Human Microglia-Like Cells Differentiated from Monocytes with GM-CSF and IL-34 Show Phagocytosis of α-Synuclein Aggregates and C/EBPß-Dependent Proinflammatory Activation.

Microglia, the main resident immune cells in the central nervous system, are implicated in the pathogenesis of various neurological disorders. Much of our knowledge on microglial biology was obtained using rodent microglial cultures. To understand the role of microglia in human disease, reliable in vitro models of human microglia are necessary. Monocyte-derived microglia-like cells (MDMi) are a promising approach. This study aimed to characterize MDMi cells generated from adult human monocytes using granulocyte-macrophage colony-stimulating factor and interleukin-34. To this end, 49 independent cultures of MDMI were prepared, and various methodological and functional studies were performed. We show that with this protocol, adult human monocytes develop into microglia-like cells, a coating is unnecessary, and high cell density seeding is preferable. When compared to monocytes, MDMi upregulate the expression of many, but not all, microglial markers, indicating that, although these cells display a microglia-like phenotype, they cannot be considered bona fide human microglia. At the functional level, MDMi phagocytose α-synuclein aggregates and responds to lipopolysaccharide (LPS) by nuclear translocation of the transcription factor nuclear factor-kappaB (NFkappaB) and the upregulation of proinflammatory genes. Finally, a long-lasting silencing of the transcription factor CCAAT/enhancer protein ß (C/EBPß) was achieved by small interfering RNA, resulting in the subsequent downregulation of proinflammatory genes. This supports the hypothesis that C/EBPß plays a key role in proinflammatory gene program activation in human microglia. Altogether, this study sheds new light on the properties of MDMi cells and supports these cells as a promising in vitro model for studying adult human microglia-like cells.

Biofluid Biomarkers in the Prognosis of Amyotrophic Lateral Sclerosis: Recent Developments and Therapeutic Applications.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the degeneration of motor neurons for which effective therapies are lacking. One of the most explored areas of research in ALS is the discovery and validation of biomarkers that can be applied to clinical practice and incorporated into the development of innovative therapies. The study of biomarkers requires an adequate theoretical and operational framework, highlighting the "fit-for-purpose" concept and distinguishing different types of biomarkers based on common terminology. In this review, we aim to discuss the current status of fluid-based prognostic and predictive biomarkers in ALS, with particular emphasis on those that are the most promising ones for clinical trial design and routine clinical practice. Neurofilaments in cerebrospinal fluid and blood are the main prognostic and pharmacodynamic biomarkers. Furthermore, several candidates exist covering various pathological aspects of the disease, such as immune, metabolic and muscle damage markers. Urine has been studied less often and should be explored for its possible advantages. New advances in the knowledge of cryptic exons introduce the possibility of discovering new biomarkers. Collaborative efforts, prospective studies and standardized procedures are needed to validate candidate biomarkers. A combined biomarkers panel can provide a more detailed disease status.

Mesoporous silica particles are phagocytosed by microglia and induce a mild inflammatory response in vitro.

Aim: Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses to MSPs of astrocytes and microglia, the two main cellular players in neuroinflammation. Materials & methods: Primary murine cortical mixed glial cultures were treated with rhodamine B-labeled MSPs. Results: MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. MSPs also do not affect mRNA levels of key proinflammatory genes; however, in combination with lipopolysaccharide, they significantly increase extracellular IL-1ß levels. Conclusion: These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells.

Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses of two types of brain cells, astrocytes and microglia, to MSPs. Mouse astrocytes and microglia were kept alive in cultures and were treated with MSPs that were labeled with a red fluorescent agent to facilitate visualization under the microscope. MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. When given alone, MSPs do not affect mRNA levels of key proinflammatory genes. However, MSPs given in combination with lipopolysaccharide, a strong proinflammatory agent, significantly increase extracellular levels of IL-1ß, one of the proinflammatory mediators studied. These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells.

HLA-DQB1*05:02, *05:03, and *03:01 alleles as risk factors for myasthenia gravis in a Spanish cohort.

