CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in 'regulation of RNA splicing' and 'mRNA catabolic process'. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.

Ratio Aß1-42:p-Tau: a Possible Diagnostic Tool in Differentiating Dementias.

BACKGROUND: Patients with Alzheimer's disease (AD) present a typical biochemical profile of biomarkers: low concentration of ß amyloid 1-42 (Aß1-42), high concentration of total Tau (t-Tau) and phosphorylated Tau at threonine 181 (p-Tau). Several neurodegenerative diseases may overlap with AD, both in regards to clinical symptoms and neuropathology. Many data suggest that Alzheimer's disease (AD) pathophysiology can be identified using biomarkers. It has been hypothesized that subjects with dementia due to AD showed low levels of Aß1-42 combined with the highest levels of total Tau and phosphorylated Tau; moreover, it has been hypothesized that the ratio Aß1-42:p-Tau further help in discriminating Alzheimer's disease from other diagnoses. The aim of this work is to verify this hypothesis in our cohort of patients and to investigate if the same ratio could be a sensitive index able to discriminate MCI due to neurodegenerative factors (MCId) from MCI due to vascular factors (MCIv). METHODS: Two hundred sixty-two patients meeting the NIA-AA and NINDS-AIREN criteria were diagnosed as follow: AD in 120 patients [mean age 71.6 (42 - 87)], FTD in 23 patients [mean age 67.3 (46 - 78)], LBD in 17 patients [mean age 73.2 (58 - 83)], VAD in 9 patients [mean age 71.2 (60 - 81)]. According to the criteria proposed by Petersen RC, 24 patients had the diagnosis of MCId [mean age 71.8 (59 - 81)], 38 MCIv [mean age 69.3 (55-82). The comparison between the ratio of Aß1-42/p-Tau among the six groups was done using t-test for independent samples. A p-value < 0.05 was considered to represent statistical significance. The ROC (Receiver Operating Characteristic) curve analysis was made using R-studio software. RESULTS: The ratio Aß1-42:p-Tau was significantly lower in AD and MCId with respect to all the other groups and the difference was also statistically significant between MCId and MCIv. CONCLUSIONS: Aß1-42:p-Tau ratio has potential for being implemented in the clinical routine for differential diagnosis between AD and other dementias and to distinguish underling pathology such as neurodegenerative or vascular disease.

Nanopore-Enabled Microbiome Analysis: Investigating Environmental and Host-Associated Samples in Rainbow Trout Aquaculture.

Microbiome sequencing is at the forefront of health management development, and as such, it is becoming of great interest to monitor the microbiome in the aquaculture industry as well. Oxford Nanopore Technologies (ONT) platforms are gaining popularity to study microbial communities, enabling faster sequencing, extended read length, and therefore, improved taxonomic resolution. Despite this, there is a lack of clear guidelines to perform a metabarcoding study, especially when dealing with samples from non-mammalian species, such as aquaculture-related samples. In this article, we provide general guidelines for sampling, nucleic acid extraction, and ONT-based library preparation for both environmental (water, sediment) and host-associated (gill or skin mucus, skin, gut content, or gut mucosa) microbiome analysis. Our procedures focus specifically on rainbow trout (Oncorhynchus mykiss) reared in experimental facilities. However, these protocols can also be transferred to alternative types of samples, such as environmental DNA (eDNA) monitoring from alternative water sources, or to different fish species. The additional challenge posed by the low biomass and limited bacterial diversity inherent in fish-associated microbiomes is addressed through the implementation of troubleshooting solutions. Furthermore, we describe a bioinformatic pipeline starting from raw reads and leading to taxonomic abundance tables using currently available tools and software. Finally, we provide a set of specific guidelines and considerations related to the strategic planning of a microbiome study within the context of aquaculture. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Environmental sample collection Basic Protocol 2: Host-associated sample collection Alternate Protocol: Host-associated sample collection: Alternative sample types Basic Protocol 3: Sample pre-treatment and nucleic acid extraction Basic Protocol 4: Quality control and preparation for 16S rRNA gene sequencing Support Protocol 1: Assessment of inhibition by quantitative PCR Support Protocol 2: Bioinformatic analysis from raw files to taxonomic abundance tables.

Clinical and cognitive correlates of visual hallucinations in dementia with Lewy bodies.

BACKGROUND: The presence of recurrent complex visual hallucinations (VHs) is a core feature of dementia with Lewy bodies (DLB). The aim of this study was to investigate which clinical and neuropsychological characteristics are associated with VHs and their predictive value over a 1 year follow-up. METHODS: 81 DLB patients, 41 with (VH+) and 36 without (VH-) VHs, and 45 patients with Alzheimer's disease (AD), were enrolled. All participants underwent extensive neuropsychological testing. Visual-spatial and perceptual abilities were evaluated with the Visual and Object Space Perception (VOSP) battery. Fluctuations in attention, rapid eye movement sleep behaviour disorder (RBD) symptoms, extrapyramidal signs and behavioural disturbances were studied with dedicated clinical scales. RESULTS: The presence of VHs was associated with older age and later disease onset, but not with disease duration or with fluctuations, RBD or parkinsonism severity. Cognitive correlates of VHs were deficits in visual attention (digit cancellation: p<0.005) and executive functions (clock drawing: p<0.05; digit span forward: p<0.05) on a background of a slightly worse global cognitive performance (Mini-Mental State Examination: p=0.05). Visual-perceptual and visual-spatial deficits were significantly worse in DLB than in AD patients (VOSP subtests scores 1, 6, 7 and 8) but were not different in DLB VH+ and VH-, except for subtest 6. Poor performance in the visual attention task was an independent predictor of VHs. DISCUSSION: Impairment of visual-spatial and perceptual abilities in DLB represents a disease related cognitive signature, independent of the presence of VHs, for which it may represent a predisposing condition. Visual attention, instead, is the main cognitive determinant for the genesis of VHs.

