Cholinergic Surveillance over Hippocampal RNA Metabolism and Alzheimer's-Like Pathology.

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aß1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.

Long non-coding RNA and alternative splicing modulations in Parkinson's leukocytes identified by RNA sequencing.

The continuously prolonged human lifespan is accompanied by increase in neurodegenerative diseases incidence, calling for the development of inexpensive blood-based diagnostics. Analyzing blood cell transcripts by RNA-Seq is a robust means to identify novel biomarkers that rapidly becomes a commonplace. However, there is lack of tools to discover novel exons, junctions and splicing events and to precisely and sensitively assess differential splicing through RNA-Seq data analysis and across RNA-Seq platforms. Here, we present a new and comprehensive computational workflow for whole-transcriptome RNA-Seq analysis, using an updated version of the software AltAnalyze, to identify both known and novel high-confidence alternative splicing events, and to integrate them with both protein-domains and microRNA binding annotations. We applied the novel workflow on RNA-Seq data from Parkinson's disease (PD) patients' leukocytes pre- and post- Deep Brain Stimulation (DBS) treatment and compared to healthy controls. Disease-mediated changes included decreased usage of alternative promoters and N-termini, 5'-end variations and mutually-exclusive exons. The PD regulated FUS and HNRNP A/B included prion-like domains regulated regions. We also present here a workflow to identify and analyze long non-coding RNAs (lncRNAs) via RNA-Seq data. We identified reduced lncRNA expression and selective PD-induced changes in 13 of over 6,000 detected leukocyte lncRNAs, four of which were inversely altered post-DBS. These included the U1 spliceosomal lncRNA and RP11-462G22.1, each entailing sequence complementarity to numerous microRNAs. Analysis of RNA-Seq from PD and unaffected controls brains revealed over 7,000 brain-expressed lncRNAs, of which 3,495 were co-expressed in the leukocytes including U1, which showed both leukocyte and brain increases. Furthermore, qRT-PCR validations confirmed these co-increases in PD leukocytes and two brain regions, the amygdala and substantia-nigra, compared to controls. This novel workflow allows deep multi-level inspection of RNA-Seq datasets and provides a comprehensive new resource for understanding disease transcriptome modifications in PD and other neurodegenerative diseases.

Deep brain stimulation modulates nonsense-mediated RNA decay in Parkinson's patients leukocytes.

BACKGROUND: Nonsense-Mediated decay (NMD) selectively degrades mRNA transcripts that carry premature stop codons. NMD is often triggered by alternative splicing (AS) modifications introducing such codons. NMD plays an important regulatory role in brain neurons, but the in vivo dynamics of AS and NMD changes in neurological diseases and under treatment were scarcely explored. RESULTS: Here, we report exon arrays analysis of leukocyte mRNA AS events prior to and following Deep Brain Stimulation (DBS) neurosurgery, which efficiently improves the motor symptoms of Parkinson's disease (PD), the leading movement disorder, and is increasingly applied to treat other diseases. We also analyzed publicly available exon array dataset of whole blood cells from mixed early and advanced PD patients. Our in-house exon array dataset of leukocyte transcripts was derived from advanced PD patients' pre- and post-DBS stimulation and matched healthy control volunteers. The mixed cohort exhibited 146 AS changes in 136 transcripts compared to controls, including 9 NMD protein-level assessed events. In comparison, PD patients from our advanced cohort differed from healthy controls by 319 AS events in 280 transcripts, assessed as inducing 27 protein-level NMD events. DBS stimulation induced 254 AS events in 229 genes as compared to the pre-DBS state including 44 NMD inductions. A short, one hour electrical stimulus cessation caused 234 AS changes in 125 genes compared to ON-stimulus state, 22 of these were assessed for NMD. Functional analysis highlighted disease-induced DNA damage and inflammatory control and its reversal under ON and OFF stimulus as well as alternative splicing in all the tested states. CONCLUSIONS: The study findings indicate a potential role for NMD both in PD and following electrical brain stimulation. Furthermore, our current observations entail future implications for developing therapies for PD, and for interfering with the impaired molecular mechanisms that underlie PD and other neurodegenerative and neurological disorders, as well as DBS-treatable conditions in general.

Single-cell RNA sequencing analysis and Alzheimer's disease: a bibliometric analysis.