BACKGROUND: Myasthenia gravis (MG) is a very heterogenic chronic autoimmune disease caused by the failure of neuromuscular transmission. The HLA gene complex has conventionally been recognized as its main genetic risk and phenotype modifying factor. Our aim was to investigate the prevalence of HLA class I and II alleles and to identify possible risk factors for sporadic MG in a Spanish cohort. METHODS: We designed a clinical case-control study comparing HLA alleles and haplotype frequencies in a cohort of 234 patients with sporadic autoimmune MG with data from a group of 492 randomly selected healthy subjects. Using a high-resolution next-generation sequencing (NGS)-based HLA genotyping assay, we investigated the contribution of HLA genotypes and haplotypes in the resulting phenotype, especially, the age at onset, sex, onset MGFA class, thymic histopathology, and serological status. RESULTS: We found that the DQB1*05:02 and DQB1*05:03 alleles could be novel risk factors for Spanish MG cases. The HLA alleles A*01:01, B*08:01, DRB1*03:01, DRB1*14:54, and DQB1*02:01 were also risk factors for the disease. DQB1*03:01 acted as a risk factor for EOMG in women with AChR-positive antibodies and thymus hyperplasia. Additionally, several alleles were identified as potential phenotype-modifying factors that could exert a protective effect: HLA-B*35:08, DRB1*13:01, and DQB1*06:03 in MG; HLA-A*24:02 in women and DRB1*07:01 and DQB1*02:02 for early onset. HLA-C*07:01 and haplotype A1-B8-C7-DR3-DQ2 were associated with an early-onset phenotype.

Altered expression of the immunoregulatory ligand-receptor pair CD200-CD200R1 in the brain of Parkinson's disease patients.

Neuroinflammation, in which activated microglia are involved, appears to contribute to the development of Parkinson's disease (PD). However, the role of microglial activation and the mechanisms governing this process remain uncertain. We focused on one inhibitory mechanism involved in the control of microglial activation, the microglia inhibitory receptor CD200R1, and its ligand CD200, mainly expressed by neurons. The human CD200R1 gene encodes two membrane-associated and two soluble protein isoforms and the human CD200 gene encodes full-length proteins (CD200full) but also truncated (CD200tr) proteins which act as CD200R1 antagonists. Little is known about their expression in the human brain under pathological conditions. We used human peripheral blood monocytes and monocyte-derived microglia-like cells from control subjects to characterize the expression of the CD200R1 mRNA variants, which showed stimulus-specific responses. We provide evidence of increased CD200R1 (mRNA variants and protein isoforms) and CD200 expression (CD200tr mRNA) in brain tissue of PD patients, mainly in the hippocampus, as well as increased CD200 expression (CD200full and CD200tr mRNAs) in iPSCs-derived dopaminergic neurons generated from skin fibroblasts of PD patients. Our results suggest that CD200-CD200R1 signalling is altered in PD, which may affect the microglial function and constitute a potential target in therapeutic strategies for PD.

Haplotype Analysis of the First A4V-SOD1 Spanish Family: Two Separate Founders or a Single Common Founder?

Despite the genetic heterogeneity reported in familial amyotrophic lateral sclerosis (ALS) (fALS), Cu/Zn superoxide-dismutase (SOD1) gene mutations are the second most common cause of the disease, accounting for around 20% of all families (ALS1) and isolated sporadic cases (sALS). At least 186 different mutations in the SOD1 gene have been reported to date. The possibility of a single founder and separate founders have been investigated for D90A (p.D91A) and A4V (p.A5V), the most common mutations worldwide. High-throughput single nucleotide polymorphism genotyping studies have suggested two founders for A4V (one for the Amerindian population and another for the European population) although the possibility that the two populations are descended from a single ancient founder cannot be ruled out. We used 15 genetic variants spanning the human chromosome 21 from the SOD1 gene to the SCAF4 gene, comparing them with the population reference panels, to demonstrate that the first A4V Spanish pedigree shared the genetic background reported in the European population.