Design and Delivery of SINEUP: A New Modular Tool to Increase Protein Translation.

SINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouse AS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)-containing the invSINEB2 element-able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.

Natural SINEUP RNAs in Autism Spectrum Disorders: RAB11B-AS1 Dysregulation in a Neuronal CHD8 Suppression Model Leads to RAB11B Protein Increase.

CHD8 represents one of the highest confidence genetic risk factors implied in Autism Spectrum Disorders, with most mutations leading to CHD8 haploinsufficiency and the insurgence of specific phenotypes, such as macrocephaly, facial dysmorphisms, intellectual disability, and gastrointestinal complaints. While extensive studies have been conducted on the possible consequences of CHD8 suppression and protein coding RNAs dysregulation during neuronal development, the effects of transcriptional changes of long non-coding RNAs (lncRNAs) remain unclear. In this study, we focused on a peculiar class of natural antisense lncRNAs, SINEUPs, that enhance translation of a target mRNA through the activity of two RNA domains, an embedded transposable element sequence and an antisense region. By looking at dysregulated transcripts following CHD8 knock down (KD), we first identified RAB11B-AS1 as a potential SINEUP RNA for its domain configuration. Then we demonstrated that such lncRNA is able to increase endogenous RAB11B protein amounts without affecting its transcriptional levels. RAB11B has a pivotal role in vesicular trafficking, and mutations on this gene correlate with intellectual disability and microcephaly. Thus, our study discloses an additional layer of molecular regulation which is altered by CHD8 suppression. This represents the first experimental confirmation that naturally occurring SINEUP could be involved in ASD pathogenesis and underscores the importance of dysregulation of functional lncRNAs in neurodevelopment.

Accuracy of diagnostic criteria for sporadic creutzfeldt-jakob disease among rapidly progressive dementia.

Rapidly progressive dementia (RPD) is a rare presentation of different neurological disorders characterized by cognitive impairment leading to loss of functional independence within 24 months or less. The increasing recognition of treatable non-prion causes of RPD has made the differential diagnosis with sporadic Creutzfeldt-Jakob disease (sCJD) of crucial importance. We therefore assessed the frequency of different etiologies of RPD and evaluated the accuracy of newly proposed diagnostic criteria for sCJD. Clinical records of patients with RPD referred to Memory Clinic between 2007 and 2012 were retrospectively analyzed. The accuracy of diagnostic criteria for sCJD was evaluated by: a) MRI images in DWI and FLAIR sequences; and (b) CSF 14-3-3 protein. In addition, CSF total tau protein level was also assessed. Final diagnosis was obtained after a 1-year follow-up or after autopsy. Among 37 patients with RPD, the most frequent causes were non-prion diseases, either untreatable (38%) or potentially treatable (32%), thus leaving sCJD as a less frequent cause (30%). DWI images had a sensitivity of 73% and specificity of 96%, while FLAIR yielded a very low sensitivity (40%). CSF 14-3-3 protein had a sensitivity of 100%, but a very low specificity (43%). The strongest independent predictor of sCJD diagnosis was the CSF tau level (p = 0.002) (91% sensitivity, 83% specificity). Treatable causes of RPD are as frequent as sCJD and a rapid differential diagnosis is mandatory. We suggest that DWI images and CSF analysis combining 14-3-3 and total tau protein determination hold the best informative diagnostic values.

LDL density and oxidation are modulated by PON1 promoter genotype in patients with Alzheimer's disease.

Cholesterol metabolism alteration is a hot topic in the field of Alzheimer's disease (AD). However, data on plasma lipoproteins cholesterol distribution and oxidation in AD and their possible genetic determinants are lacking. The paraoxonase-1 (PON1) gene -107C/T promoter polymorphisms have been found associated with AD. One of the fundamental functions of PON1 enzyme is the inhibition of low-density lipoproteins (LDL) oxidation. We therefore evaluated plasma lipoprotein profile and LDL density and oxidation in late-onset AD patients and healthy elderly subjects, without neuroimaging evidence of cerebrovascular lesions and not on lipid-lowering treatment, and their interaction with PON1 -107C/T and apolipoprotein E (APOE) genotypes. Mean plasma total cholesterol and LDL levels were higher in AD than controls (p < 0.05). Lipoproteins cholesterol distribution shifted toward a greater prevalence of smaller, denser LDL (sd-LDL, p < 0.05) only in AD patients with PON1 -107TT genotype, who also showed increased plasma levels of oxidized LDL (ox-LDL, p = 0.02). A significant association was observed between sd-LDL and ox-LDL levels (p < 005) in AD patients. APOE genotype did not modulate lipoprotein distribution. Increased levels of sd-LDL and ox-LDL particles in the AD PON1 TT patients could be explained by the combined effect of an AD-related pro-oxidant milieu and an ineffective PON1 gene polymorphism-related antioxidant capacity. The functional correlate of the association between PON1 -107C/T polymorphism and AD may be the abnormal modulation of LDL oxidation. Ox-LDL may amplify the processes of endothelial injury promoted by vascular amyloid deposition, which represents one of the potential pathways explaining the cross-road between vascular and neurodegenerative pathomechanisms in AD.