Alzheimer's disease (AD) is a devastative disease, the 1st most frequent neurodegenerative disease worldwide. Its prevalence is increasing and early detection methods as well as potential genomic based therapeutics are urgently needed. OBJECTIVES: To better characterize recent seq studies of AD and site recent relevant literature. Using single-cell RNA sequencing, the characteristics of neuronal cell populations in Alzheimer's disease (AD) have not been completely elucidated. METHODS: We conducted a dynamic and longitudinal bibliometric analysis to investigate existing studies on Single-cell RNA sequencing analysis and Alzheimer's Disease and identify data gaps and possible new research avenues. RESULTS: All AD papers concentrating on Single-cell RNA sequencing analysis were found using the search terms "Alzheimer's Disease", and "Single-cell RNA sequencing analysis" in the PubMed/MEDLINE database. Only English publications published between 2015 and 2023 were chosen using filters. CONCLUSIONS: Original English-language research publications disclosing Single-cell RNA sequencing analysis and Alzheimer's Disease were examined for inclusion. Two sets of independent reviewers discovered and extracted pertinent data. The bibliometric study was carried out using the R software packages Bibliometrix and Biblioshiny. The narrowed search yielded 158 publications, all published between 2015 and 2023. Yet, after applying filters and considering the inclusion requirements, the search results comprise just 51 original articles out of 158 articles. There were 107 articles eliminated. The importance of the discovery of Single-cell RNA sequencing analysis and Alzheimer's Disease a decade ago only grows with time. Our results have important implications for future studies of AD and may help researchers across the world better understand the global context of the Single-cell RNA sequencing analysis and Alzheimer's Disease link. This study puts emphasis on the critical need for more diverse participant demographics in Alzheimer's disease investigations.

Single-cell RNA sequencing analysis of human Alzheimer's disease brain samples reveals neuronal and glial specific cells differential expression.

Alzheimer's disease is the most common neurological disease worldwide. Unfortunately, there are currently no effective treatment methods nor early detection methods. Furthermore, the disease underlying molecular mechanisms are poorly understood. Global bulk gene expression profiling suggested that the disease is governed by diverse transcriptional regulatory networks. Thus, to identify distinct transcriptional networks impacted into distinct neuronal populations in Alzheimer, we surveyed gene expression differences in over 25,000 single-nuclei collected from the brains of two Alzheimer's in disease patients in Braak stage I and II and age- and gender-matched controls hippocampal brain samples. APOE status was not measured for this study samples (as well as CERAD and THAL scores). Our bioinformatic analysis identified discrete glial, immune, neuronal and vascular cell populations spanning Alzheimer's disease and controls. Astrocytes and microglia displayed the greatest transcriptomic impacts, with the induction of both shared and distinct gene programs.

Age-related telomere attrition in the human putamen.

Age is a major risk factor for neurodegenerative diseases. Shortening of leucocyte telomeres with advancing age, arguably a measure of "biological" age, is a known phenomenon and epidemiologically correlated with age-related disease. The main mechanism of telomere shortening is cell division, rendering telomere length in post-mitotic cells presumably stable. Longitudinal measurement of human brain telomere length is not feasible, and cross-sectional cortical brain samples so far indicated no attrition with age. Hence, age-related changes in telomere length in the brain and the association between telomere length and neurodegenerative diseases remain unknown. Here, we demonstrate that mean telomere length in the putamen, a part of the basal ganglia, physiologically shortens with age, like leukocyte telomeres. This was achieved by using matched brain and leukocyte-rich spleen samples from 98 post-mortem healthy human donors. Using spleen telomeres as a reference, we further found that mean telomere length was brain region-specific, as telomeres in the putamen were significantly shorter than in the cerebellum. Expression analyses of genes involved in telomere length regulation and oxidative phosphorylation revealed that both region- and age-dependent expression pattern corresponded with region-dependent telomere length dynamics. Collectively, our results indicate that mean telomere length in the human putamen physiologically shortens with advancing age and that both local and temporal gene expression dynamics correlate with this, pointing at a potential mechanism for the selective, age-related vulnerability of the nigro-striatal network.

Deep brain stimulation induces rapidly reversible transcript changes in Parkinson's leucocytes.