Two novel ligand-independent variants of the VEGFR-1 receptor are expressed in human testis and spermatozoa, one of them with the ability to activate SRC proto-oncogene tyrosine kinases.

The vascular endothelial growth factor receptor 1 (VEGFR-1) family of receptors is preferentially expressed in endothelial cells, with the full-length and mostly the soluble (sVEGFR-1) isoforms being the most expressed ones. Surprisingly, cancer cells (MDA-MB-231) express, instead, alternative intracellular VEGFR-1 variants. We wondered if these variants, that are no longer dependent on ligands for activation, were expressed in a physiological context, specifically in spermatogenic cells, and whether their expression was maintained in spermatozoa and required for human fertility. By interrogating a human library of mature testis cDNA, we characterized two new truncated intracellular variants different from the ones previously described in cancer cells. The new isoforms were transcribed from alternative transcription start sites (aTSS) located respectively in intron-19 (i19VEGFR-1) and intron-28 (i28VEGFR-1) of the VEGFR-1 gene (GenBank accession numbers JF509744 and JF509745) and expressed in mature testis and spermatozoa. In this paper, we describe the characterization of these isoforms by RT-PCR, northern blot, and western blot, their preferential expression in human mature testis and spermatozoa, and the elements that punctuate their proximal promoters and suggest cues for their expression in spermatogenic cells. Mechanistically, we show that i19VEGFR-1 has a strong ability to phosphorylate and activate SRC proto-oncogene non-receptor tyrosine kinases and a significant bias toward a decrease in expression in patients considered infertile by WHO criteria.

Evaluación del aprendizaje de los estudiantes en los tres primeros cursos del Grado de Medicina de la Universitat de Barcelona / Student's learning assessment in the first three years of the Degree of Medicine in the University of Barcelona

Introducción: El proceso de adaptación al Espacio Europeo de Educación Superior ha comportado cambios en los sistemas de evaluación de los aprendizajes en las facultades de medicina y ha introducido los conceptos de evaluación de competencias y evaluación continuada. Objetivos: Describir y analizar, después de seis años de implementación del nuevo plan de estudios de medicina, cómo se está realizando el proceso de evaluación continuada en las diferentes asignaturas de los tres primeros cursos del Grado de Medicina de la Universitat de Barcelona, comparándolo con lo recomendado por los expertos en evaluación en educación médica, y establecer posibles mejoras. Sujetos y métodos: Se describen las actividades evaluativas e instrumentos utilizados por las diferentes asignaturas que demuestran una gran variabilidad y diversificación y una evaluación compartimentada. Se recogen las opiniones y el grado de satisfacción de los coordinadores sobre las actividades evaluativas utilizadas. Asimismo, se evidencia el esfuerzo realizado por los profesores de las distintas asignaturas para promover mejoras en el sistema. Resultados: Se comparan los resultados obtenidos con las recomendaciones establecidas por los expertos en educación médica, con especial referencia a los programas de formación institucional y al paradigma de la evaluación para el aprendizaje. Se discuten las dificultades existentes para desarrollar un mejor sistema de evaluación continuada. Conclusiones: A pesar del esfuerzo realizado y de las mejoras introducidas en las actividades de evaluación, estas no se ajustan totalmente a lo que debería ser una evaluación continuada real; se aboga por promover programas de formación del profesorado sobre evaluación en educación médica

