Integrated DNA methylation and gene expression profiling across multiple brain regions implicate novel genes in Alzheimer's disease.

Late-onset Alzheimer's disease (AD) is a complex age-related neurodegenerative disorder that likely involves epigenetic factors. To better understand the epigenetic state associated with AD, we surveyed 420,852 DNA methylation (DNAm) sites from neurotypical controls (N = 49) and late-onset AD patients (N = 24) across four brain regions (hippocampus, entorhinal cortex, dorsolateral prefrontal cortex and cerebellum). We identified 858 sites with robust differential methylation collectively annotated to 772 possible genes (FDR < 5%, within 10 kb). These sites were overrepresented in AD genetic risk loci (p = 0.00655) and were enriched for changes during normal aging (p < 2.2 × 10-16), and nearby genes were enriched for processes related to cell-adhesion, immunity, and calcium homeostasis (FDR < 5%). To functionally validate these associations, we generated and analyzed corresponding transcriptome data to prioritize 130 genes within 10 kb of the differentially methylated sites. These 130 genes were differentially expressed between AD cases and controls and their expression was associated with nearby DNAm (p < 0.05). This integrated analysis implicates novel genes in Alzheimer's disease, such as ANKRD30B. These results highlight DNAm differences in Alzheimer's disease that have gene expression correlates, further implicating DNAm as an epigenetic mechanism underlying pathological molecular changes associated with AD. Furthermore, our framework illustrates the value of integrating epigenetic and transcriptomic data for understanding complex disease.

A manual multiplex immunofluorescence method for investigating neurodegenerative diseases.

BACKGROUND: Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ is critical to facilitate discovery and validation of pathogenic molecular pathways. Immunostaining methods enable in situ detection of specific targets. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue adhesion and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Furthermore, stripping techniques require antibody-specific optimization, validation, and quality control steps. NEW METHOD: Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives based on extensive testing. RESULTS: A protocol using ß-mercaptoethanol and SDS consistently enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue adhesion and antigen integrity in human postmortem tissue. COMPARISON WITH EXISTING METHODS: Our proposed method is optimal for standard lab settings and shows consistent efficacy despite the intricacies of suboptimal human postmortem tissue and the need to strip markers bound to highly aggregated proteins. Additionally, it incorporates quality control steps to validate antibody stripping. CONCLUSIONS: Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters during optimization and validation phases of experiments is prudent.

Human Brain Slice Culture: A Useful Tool to Study Brain Disorders and Potential Therapeutic Compounds.

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

TGFß pathway deregulation and abnormal phospho-SMAD2/3 staining in hereditary cerebral hemorrhage with amyloidosis-Dutch type.

Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) pathology, caused by the E22Q mutation in the amyloid ß (Aß) peptide. Transforming growth factor ß1 (TGFß1) is a key player in vascular fibrosis and in the formation of angiopathic vessels in transgenic mice. Therefore, we investigated whether the TGFß pathway is involved in HCHWA-D pathogenesis in human postmortem brain tissue from frontal and occipital lobes. Components of the TGFß pathway were analyzed with quantitative RT-PCR. TGFß1 and TGFß Receptor 2 (TGFBR2) gene expression levels were significantly increased in HCHWA-D in comparison to the controls, in both frontal and occipital lobes. TGFß-induced pro-fibrotic target genes were also upregulated. We further assessed pathway activation by detecting phospho-SMAD2/3 (pSMAD2/3), a direct TGFß down-stream signaling mediator, using immunohistochemistry. We found abnormal pSMAD2/3 granular deposits specifically on HCHWA-D angiopathic frontal and occipital vessels. We graded pSMAD2/3 accumulation in angiopathic vessels and found a positive correlation with the CAA load independent of the brain area. We also observed pSMAD2/3 granules in a halo surrounding occipital vessels, which was specific for HCHWA-D. The result of this study indicates an upregulation of TGFß1 in HCHWA-D, as was found previously in AD with CAA pathology. We discuss the possible origins and implications of the TGFß pathway deregulation in the microvasculature in HCHWA-D. These findings identify the TGFß pathway as a potential biomarker of disease progression and a possible target of therapeutic intervention in HCHWA-D.

Human Brain Slice Culture: A Useful Tool to Study Brain Disorders and Potential Therapeutic Compounds / 神经科学通报·英文版

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

Influence of external factors on the preservation of human nervous tissue for histological studies: review article / Influência dos fatores externos sobre a preservação do tecido nervoso humano nos estudos histológicos: artigo de revisão

Neuropathological studies are crucial for the new knowledge on pathophysiology and treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. The postmortem brain tissue processing method directly impacts on both the appropriate integrity and the biomolecules detection by different histological and molecular biology techniques. In this review we will discuss topics on the influence of some external factors on the preservation of the brain tissue for histological studies (histochemistry and immunohistochemistry), such as factors either prior or after the death, and the chosen method for the preservation of nervous tissue. By means of a specific example, we propose a strict record of various conditions involved in the method of preservation of nervous tissue, and its correlation with variables that measure the quality of the histological sample as markers of preservation of biological material for further studies.

Os estudos neuropatológicos são fundamentais para novas descobertas sobre a fisiopatologia e o tratamento de doenças neurodegenerativas, como a doença de Alzheimer e a doença de Parkinson. O modo como o encéfalo pós-morte é processado influencia diretamente na adequada integridade e na detecção de biomoléculas por diferentes técnicas histológicas e de biologia molecular. Nesta revisão, abordaremos tópicos sobre a influência de determinados fatores externos sobre a preservação do encéfalo para estudos histológicos (histoquímica e imuno-histoquímica), como as condições anteriores e posteriores ao óbito, e o método escolhido de conservação do tecido nervoso. Por meio de um exemplo específico, propomos um rigoroso registro das diversas condições envolvidas no processo de preservação do tecido nervoso e sua correlação com variáveis que avaliam a qualidade da amostra histológica, como marcadores da preservação do material biológico para estudos posteriores..