Subthalamic deep brain stimulation (DBS) reversibly modulates Parkinson's disease (PD) motor symptoms, providing an unusual opportunity to compare leucocyte transcripts in the same individuals before and after neurosurgery and 1 hr after stimulus cessation (ON- and OFF-stimulus). Here, we report DBS-induced reversibility and OFF-stimulus restoration in 12 of 16 molecular functions and 3 of 4 biological processes shown in exon microarrays to be differentially expressed between PD patients and controls, post-DBS from pre-DBS and OFF from ON states. Intriguingly, 6 of 18 inflammation and immune-related functions exhibited reversibility, and the extent of stimulus-induced changes correlated with the neurological DBS efficacy, suggesting mechanistic implications. A minimal list of 29 transcripts that changed in all three comparisons between states discriminated pre-surgery and OFF states from post-surgery and controls. Six of these transcripts were found to be able to distinguish between PD patients and both healthy controls and patients with other neurological diseases in a previously published whole blood 3' array data study of early PD patients. Our findings support the future use of this approach for identifying targets for therapeutic intervention and assessing the efficacy of current and new treatments in this and other neurological diseases.

Meta-analysis of genetic and environmental Parkinson's disease models reveals a common role of mitochondrial protection pathways.

Both genetic and environmental factors trigger risks of and protection from Parkinson's disease, the second most common neurodegenerative syndrome, but possible inter-relationships between these risk and protection processes were not yet explored. By examining gene expression changes in the brains of mice under multiple treatments that increase or attenuate PD symptoms we detected underlying disease and protection-associated genes and pathways. In search for potential links between these different genes and pathways, we conducted meta-analysis on 131 brain region transcriptomes from mice over-expressing native or mutated α-synuclein (SNCA) with or without the protective HSP70 chaperone, or exposed to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), with or without the protective acetylcholinesterase (AChE-R) variant. All these models showed shared risk-inducible and protection-suppressible transcript modifications. Self-organized map (SOM) classification revealed risk- and protection-associated alterations in nuclear and mitochondrial metal ion-regulated transcripts, respectively; Gene Ontology based analysis validated these pathways. To complement this approach, and identify potential outcome damages, we further searched for shared functional enrichments in the lists of genes detected in young SNCA mutant or in old SNCA mutants and MPTP-exposed mice. This post-hoc functional analysis identified early-onset changes in Parkinsonian, immune and alternative splicing pathways which shifted into late-onset or exposure-associated NFkB-mediated neuro-inflammation. Our study suggests metal ions-mediated cross-talk between nuclear and mitochondrial pathways by both environmental and genetic risk and protective factors involved in Parkinson's disease, which eventually culminates in neuro-inflammation. Together, these findings offer new insights and novel targets for therapeutic interference with the gene-environment interactions underlying sporadic PD.

Exon arrays reveal alternative splicing aberrations in Parkinson's disease leukocytes.

BACKGROUND: Parkinson's disease (PD) is the second most frequent neurodegenerative disease worldwide. Clinical diagnosis can only be made when the vast majority of the dopaminergic cell population has died. However, the cause(s) for sporadic PD is/are yet unclear. Transcript changes have recently been described in PD patients' whole blood cells, but corresponding splicing patterns remained unknown. OBJECTIVE: To search for alternative splicing aberrations in PD patients' blood leukocytes. METHODS: We applied exon microarrays to profile PD patients' blood leukocyte mRNA. Exon level splicing analysis served as a basis for downstream classification and functional analyses. RESULTS: Patients and carefully matched controls were classified by the splicing exon profiles of their leukocyte transcripts. Specifically, many exons were downregulated in PD patients compared to controls. Functional analysis highlighted aberrant splicing of PD-related transcripts and impaired NF-κB cascade and immune response. CONCLUSION: PD patient's blood leukocytes exhibit alternative splicing of numerous transcripts. The aberrant alternative splicing in PD patients' blood cells has potential implications for early diagnosis and future therapeutics.

Adaptive alternative splicing correlates with less environmental risk of parkinsonism.

BACKGROUND/OBJECTIVE: Environmental exposure to anti-acetylcholinesterases (AChEs) aggravates the risk of Parkinsonism due to currently unclear mechanism(s). We explored the possibility that the brain's capacity to induce a widespread adaptive alternative splicing response to such exposure may be involved. METHODS: Following exposure to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), brain region transcriptome profiles were tested. RESULTS: Changes in transcript profiles, alternative splicing patterns and splicing-related gene categories were identified. Engineered mice over-expressing the protective AChE-R splice variant showed less total changes but more splicing-related ones than hypersensitive AChE-S over-expressors with similarly increased hydrolytic activities. Following MPTP exposure, the substantia nigra and prefrontal cortex (PFC) of both strains showed a nuclear increase in the splicing factor ASF/SF2 protein. Furthermore, intravenous injection with highly purified recombinant human AChE-R changed transcript profiles in the striatum. CONCLUSIONS: Our findings are compatible with the working hypothesis that inherited or acquired alternative splicing deficits may promote parkinsonism, and we propose adaptive alternative splicing as a strategy for attenuating its progression.