Introduction: The process of adaptation to the European Higher Education Area has brought changes in the assessment of learning outcomes in medical schools and has introduced the concepts of competence assessment and continuous assessment. Aims: To describe and analyze, six years after the implementation of the new curriculum, how the continuous assessment process is being carried out in the different subjects of the first three years of the Degree of Medicine at the University of Barcelona, comparing it with what is recommended by the experts in assessment and to propose ways to improve it. Subjects and methods: The different assessment activities and tools used in the different subjects are described, showing a great variability and diversification and a compartmentalized assessment. The opinions and level of satisfaction of the coordinators of different subjects regarding the assessment activities used are also shown. Likewise, it is evidenced the effort made by the teachers in promoting improvements in the system. Results: The results obtained are compared with the recommendations established in the literature by the experts in medical education, with special reference to the concept of programmatic and institutional assessment and the paradigm of the assessment for learning. The existing difficulties to develop a better continuous assessment are discussed. Conclusions: Despite the efforts made and the improvements introduced in the assessment activities, these do not totally conform to what a real continuous assessment must be and we advocate to promote teacher's training programs on assessment in medical education

RNA-Seq transcriptomic profiling of primary murine microglia treated with LPS or LPS + IFNγ.

Microglia, the main resident immune cells in the CNS, are thought to participate in the pathogenesis of various neurological disorders. LPS and LPS + IFNγ are stimuli that are widely used to activate microglia. However, the transcriptomic profiles of microglia treated with LPS and LPS + IFNγ have not been properly compared. Here, we treated murine primary microglial cultures with LPS or LPS + IFNγ for 6 hours and then performed RNA-Sequencing. Gene expression patterns induced by the treatments were obtained by WGCNA and 11 different expression profiles were found, showing differential responses to LPS and LPS + IFNγ in many genes. Interestingly, a subset of genes involved in Parkinson's, Alzheimer's and Huntington's disease were downregulated by both treatments. By DESeq analysis we found differentially upregulated and downregulated genes that confirmed LPS and LPS + IFNγ as inducers of microglial pro-inflammatory responses, but also highlighted their involvement in specific cell functions. In response to LPS, microglia tended to be more proliferative, pro-inflammatory and phagocytic; whereas LPS + IFNγ inhibited genes were involved in pain, cell division and, unexpectedly, production of some inflammatory mediators. In summary, this study provides a detailed description of the transcriptome of LPS- and LPS + IFNγ treated primary microglial cultures. It may be useful to determine whether these in vitro phenotypes resemble microglia in in vivo pathological conditions.

KATP Channel Expression and Genetic Polymorphisms Associated with Progression and Survival in Amyotrophic Lateral Sclerosis.

The ATP-sensitive potassium (KATP) channel directly regulates the microglia-mediated inflammatory response following CNS injury. To determine the putative role of the KATP channel in amyotrophic lateral sclerosis (ALS) pathology, we investigated whether ALS induces changes in KATP channel expression in the spinal cord and motor cortex. We also characterized new functional variants of human ABCC8, ABCC9, KCNJ8, and KCNJ11 genes encoding for the KATP channel and analyzed their association with ALS risk, rate of progression, and survival in a Spanish ALS cohort. The expression of ABCC8 and KCNJ8 genes was enhanced in the spinal cord of ALS samples, and KCNJ11 increased in motor cortex of ALS samples, as determined by real-time polymerase chain reaction. We then sequenced the exons and regulatory regions of KATP channel genes from a subset of 28 ALS patients and identified 50 new genetic variants. For the case-control association analysis, we genotyped five selected polymorphisms with predicted functional relevance in 185 Spanish ALS (134 spinal ALS and 51 bulbar ALS) patients and 493 controls. We found that bulbar ALS patients presenting the G/G genotype of the rs4148646 variant of ABCC8 and the T/T genotype of the rs5219 variant of KCNJ11 survived longer than other ALS patients presenting other genotypes. Also, the C/C genotype of the rs4148642 variant of ABCC8 and the T/C genotype of the rs148416760 variant of ABCC9 modified the progression rate in spinal ALS patients. Our results suggest that the KATP channel plays a role in the pathophysiological mechanisms of ALS.

Association of the CX3CR1-V249I Variant with Neurofibrillary Pathology Progression in Late-Onset Alzheimer's Disease.

