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RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis.

The generation of new neurons at the hippocampal neurogenic niche, known as adult hippocampal neurogenesis (AHN), and its impairment, have been implicated in Alzheimer's disease (AD). MicroRNA-132 (miR-132), the most consistently downregulated microRNA (miRNA) in AD, was recently identified as a potent regulator of AHN, exerting multilayered proneurogenic effects in adult neural stem cells (NSCs) and their progeny. Supplementing miR-132 in AD mouse brain restores AHN and relevant memory deficits, yet the exact mechanisms involved are still unknown. Here, we identify NACC2 as a novel miR-132 target implicated in both AHN and AD. miR-132 deficiency in mouse hippocampus induces Nacc2 expression and inflammatory signaling in adult NSCs. We show that miR-132-dependent regulation of NACC2 is involved in the initial stages of human NSC differentiation towards astrocytes and neurons. Later, NACC2 function in astrocytic maturation becomes uncoupled from miR-132. We demonstrate that NACC2 is present in reactive astrocytes surrounding amyloid plaques in mouse and human AD hippocampus, and that there is an anticorrelation between miR-132 and NACC2 levels in AD and upon induction of inflammation. Unraveling the molecular mechanisms by which miR-132 regulates neurogenesis and cellular reactivity in AD, will provide valuable insights towards its possible application as a therapeutic target. [ABSTRACT FROM AUTHOR]

This study explores the seismotectonics of Kachchh in western India, a region with a low-to-moderate strain rate and a history of significant earthquakes, notably the 1819, Mw 7.8 Allah Bund, and the 2001, Mw 7.6 Bhuj. Despite its substantial seismic risk, comprehensive studies on Kachchh's seismogenic sources are scarce. This is attributed to the concealed nature of active structures, hindering definitive age constraints in paleoseismological research. Our research comprises a detailed paleoseismic analysis of the north-verging, reverse Jhura Fault underlying the Jhura anticline, a segment of the Kachchh Mainland Fault. This fault segment shows evidence of surface-rupturing earthquakes in the area south of the Great Rann of Kachchh. The investigation reveals three paleoseismic events: Event I before 9.72 ka B.P., Event II between 8.63–8.20 ka B.P., and Event III between 6.20–6.09 ka B.P. The elapsed time since the last event on this fault is > 8000 years, suggesting that the area is exposed to a significant earthquake hazard. This highlights the need for more precise characterization of individual seismogenic sources for future earthquake preparedness. [ABSTRACT FROM AUTHOR]

Local cell densities and positioning within cellular monolayers and stratified epithelia have important implications for cell interactions and the functionality of various biological processes. To analyze the relationship between cell localization and tissue physiology, density-based clustering algorithms, such as DBSCAN, allow for a detailed characterization of the spatial distribution and positioning of individual cells. However, these methods rely on predefined parameters that influence the outcome of the analysis. With varying cell densities in cell cultures or tissues impacting cell sizes and, thus, cellular proximities, these parameters need to be carefully chosen. In addition, standard DBSCAN approaches generally come short in appropriately identifying individual cell positions. We therefore developed three extensions to the standard DBSCAN-algorithm that provide: (i) an automated parameter identification to reliably identify cell clusters, (ii) an improved identification of cluster edges; and (iii) an improved characterization of the relative positioning of cells within clusters. We apply our novel methods, which are provided as a user-friendly OpenSource-software package (DBSCAN-CellX), to cellular monolayers of different cell lines. Thereby, we show the importance of the developed extensions for the appropriate analysis of cell culture experiments to determine the relationship between cell localization and tissue physiology. [ABSTRACT FROM AUTHOR]

The role of glia in amyotrophic lateral sclerosis (ALS) is undeniable. Their disease-related activity has been extensively studied in the spinal cord, but only partly in the brain. We present herein a comprehensive study of glia in the cortex of SOD1(G93A) mice—a widely used model of ALS. Using single-cell RNA sequencing (scRNA-seq) and immunohistochemistry, we inspected astrocytes, microglia, and oligodendrocytes, in four stages of the disease, respecting the factor of sex. We report minimal changes of glia throughout the disease progression and regardless of sex. Pseudobulk and single-cell analyses revealed subtle disease-related transcriptional alterations at the end-stage in microglia and oligodendrocytes, which were supported by immunohistochemistry. Therefore, our data support the hypothesis that the SOD1(G93A) mouse cortex does not recapitulate the disease in patients, and we recommend the use of a different model for future studies of the cortical ALS pathology. [ABSTRACT FROM AUTHOR]