Serum cholinesterase activities distinguish between stroke patients and controls and predict 12-month mortality.

To date there is no diagnostic biomarker for mild stroke, although elevation of inflammatory biomarkers has been reported at early stages. Previous studies implicated acetylcholinesterase (AChE) involvement in stroke, and circulating AChE activity reflects inflammatory response, since acetylcholine suppresses inflammation. Therefore, carriers of polymorphisms that modify cholinergic activity should be particularly susceptible to inflammatory damage. Our study sought diagnostic values of AChE and Cholinergic Status (CS, the total capacity for acetylcholine hydrolysis) in suspected stroke patients. For this purpose, serum cholinesterase activities, butyrylcholinesterase-K genotype and inflammatory biomarkers were determined in 264 ischemic stroke patients and matched controls during the acute phase. AChE activities were lower (P<0.001), and butyrylcholinesterase activities were higher in patients than in controls (P=0.004). When normalized to sampling time from stroke occurrence, both cholinergic parameters were correlated with multiple inflammatory biomarkers, including fibrinogen, interleukin-6 and C-reactive protein (r=0.713, r=0.607; r=0.421, r=0.341; r=0.276, r=0.255; respectively; all P values<0.001). Furthermore, very low AChE activities predicted subsequent nonsurvival (P=0.036). Also, carriers of the unstable butyrylcholinesterase-K variant were more abundant among patients than controls, and showed reduced activity (P<0.001). Importantly, a cholinergic score combining the two cholinesterase activities discriminated between 94.3% matched pairs of patients and controls, compared with only 75% for inflammatory measures. Our findings present the power of circulation cholinesterase measurements as useful early diagnostic tools for the occurrence of stroke. Importantly, these were considerably more distinctive than the inflammatory biomarkers, albeit closely associated with them, which may open new venues for stroke diagnosis and treatment.

Advanced microarray analysis highlights modified neuro-immune signaling in nucleated blood cells from Parkinson's disease patients.

Laboratory tests for Parkinson's disease (PD) were recently extended to microarray analyses of nucleated blood cells. Here, we report the use of statistical and gene ontology tools to re-examine these microarray data. Distribution plots and PCA mapping enabled removal of several outliers out of the 105 analyzed PD and control samples, which improved the discriminative power for PD blood cells compared to healthy and neurological disease controls. Combined with gene ontology tests, our findings point at neuro-immune signaling-related transcripts as distinctly expressed early in PD progress and call for exploiting microarray tests also for follow-up of PD treatment efficacy.

Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in mice.

Microglia, the resident immune cell of the brain, can be eliminated via pharmacological inhibition of the colony-stimulating factor 1 receptor (CSF1R). Withdrawal of CSF1R inhibition then stimulates microglial repopulation, effectively replacing the microglial compartment. In the aged brain, microglia take on a "primed" phenotype and studies indicate that this coincides with age-related cognitive decline. Here, we investigated the effects of replacing the aged microglial compartment with new microglia using CSF1R inhibitor-induced microglial repopulation. With 28 days of repopulation, replacement of resident microglia in aged mice (24 months) improved spatial memory and restored physical microglial tissue characteristics (cell densities and morphologies) to those found in young adult animals (4 months). However, inflammation-related gene expression was not broadly altered with repopulation nor the response to immune challenges. Instead, microglial repopulation resulted in a reversal of age-related changes in neuronal gene expression, including expression of genes associated with actin cytoskeleton remodeling and synaptogenesis. Age-related changes in hippocampal neuronal complexity were reversed with both microglial elimination and repopulation, while microglial elimination increased both neurogenesis and dendritic spine densities. These changes were accompanied by a full rescue of age-induced deficits in long-term potentiation with microglial repopulation. Thus, several key aspects of the aged brain can be reversed by acute noninvasive replacement of microglia.

Major Shifts in Glial Regional Identity Are a Transcriptional Hallmark of Human Brain Aging.

Gene expression studies suggest that aging of the human brain is determined by a complex interplay of molecular events, although both its region- and cell-type-specific consequences remain poorly understood. Here, we extensively characterized aging-altered gene expression changes across ten human brain regions from 480 individuals ranging in age from 16 to 106 years. We show that astrocyte- and oligodendrocyte-specific genes, but not neuron-specific genes, shift their regional expression patterns upon aging, particularly in the hippocampus and substantia nigra, while the expression of microglia- and endothelial-specific genes increase in all brain regions. In line with these changes, high-resolution immunohistochemistry demonstrated decreased numbers of oligodendrocytes and of neuronal subpopulations in the aging brain cortex. Finally, glial-specific genes predict age with greater precision than neuron-specific genes, thus highlighting the need for greater mechanistic understanding of neuron-glia interactions in aging and late-life diseases.