Neuroinflammation and microglial dysfunction have a prominent role in the pathogenesis of late-onset Alzheimer's disease (LOAD). CX3CR1 is a microglia-specific gene involved in microglia-neuron crosstalk and neuroinflammation. Numerous evidence show the involvement of CX3CR1 in AD. The aim of this study was to investigate if some functional genetic variants of this gene could influence on LOAD's outcome, in a neuropathologically confirmed Spanish cohort. We designed an open, pragmatic, case-control retrospective study including a total of 475 subjects (205 pathologically confirmed AD cases and 270 controls). We analyzed the association of the two CX3CR1 functional variants (V249I, rs3732379; and T280M, rs3732378) with neurofibrillary pathology progression rate according to Braak's staging system, age at onset (AAO), survival time, and risk of suffering LOAD. We found that individuals heterozygous for CX3CR1-V249I presented a lower neurofibrillary pathology stage at death (OR = 0.42, 95%CI [0.23, 0.74], p = 0.003, adj-p = 0.013) than the other genotypes. Eighty percent of the subjects homozygous for 249I had higher neurofibrillary pathology progression (Braak's stage VI). Moreover, homozygosis for 280M and 249I could be associated with a higher AAO in the subgroups of AD with Lewy bodies and without Lewy bodies. These CX3CR1 genetic variants could represent new modifying factors of pathology progression and age at onset in LOAD. These results provide further evidence of the involvement of CX3CR1 pathway and microglia/macrophages in the pathogenesis of LOAD.

Myeloid C/EBPß deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis.

BACKGROUND: CCAAT/enhancer binding protein ß (C/EBPß) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPß show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBPß-expressing cell types is not solved. Since C/EBPß-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBPß deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBPß deficiency. METHODS: Mice with myeloid C/EBPß deficiency were generated by crossing LysMCre and C/EBPßfl/fl mice. Primary microglial cultures from C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBPß deletion were analyzed in vivo in microglia isolated from the brains of C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPßfl/fl or control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used. RESULTS: LysMCre-C/EBPßfl/fl mice showed an efficiency of C/EBPß deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBPß deficiency. Transcriptomic analysis of C/EBPß-deficient primary microglia revealed C/EBPß-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBPß deletion. CNS expression of C/EBPß was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBPßfl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis. CONCLUSION: This study provides new data that support a central role for C/EBPß in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBPß inhibition in multiple sclerosis.

C/EBPß and C/EBPδ transcription factors: Basic biology and roles in the CNS.

CCAAT/enhancer binding protein (C/EBP) ß and C/EBPδ are transcription factors of the basic-leucine zipper class which share phylogenetic, structural and functional features. In this review we first describe in depth their basic molecular biology which includes fascinating aspects such as the regulated use of alternative initiation codons in the C/EBPß mRNA. The physical interactions with multiple transcription factors which greatly opens the number of potentially regulated genes or the presence of at least five different types of post-translational modifications are also remarkable molecular mechanisms that modulate C/EBPß and C/EBPδ function. In the second part, we review the present knowledge on the localization, expression changes and physiological roles of C/EBPß and C/EBPδ in neurons, astrocytes and microglia. We conclude that C/EBPß and C/EBPδ share two unique features related to their role in the CNS: whereas in neurons they participate in memory formation and synaptic plasticity, in glial cells they regulate the pro-inflammatory program. Because of their role in neuroinflammation, C/EBPß and C/EBPδ in microglia are potential targets for treatment of neurodegenerative disorders. Any strategy to reduce C/EBPß and C/EBPδ activity in neuroinflammation needs to take into account its potential side-effects in neurons. Therefore, cell-specific treatments will be required for the successful application of this strategy.

UNC13A confers risk for sporadic ALS and influences survival in a Spanish cohort.

