Markers Initiative (PPMI, n = 360). Follow-up analyses were conducted to assess gene variant × serum urate interaction effects on magnetic resonance imaging-derived regional brain volumes and clinical status. We then attempted to replicate our primary analysis in patients who entered the Parkinson Research Examination of CEP-1347 Trial (PRECEPT) with a clinical diagnosis of PD (n = 349). RESULTS: Rs1109303 (T>G) variant within the INPP5K gene on chromosome 17p13.3 demonstrated a genome-wide significant interaction with serum urate level to predict striatal dopamine transporter density among all PPMI participants (n = 359) with possible PD (p = 2.01 × 10(-8) ; after excluding participants with SWEDD [scan without evidence of dopaminergic deficit]: p = 1.12 × 10(-9) ; n = 316). Independent of striatal dopamine transporter density, similar effects on brain atrophy, bradykinesia, anxiety, and depression were observed. No effect was present in the PRECEPT sample at baseline; however, in non-SWEDD PD participants in PRECEPT (n = 309), we observed a significant longitudinal genotype × serum urate interaction effect, consistent in direction with the PPMI sample, on progression of striatal dopamine transporter density over the 22-month follow-up. INTERPRETATION: Genetic profile combined with serum urate level can be used to predict disease severity and potential disease progression in patients with PD. These results may be relevant to therapeutic efforts targeting the urate pathway.

Impact of Vitamin A Supplementation on Disease Progression in Patients with Multiple Sclerosis.

BACKGROUND: Many studies have shown that active vitamin A derivatives suppress the formation of pathogenic T cells in multiple sclerosis (MS) patients. The aim of the present study is to determine the impact of vitamin A on disease progression in MS patients. METHODS: A total of 101 relapsing-remitting MS (RRMS) patients were enrolled in a 1-year placebo-controlled randomized clinical trial. The treated group received 25000 IU/d retinyl palmitate for six month followed by 10000 IU/d retinyl palmitate for another six month. The results of the expanded disability status scale (EDSS) and multiple sclerosis functional composite (MSFC) were recorded at the beginning and the end of the study. The relapse rate was recorded during the intervention. Patients underwent baseline and follow up brain MRIs. RESULTS: The results showed "Mean ± SD" of MSFC changes in the treated group was (-0.14 ± 0.20) and in the placebo group was (-0.31 ± 0.19). MSFC was improved significantly (P < 0.001) in the treatment group. There were no significant differences between the "Mean ± SD" of EDSS changes in the treated (0.07 ± 0.23) and placebo (0.08 ± 0.23) groups (P = 0.73). There were also no significant differences between the "Mean ± SD" of annualized relapse rate in the treated group (-0.36 ± 0.56) and placebo (-0.53 ± 0.55) groups (P = 0.20). The "Mean ± SD" of enhanced lesions in the treatment (0.4 ± 1.0) and in the placebo (0.2 ± 0.6) groups were not significantly different (P = 0.26). Volume of T2 hyperintense lesions "Mean ± SD" was not significantly different between treatment (45 ± 137) and placebo (23 ± 112) groups after intervention (P = 0.23). CONCLUSION: Vitamin A improved total MSFC score in RRMS patients, but it did not change EDSS, relapse rate and brain active lesions.

The Effect of Vitamin A Supplementation on FoxP3 and TGF-ß Gene Expression in Avonex-Treated Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoinflammatory condition of the central nervous system with impaired T helper (Th)17 and regulatory T cell (Treg) balance that is involved in disease immunopathogenesis. The vitamin A active metabolite, retinoic acid, can re-establish this imbalance through the modulation of gene expression of specific nuclear receptors including Forkhead box P3 (FoxP3). At present, few data exist on the impact of vitamin A supplementation on T cell balance. This study reports the results of a clinical trial, over a 6-month period, of 36 relapsing-remitting MS (RRMS) patients that received vitamin A (25,000 IU retinyl palmitate) or placebo (one capsule of placebo per day). Peripheral blood mononuclear cells were isolated from patients, and the expression of FoxP3 and transforming growth factor (TGF)-ß gene expression was measured using real-time PCR at the beginning and end of the study. The results of this study showed that vitamin A upregulated TGF-ß and FoxP3 gene expression. Therefore, vitamin A supplementation can be considered as a new approach in MS prevention and treatment.

Classification algorithms with multi-modal data fusion could accurately distinguish neuromyelitis optica from multiple sclerosis.