Correction: Gene Expression Differences in Peripheral Blood of Parkinson's Disease Patients with Distinct Progression Profiles.

[This corrects the article DOI: 10.1371/journal.pone.0157852.].

Math1 target genes are enriched with evolutionarily conserved clustered E-box binding sites.

The basic helix-loop-helix (bHLH) transcription factor Math1 and its orthologs are fundamental for proper development of various neuronal subpopulations, such as cerebellar granule cells, D1 interneurons in the spinal cord, and inner ear hair cells. Although crucial for neurogenesis, the mechanisms by which Math1 specifically recognizes its direct targets are not fully understood. To search for direct and indirect target genes and signaling pathways controlled by Math1, we analyzed the effect of Math1 knockout on the expression profile of multiple genes in the embryonic cerebellum. Eighteen differentially expressed transcripts were identified and found to belong to a few developmentally-related functional groups, such as transcriptional regulation, proliferation, organogenesis, signal transduction, and apoptosis. Importantly, genomic analysis of E-box motifs has identified a significant enrichment and clustering of MATH1-binding E-boxes only in a subset of differentially expressed genes (Nr2f6, Hras1, and Hes5) in both mouse and man. Moreover, Math1 was shown by chromatin immunoprecipitation (ChIP) to bind, and by a luciferase reporter assay to activate transcription, of an upstream genomic fragment of Nr2f6. Taken together, we propose that when putative direct targets of Math1 are being selected for detailed studies on DNA microarray hybridization, the enrichment and clustering of binding E-boxes in multiple species may be helpful criteria. Our findings may be useful to the study of other bHLH transcription factors, many of which control the development of the nervous system.

Transcriptome profiling in Parkinson's leukocytes: from early diagnostics to neuroimmune therapeutic prospects.

Parkinson's disease (PD) involves motor symptoms reflecting the progressive degeneration of dopaminergic neurons in the substantia nigra. However, diagnosis is only enabled late in the disease, limiting treatment to palliative assistance. Here, we review recently generated transcriptional profiling datasets from blood and brain RNA of human PD cohorts and animal models that may offer unprecedented progress in PD research. Specifically, advanced analysis techniques demonstrated functionally inter-related underlying impairments of RNA metabolism and neuroimmune signalling processes. Identifying novel biomarkers in serum and nucleated blood cells, including protein networks and non-coding RNAs can drive discovery of the molecular mechanisms involved and reveal new targets for therapeutic intervention, posing a dual diagnosis/treatment opportunity for limiting the exacerbation of neuroinflammatory events in PD.

Gene Expression Differences in Peripheral Blood of Parkinson's Disease Patients with Distinct Progression Profiles.

The prognosis of neurodegenerative disorders is clinically challenging due to the inexistence of established biomarkers for predicting disease progression. Here, we performed an exploratory cross-sectional, case-control study aimed at determining whether gene expression differences in peripheral blood may be used as a signature of Parkinson's disease (PD) progression, thereby shedding light into potential molecular mechanisms underlying disease development. We compared transcriptional profiles in the blood from 34 PD patients who developed postural instability within ten years with those of 33 patients who did not develop postural instability within this time frame. Our study identified >200 differentially expressed genes between the two groups. The expression of several of the genes identified was previously found deregulated in animal models of PD and in PD patients. Relevant genes were selected for validation by real-time PCR in a subset of patients. The genes validated were linked to nucleic acid metabolism, mitochondria, immune response and intracellular-transport. Interestingly, we also found deregulation of these genes in a dopaminergic cell model of PD, a simple paradigm that can now be used to further dissect the role of these molecular players on dopaminergic cell loss. Altogether, our study provides preliminary evidence that expression changes in specific groups of genes and pathways, detected in peripheral blood samples, may be correlated with differential PD progression. Our exploratory study suggests that peripheral gene expression profiling may prove valuable for assisting in prediction of PD prognosis, and identifies novel culprits possibly involved in dopaminergic cell death. Given the exploratory nature of our study, further investigations using independent, well-characterized cohorts will be essential in order to validate our candidates as predictors of PD prognosis and to definitively confirm the value of gene expression analysis in aiding patient stratification and therapeutic intervention.