To investigate the association of functional variants of the human UNC13A gene with the risk of ALS, survival and the disease progression rate in a Spanish ALS cohort. 136 sporadic ALS (sALS) patients and 487 healthy controls were genotyped for the UNC13A rs12608932 variant. Clinical characterization of ALS patients included gender, age at first symptom, initial topography, disease progression rate, and survival. Genetic association was analyzed under five inheritance models. The sALS patients with the rs12608932(CC) genotype had an increased risk of ALS under a recessive genetic model [OR 2.16; 95 % CI (1.23, 3.8), p = 0.009; corrected p = 0.028]. Genotypes with a C allele are also associated with increased risk [OR 1.47; 95 % CI (1.11, 1.95); p = 0.008; corrected p = 0.023] under an additive model. sALS patients with a C/C genotype had a shorter survival than patients with A/A and A/C genotypes [HR 1.44; 95 % CI (1.11, 1.873); p = 0.007] under a recessive model. In an overdominant model, heterozygous patients had a longer survival than homozygous patients [HR 0.36; 95 % CI (0.22, 0.59); p = 0.001]. The rs12608932 genotypes modify the progression of symptoms measured using the ALSFRS-R. No association with age of onset, initial topography or rate of decline in FVC was found. Our results show that rs12608932 is a risk factor for ALS in the Spanish population and replicate the findings described in other populations. The rs12608932 is a modifying factor for survival and disease progression rate in our series. Our results also corroborated that it did not influence the age of onset.

Astroglia-Microglia Cross Talk during Neurodegeneration in the Rat Hippocampus.

Brain injury triggers a progressive inflammatory response supported by a dynamic astroglia-microglia interplay. We investigated the progressive chronic features of the astroglia-microglia cross talk in the perspective of neuronal effects in a rat model of hippocampal excitotoxic injury. N-Methyl-D-aspartate (NMDA) injection triggered a process characterized within 38 days by atrophy, neuronal loss, and fast astroglia-mediated S100B increase. Microglia reaction varied with the lesion progression. It presented a peak of tumor necrosis factor-α (TNF-α) secretion at one day after the lesion, and a transient YM1 secretion within the first three days. Microglial glucocorticoid receptor expression increased up to day 5, before returning progressively to sham values. To further investigate the astroglia role in the microglia reaction, we performed concomitant transient astroglia ablation with L-α-aminoadipate and NMDA-induced lesion. We observed a striking maintenance of neuronal death associated with enhanced microglial reaction and proliferation, increased YM1 concentration, and decreased TNF-α secretion and glucocorticoid receptor expression. S100B reactivity only increased after astroglia recovery. Our results argue for an initial neuroprotective microglial reaction, with a direct astroglial control of the microglial cytotoxic response. We propose the recovery of the astroglia-microglia cross talk as a tissue priority conducted to ensure a proper cellular coordination that retails brain damage.

CX3CR1 is a modifying gene of survival and progression in amyotrophic lateral sclerosis.

The objective of this study was to investigate the association of functional variants of the human CX3CR1 gene (Fractalkine receptor) with the risk of Amyotrophic Lateral Sclerosis (ALS), the survival and the progression rate of the disease symptoms in a Spanish ALS cohort. 187 ALS patients (142 sporadic [sALS] and 45 familial) and 378 controls were recruited. We investigated CX3CR1 V249I (rs3732379) and T280M (rs3732378) genotypes and their haplotypes as predictors of survival, the progression rate of the symptoms (as measured by ALSFRS-R and FVC decline) and the risk of suffering ALS disease. The results indicated that sALS patients with CX3CR1 249I/I or 249V/I genotypes presented a shorter survival time (42.27 ± 4.90) than patients with 249V/V genotype (67.65 ± 7.42; diff -25.49 months 95%CI [-42.79,-8.18]; p = 0.004; adj-p = 0.018). The survival time was shorter in sALS patients with spinal topography and CX3CR1 249I alleles (diff =  -29.78 months; 95%CI [-49.42,-10.14]; p = 0.003). The same effects were also observed in the spinal sALS patients with 249I-280M haplotype (diff =  -27.02 months; 95%CI [-49.57, -4.48]; p = 0.019). In the sALS group, the CX3CR1 249I variant was associated with a faster progression of the disease symptoms (OR = 2.58; 95IC% [1.32, 5.07]; p = 0.006; adj-p = 0.027). There was no evidence for association of these two CX3CR1 variants with ALS disease risk. The association evidenced herein is clinically relevant and indicates that CX3CR1 could be a disease-modifying gene in sALS. The progression rate of the disease's symptoms and the survival time is affected in patients with one or two copies of the CX3CR1 249I allele. The CX3CR1 is the most potent ALS survival genetic factor reported to date. These results reinforce the role of the immune system in ALS pathogenesis.