Neuromyelitis optica (NMO) exhibits substantial similarities to multiple sclerosis (MS) in clinical manifestations and imaging results and has long been considered a variant of MS. With the advent of a specific biomarker in NMO, known as anti-aquaporin 4, this assumption has changed; however, the differential diagnosis remains challenging and it is still not clear whether a combination of neuroimaging and clinical data could be used to aid clinical decision-making. Computer-aided diagnosis is a rapidly evolving process that holds great promise to facilitate objective differential diagnoses of disorders that show similar presentations. In this study, we aimed to use a powerful method for multi-modal data fusion, known as a multi-kernel learning and performed automatic diagnosis of subjects. We included 30 patients with NMO, 25 patients with MS and 35 healthy volunteers and performed multi-modal imaging with T1-weighted high resolution scans, diffusion tensor imaging (DTI) and resting-state functional MRI (fMRI). In addition, subjects underwent clinical examinations and cognitive assessments. We included 18 a priori predictors from neuroimaging, clinical and cognitive measures in the initial model. We used 10-fold cross-validation to learn the importance of each modality, train and finally test the model performance. The mean accuracy in differentiating between MS and NMO was 88%, where visible white matter lesion load, normal appearing white matter (DTI) and functional connectivity had the most important contributions to the final classification. In a multi-class classification problem we distinguished between all of 3 groups (MS, NMO and healthy controls) with an average accuracy of 84%. In this classification, visible white matter lesion load, functional connectivity, and cognitive scores were the 3 most important modalities. Our work provides preliminary evidence that computational tools can be used to help make an objective differential diagnosis of NMO and MS.

Impact of Melatonin on Motor, Cognitive and Neuroimaging Indices in Patients with Multiple Sclerosis.

A series of preclinical and clinical studies have shown the immunomodulatory effect of  melatonin, especially in the state of chronic inflammation. A double-blind, randomized, parallel-group, placebo-controlled clinical trial was designed to study the tolerability and efficacy of supplemental therapy with melatonin (3 mg/day) in comparison to placebo in relapsing-remitting MS (RRMS) patients receiving once weekly interferon beta. Patients were followed up for 12 months. Primary outcomes consisted of the number of relapses, change in Extended Disability Status Scale (EDSS), and the number and volume of new T2 and gadolinium-enhancing brain lesions. Secondary outcomes included change in performance on Multiple Sclerosis Functional Composite (MSFC) as well as change in fatigue and depression. The outcomes were evaluated every three months. Twenty-six patients (13 in each group) were recruited in the study. All participants, except for one patient in the placebo group, completed the study. No patient reported serious adverse events. There was no significant difference either in primary or secondary outcomes between melatonin and placebo arm. However, a trend for beneficial effect was observed for melatonin on change in MSFC performance and the cognitive subscore of the Modified Fatigue Impact Scale (p=0.05 and 0.006, respectively, not corrected for multiple comparisons). We found no significant effect for treatment with melatonin on measures of clinical and functional disability and development of brain lesions in our small sample-size study. Studies with higher statistical power and longer follow up are needed to further evaluate the potential immunomodulatory effect of melatonin in RRMS treatment.

The human cerebellum: a review of physiologic neuroanatomy.

The cerebellum resides in the posterior cranial fossa dorsal to the brainstem and has diverse connections to the cerebrum, brain stem, and spinal cord. It is anatomically and physiologically divided into distinct functional compartments and is composed of highly regular arrays of neuronal units, each sharing the same basic cerebellar microcircuitry. Its circuitry is critically involved in motor control and motor learning, and its role in nonmotor cognitive and affective functions is becoming increasingly recognized. This article describes the cerebellar gross and histologic neuroanatomy in relation to its function, and the relevance of cerebellar circuitry and firing patterns to motor learning.

Imaging proteomics for diagnosis, monitoring and prediction of Alzheimer's disease.

Proteomic and imaging markers have been widely studied as potential biomarkers for diagnosis, monitoring and prognosis of Alzheimer's disease. In this study, we used Alzheimer Disease Neuroimaging Initiative dataset and performed parallel independent component analysis on cross sectional and longitudinal proteomic and imaging data in order to identify the best proteomic model for diagnosis, monitoring and prediction of Alzheimer disease (AD). We used plasma proteins measurement and imaging data from AD and healthy controls (HC) at the baseline and 1 year follow-up. Group comparisons at baseline and changes over 1 year were calculated for proteomic and imaging data. The results were fed into parallel independent component analysis in order to identify proteins that were associated with structural brain changes cross sectionally and longitudinally. Regression model was used to find the best model that can discriminate AD from HC, monitor AD and to predict MCI converters from non-converters. We showed that five proteins are associated with structural brain changes in the brain. These proteins could discriminate AD from HC with 57% specificity and 89% sensitivity. Four proteins whose change over 1 year were associated with brain structural changes could discriminate AD from HC with sensitivity of 93%, and specificity of 92%. This model predicted MCI conversion to AD in 2 years with 94% accuracy. This model has the highest accuracy in prediction of MCI conversion to AD within the ADNI-1 dataset. This study shows that combination of selected plasma protein levels and MR imaging is a useful method in identifying potential biomarker.