TF-ChIP Method for Tissue-Specific Gene Targets

Chromatin immunoprecipitation (ChIP) is an assay developed in order to define the dynamic nature of transcription processes. This method has been widely employed to identify methylated and acetylated DNA sequences in a variety of organs both in animals and humans. Nevertheless, this technique is significantly less employed to study transcriptional targets of specific nuclear signaling factors (TFs) and the data published so far have mainly used cell culture material and have been hardly reproduced in ex-vivo tissue. As nuclear signaling underlies important adaptive and maladaptive responses in chronic conditions such as cancer and neurodegeneration, there is a need for streamlining the upfront workflow of TF-ChIP for subsequent target sequencing. Based on the typical low concentration of the signaling transcriptional complex and the complexity/length of the ChIP Seq protocol, the field of cellular signaling has been confronted with a major roadblock in identifying clinically relevant targets of pathological and physiological signaling pathways. The present protocol offers a standardized procedure for detecting signaling targets in any whole tissue or specific dissected regions. The advantages of the protocol compared to the existing published methods are: (1) the small amount of starting material; appropriate for tissue subregions; (2) the optimization of DNA fragmentation from whole tissue; (3) suitability for sparsely populated tissues (i.e., brain); (4) the specificity of the TF-targeting readout; and (5) high DNA quality for sequencing or hybridization. The present protocol is highly detailed and can be reproduced using both fresh and fresh-frozen tissue. This is particularly relevant in the clinical setting, where specimen integrity is often the limiting step and where transcriptional target profiling is therapeutically relevant. The method is centered on Notch signaling but can be applied to a variety of nuclear signaling pathways as long as specific antibodies are available for pull down. Taken the superior yield/readout of this procedure, ChIP may finally provide relevant information about dynamic downstream gene changes in vivo for use in both basic research and clinical applications

Olfactory dysfunction in the pathophysiological continuum of dementia

Sensory capacities like smell, taste, hearing, vision decline with aging, but increasing evidence show that sensory dysfunctions are one of the early signs diagnosing the conversion from physiological to pathological brain state. Smell loss represents the best characterized sense in clinical practice and is considered as one of the first preclinical signs of Alzheimer’s and Parkinson’s disease, occurring a decade or more before the onset of cognitive and motor symptoms. Despite the numerous scientific reports and the adoption in clinical practice, the etiology of sensory damage as prodromal of dementia remains largely unexplored and more studies are needed to resolve the mechanisms underlying sensory network dysfunction. Although both cognitive and sensory domains are progressively affected, loss of sensory experience in early stages plays a major role in reducing the autonomy of demented people in their daily tasks or even possibly contributing to their cognitive decline. Interestingly, the chemosensory circuitry is devoid of a blood brain barrier, representing a vulnerable port of entry for neurotoxic species that can spread to the brain. Furthermore, the exposure of the olfactory system to the external environment make it more susceptible to mechanical injury and trauma, which can cause degenerative neuroinflammation. In this review, we will summarize several findings about chemosensory impairment signing the conversion from healthy to pathological brain aging and we will try to connect those observations to the promising research linking environmental influences to sporadic dementia. The scientific body of knowledge will support the use of chemosensory diagnostics in the presymptomatic stages of AD and other biomarkers with the scope of finding treatment strategies before the onset of the disease

Periodontal microorganisms and Alzheimer disease - A causative relationship?

In the initiation or exacerbation of Alzheimer disease, the dissemination of oral microorganisms into the brain tissue or the low-level systemic inflammation have been speculated to play a role. However, the impact of oral microorganisms, such as Porphyromonas gingivalis, on the pathogenesis of Alzheimer disease and the potential causative relationship is still unclear. The present review has critically reviewed the literature by examining the following aspects: (a) the oral microbiome and the immune response in the elderly population, (b) human studies on the association between periodontal and gut microorganisms and Alzheimer disease, (c) animal and in vitro studies on microorganisms and Alzheimer disease, and (d) preventive and therapeutic approaches. Factors contributing to microbial dysbiosis seem to be aging, local inflammation, systemic diseases, wearing of dentures, living in nursing homes and no access to adequate oral hygiene measures. Porphyromonas gingivalis was detectable in post-mortem brain samples. Microbiome analyses of saliva samples or oral biofilms showed a decreased microbial diversity and a different composition in Alzheimer disease compared to cognitively healthy subjects. Many in-vitro and animal studies underline the potential of P gingivalis to induce Alzheimer disease-related alterations. In animal models, recurring applications of P gingivalis or its components increased pro-inflammatory mediators and β-amyloid in the brain and deteriorated the animals' cognitive performance. Since periodontitis is the result of a disturbed microbial homoeostasis, an effect of periodontal therapy on the oral microbiome and host response related to cognitive parameters may be suggested and should be elucidated in further clinical trials

Histone H3 Lysine 4 and 27 Trimethylation Landscape of Human Alzheimer's Disease

none9sìBackground: Epigenetic remodeling is emerging as a critical process for both the onset and progression of Alzheimer's disease (AD), the most common form of neurodegenerative dementia. However, it is not clear to what extent the distribution of histone modifications is involved in AD. Methods: To investigate histone H3 modifications in AD, we compared the genome-wide distributions of H3K4me3 and H3K27me3 in entorhinal cortices from severe sporadic AD patients and from age-matched healthy individuals of both sexes. Results: AD samples were characterized by typical average levels and distributions of the H3K4me3 and H3K27me3 signals. However, AD patients showed a lower H3K4me3 and higher H3K27me3 signal, particularly in males. Interestingly, the genomic sites found differentially trimethylated at the H3K4 between healthy and AD samples involve promoter regions of genes belonging to AD-related pathways such as glutamate receptor signaling. Conclusions: The signatures of H3K4me3 and H3K27me3 identified in AD patients validate the role of epigenetic chromatin remodeling in neurodegenerative disease and shed light on the genomic adaptive mechanisms involved in AD.openPersico, Giuseppe; Casciaro, Francesca; Amatori, Stefano; Rusin, Martina; Cantatore, Francesco; Perna, Amalia; Auber, Lavinia Alberi; Fanelli, Mirco; Giorgio, MarcoPersico, Giuseppe; Casciaro, Francesca; Amatori, Stefano; Rusin, Martina; Cantatore, Francesco; Perna, Amalia; Auber, Lavinia Alberi; Fanelli, Mirco; Giorgio, Marc

Establishment of a consensus protocol to explore the brain pathobiome in patients with mild cognitive impairment and Alzheimer\u27s disease: Research outline and call for collaboration.

Microbial infections of the brain can lead to dementia, and for many decades microbial infections have been implicated in Alzheimer\u27s disease (AD) pathology. However, a causal role for infection in AD remains contentious, and the lack of standardized detection methodologies has led to inconsistent detection/identification of microbes in AD brains. There is a need for a consensus methodology; the Alzheimer\u27s Pathobiome Initiative aims to perform comparative molecular analyses of microbes in post mortem brains versus cerebrospinal fluid, blood, olfactory neuroepithelium, oral/nasopharyngeal tissue, bronchoalveolar, urinary, and gut/stool samples. Diverse extraction methodologies, polymerase chain reaction and sequencing techniques, and bioinformatic tools will be evaluated, in addition to direct microbial culture and metabolomic techniques. The goal is to provide a roadmap for detecting infectious agents in patients with mild cognitive impairment or AD. Positive findings would then prompt tailoring of antimicrobial treatments that might attenuate or remit mounting clinical deficits in a subset of patients