Single quantum dots (Qdots) are often used in the field of single-molecule imaging. Qdots are sensitive to changes in the physical interactions between the Qdots and the surrounding materials. However, the spectral changes in a single Qdot emission have not been studied in detail. Low-temperature plasma treatment of glass surfaces reduced the intensity of the 655 nm emission peak of Qdot655 on glass surfaces, but did not significantly change the intensity of the 580 nm emission. Silanization of the glass surface increases the thickness of the silane layer, and the 655 nm emission peak increased. When single Qdots on the untreated glass were imaged, plasma treatment decreased the intensity of red emission and increased yellow emission. When Qdots were brought close to the glass surface in the range of 28–0 nm, the red emission intensity decreased and the yellow emission intensity increased slightly. When single actin filaments were labeled with Qdots, fluctuations of the yellow and red emission of the Qdot were detected, which reflected the very small distance changes. Our results indicate that the local interaction of Qdots with the glass surface improves the spatial and temporal resolution of optical measurements of biomolecules labeled with Qdots. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that glutathione loss is closely associated with the progression of neurodegenerative disorders. Here, we found that the neuronal conditional-knockout (KO) of glutamyl-cysteine-ligase catalytic-subunit (GCLC), a rate-limiting enzyme for glutathione synthesis, induced brain atrophy accompanied by neuronal loss and neuroinflammation. GCLC-KO mice showed activation of C1q, which triggers engulfment of neurons by microglia, and disease-associated-microglia (DAM), suggesting that activation of microglia is linked to the neuronal loss. Furthermore, gasdermins, which regulate inflammatory form of cell death, were upregulated in the brains of GCLC-KO mice, suggesting the contribution of pyroptosis to neuronal cell death in these animals. In particular, GSDME-deficiency significantly attenuated the hippocampal atrophy and changed levels of DAM markers in GCLC-KO mice. Finally, we found that the expression of GCLC was decreased around amyloid plaques in AppNL-G-F AD model mice. AppNL-G-F mouse also exhibited inflammatory events similar to GCLC-KO mouse. We propose a mechanism by which a vicious cycle of oxidative stress and neuroinflammation enhances neurodegenerative processes. Furthermore, GCLC-KO mouse will serve as a useful tool to investigate the molecular mechanisms underlying neurodegeneration and in the development of new treatment strategies to address neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the leading cause of dementia in aged population. Oxidative stress and neuroinflammation play important roles in the pathogenesis of AD. Investigation of hub genes for the development of potential therapeutic targets and candidate biomarkers is warranted. The differentially expressed genes (DEGs) in AD were screened in GSE48350 dataset. The differentially expressed oxidative stress genes (DEOSGs) were analyzed by intersection of DEGs and oxidative stress-related genes. The immune-related DEOSGs and hub genes were identified by weighted gene co-expression network analysis (WGCNA) and protein–protein interaction (PPI) analysis, respectively. Enrichment analysis was performed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The diagnostic value of hub genes was assessed by receiver operating characteristic analysis and validated in GSE1297. The mRNA expression of diagnostic genes was determined by qRT-PCR analysis. Finally, we constructed the drug, transcription factors (TFs), and microRNA network of the diagnostic genes. A total of 1160 DEGs (259 up-regulated and 901 down-regulated) were screened in GSE48350. Among them 111 DEOSGs were identified in AD. Thereafter, we identified significant difference of infiltrated immune cells (effector memory CD8 T cell, activated B cell, memory B cell, natural killer cell, CD56 bright natural killer cell, natural killer T cell, plasmacytoid dendritic cell, and neutrophil) between AD and control samples. 