Genetic variation in APOE cluster region and Alzheimer's disease risk.

We report the fine mapping/sequencing results of promoter and regulatory regions of APOE cluster genes (APOE, APOC1, APOC4, APOC2, and TOMM40) in Alzheimer's disease (AD) risk as well as in the progression from mild cognitive impairment (MCI) to AD. Long-range sequencing in 29 MCI subjects who progressed to dementia revealed 7 novel variants. Two potentially relevant novel variants and 34 single nucleotide polymorphisms (SNPs) were genotyped in a large sample of AD, MCI, and control subjects (n = 1453). Globally, very little association signal was observed in our sample in the absence of APOE ε4. Rs5158 (APOC4 intron 1) and rs10413089 (3' to APOC2) showed a trend toward an increase in AD risk independently from APOE ε4 associated risk though it did not survive multiple test correction (uncorrected p = 0.0099 and 0.01, respectively). Interestingly, rs10413089 showed a similar effect in an independent series. The analysis of the discovery sample showed an association of TOMM40 single nucleotide polymorphisms with progression from MCI stage to AD (rs59007384 and rs11556510), as well as with a shorter time to progression from MCI status to AD (rs10119), though these results could not be replicated in independent series. Further studies are needed to investigate the role of APOE cluster variants in AD risk.

Different MAPT haplotypes are associated with Parkinson's disease and progressive supranuclear palsy.

The H1 MAPT haplotype in the 17q21 chromosomal region has been associated with several neurodegenerative diseases. Some reports have suggested that there is an association between genetic variants within the H1 haplotype with Parkinson's disease (PD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Here we report a genetic association study using seven SNPs located along the 17q21 region, in PD patients and controls. In addition, we compared these results with a dataset of previously published PSP/CBD patients from the same population. Our results show that the H1-rs242557(G) allele sub-haplotype is increased in PD (p=0.005), while the H1-rs242557(A) allele sub-haplotype is increased in PSP/CBD (p=0.0002), comparing to controls. The rs242557 polymorphism could act modulating the phenotypic expressivity of the H1 risk on these parkinsonisms. The location of this polymorphism in the 5' regulatory region of MAPT gene suggests the presence of a functional mechanism involved in the variation of MAPT expression levels.

5'-Upstream variants of CRHR1 and MAPT genes associated with age at onset in progressive supranuclear palsy and cortical basal degeneration.

Two different H1 sub-haplotypes at chromosome 17q21, H1C and H1E'A, have been associated with progressive supranuclear palsy (PSP) and cortical basal degeneration (CBD). We analyzed the SNPs included in the H1C and H1E'A haplotypes in a large Spanish PSP/CBD series and their interaction with age at onset (AAO). Survival analysis of rs1880753 marker was consistently associated with disease risk and with an earlier age at onset under an additive model. Its location at 160 kb and 50 kb upstream of tau and CRHR1 genes, respectively, suggests that it might act as a cis-element that regulates gene expression. Rs45502095(H1) was also associated with AAO under a recessive model. Haplotype analysis failed to replicate the association of H1C and H1E'A haplotypes with PSP/CBD. However, we found a strong association of two H1 sub-haplotypes with PSP and CBD (H1E'C and H1Q), which include MAPT and CRHR1 genes where the risk variant for PSP/CBD could lie.

A common protamine 1 promoter polymorphism (-190 C->A) correlates with abnormal sperm morphology and increased protamine P1/P2 ratio in infertile patients.

It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; -190 C-->A). The -190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (A change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 -190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 -190 C-->A polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.