27 gene modules were obtained through WGCNA and turquoise module was the most relevant module. We obtained 66 immune-related DEOSGs by intersecting turquoise module with the DEOSGs and identified 15 hub genes through PPI analysis. Among them, 9 hub genes (CCK, CNR1, GAD1, GAP43, NEFL, NPY, PENK, SST, and TAC1) were identified with good diagnostic values and verified in GSE1297. qRT-PCR analysis revealed the downregulation of SST, NPY, GAP43, CCK, and PENK and upregulation of NEFL in AD. Finally, we identified 76 therapeutic agents, 152 miRNAs targets, and 91 TFs regulatory networks. Our study identified 9 key genes associated with oxidative stress and immune reaction in AD pathogenesis. The findings may help to provide promising candidate biomarkers and therapeutic targets for AD. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disease that eventually affects memory and behavior. The identification of biomarkers based on risk factors for AD provides insight into the disease since the exact cause of AD remains unknown. Several studies have proposed microRNAs (miRNAs) in blood as potential biomarkers for AD. Exposure to heavy metals is a potential risk factor for onset and development of AD. Blood cells of subjects that are exposed to lead detected in the circulatory system, potentially reflect molecular responses to this exposure that are similar to the response of neurons. In this study we analyzed blood cell-derived miRNAs derived from a general population as proxies of potentially AD-related mechanisms triggered by lead exposure. Subsequently, we analyzed these mechanisms in the brain tissue of AD subjects and controls. A total of four miRNAs were identified as lead exposure-associated with hsa-miR-3651, hsa-miR-150-5p and hsa-miR-664b-3p being negatively and hsa-miR-627 positively associated. In human brain derived from AD and AD control subjects all four miRNAs were detected. Moreover, two miRNAs (miR-3651, miR-664b-3p) showed significant differential expression in AD brains versus controls, in accordance with the change direction of lead exposure. The miRNAs' gene targets were validated for expression in the human brain and were found enriched in AD-relevant pathways such as axon guidance. Moreover, we identified several AD relevant transcription factors such as CREB1 associated with the identified miRNAs. These findings suggest that the identified miRNAs are involved in the development of AD and might be useful in the development of new, less invasive biomarkers for monitoring of novel therapies or of processes involved in AD development. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS), the most prevalent motor neuron disease characterized by its complex genetic structure, lacks a single diagnostic test capable of providing a conclusive diagnosis. In order to demonstrate the potential for genetic diagnosis and shed light on the pathogenic role of miRNAs in ALS, we developed an ALS diagnostic rule by training the model using 80% of a miRNA profiling dataset consisting of 253 ALS samples and 103 control samples. Subsequently, we validated the diagnostic rule using the remaining 20% of unseen samples. The diagnostic rule we developed includes miR-205-5p, miR-206, miR-376a-5p, miR-412-5p, miR-3927-3p, miR-4701-3p, miR-6763-5p, and miR-6801-3p. Remarkably, the rule achieved an 82% true positive rate and a 73% true negative rate when predicting the unseen samples. Furthermore, the identified miRNAs target 21 genes in the PI3K-Akt pathway and 27 genes in the ALS pathway, including notable genes such as BCL2, NEFH, and OPTN. We propose that miRNA profiling may serve as a complementary diagnostic tool to supplement the clinical presentation and aid in the early recognition of ALS., (© 2023. Springer Nature Limited.)

There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases. However, their detection using massive parallel sequencing methods still has important limitations. In addition, the relative importance and dynamics of somatic variation in healthy tissues are not fully understood. We performed high-depth whole-exome sequencing in 16 samples from patients with a previously determined pathogenic somatic variant for a primary immunodeficiency and tested different variant callers detection ability. Subsequently, we explored the load of somatic variants in the whole blood of these individuals and validated it by amplicon-based deep sequencing. Variant callers allowing low frequency read thresholds were able to detect most of the variants, even at very low frequencies in the tissue. The genetic load of somatic coding variants detectable in whole blood is low, ranging from 1 to 2 variants in our dataset, except for one case with 17 variants compatible with clonal haematopoiesis under genetic drift. Because of the ability we demonstrated to detect this type of genetic variation, and its relevant role in disorders such as primary immunodeficiencies, we suggest considering this model of gene mosaicism in future genetic studies and considering revisiting previous massive parallel sequencing data in patients with negative results. [ABSTRACT FROM AUTHOR]

Neuroinflammation and accompanying microglial dysfunction are now appreciated to be involved in Alzheimer’s disease (AD) pathogenesis. Critical to the process of neuroinflammation are the type-I interferon (IFN) family of cytokines. Efforts to phenotypically characterize microglia within AD identify distinct populations associated with type-I IFN signalling, yet how this affects underlying microglial function is yet to be fully elucidated. Here we demonstrate that Aβ1–42 exposure increases bioactive levels of type-I IFN produced by primary microglia alongside increased expression of type-I IFN related genes. Primary microglia isolated from brains of APPswePS1ΔE9 mice with ablated type-I IFN signalling show an increased phagocytic ability to uptake FITC-Aβ1–42. Correlative assessment of plaque sizes in aged APPswePS1ΔE9 mice with abrogated type-I IFN signalling show unchanged deposition levels. Microglia from these mice did however show alterations in morphology. This data further highlights the role of type-I IFN signalling within microglia and identifies a role in phagocytosis. As such, targeting both microglial and global type-I IFN signalling presents as a novel therapeutic strategy for AD management. [ABSTRACT FROM AUTHOR]

Hepatitis B is one of most etiologies of Liver cirrhosis in China, and clinically lacks the effective strategy for Hepatitis B caused cirrhosis (HBC) therapy. As a complementary and alternative medicine, Chinese Traditional Medicine (TCM) has special therapeutic effects for HBC. Here, we focus on the evolution process of HBC TCM syndromes, which was from Excessive (Liver-Gallbladder Dampness-Heat Syndrome, LGDHS) to Deficient (Liver-Kidney Deficiency Syndrome, LKYDS) via Excessive-Deficient syndrome (Liver-Depression and Spleen-Deficiency Syndrome, LDSDS). Using R package, 16 miRNAs in LGDHS/Normal, 48 miRNAs in LDSDS/LGDHS, and 16 miRNAs in LKYDS/LDSDS were identified, respectively. The miRNA-target networks show that the LDSDS was most stability and complicated. Subsequently, 4 kernel miRNAs with LGDHS-LDSDS process, and 5 kernel miRNAs with LDSDS-LKYDS process were screened. Using RT-qPCR data, p1 (hsa-miR-17-3p, -377-3p, -410-3p and -495) and p2 miRNA panel (hsa-miR-377-3p, -410-3p, -27a-3p, 149-5p and 940) were identified by Logistic Regression Model, which clearly improve the accuracy of TCM syndrome classification. The rebuilt miRNA-target network shows that the LDSDS is a critical point and might determine the evolution directions of HBC TCM syndrome. This study suggests that the identified kernel miRNAs act as potential biomarkers and benefit to evaluate the evolution tendency of HBC TCM syndromes. [ABSTRACT FROM AUTHOR]

Neuronal activity can modify Alzheimer's disease pathology. Overexcitation of neurons can facilitate disease progression whereas the induction of cortical gamma oscillations can reduce amyloid load and improve cognitive functions in mouse models. Although previous studies have induced cortical gamma oscillations by either optogenetic activation of cortical parvalbumin-positive (PV+) neurons or sensory stimuli, it is still unclear whether other approaches to induce gamma oscillations can also be beneficial. Here we show that optogenetic activation of PV+ neurons in the basal forebrain (BF) increases amyloid burden, rather than reducing it. We applied 40 Hz optical stimulation in the BF by expressing channelrhodopsin-2 (ChR2) in PV+ neurons of 5xFAD mice. After 1-h induction of cortical gamma oscillations over three days, we observed the increase in the concentration of amyloid-β42 in the frontal cortical region, but not amyloid-β40. Amyloid plaques were accumulated more in the medial prefrontal cortex and the septal nuclei, both of which are targets of BF PV+ neurons. These results suggest that beneficial effects of cortical gamma oscillations on Alzheimer's disease pathology can depend on the induction mechanisms of cortical gamma oscillations. [ABSTRACT FROM AUTHOR]

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms. [ABSTRACT FROM AUTHOR]