bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating circular RNA‐mediated neurotoxicity in tauopathies
Authors: Atrian Farzaneh , Ramirez Paulino , Frost Bess
Published: December 2021

Background

Circular RNAs (circRNA) are a subclass of non‐coding RNAs with a covalently closed loop structure that are formed via non‐canonical splicing1. CircRNAs are specifically enriched in the brain among different species and further accumulate in the brain as a function of physiological aging2. CircRNA biogenesis is regulated by N6‐methyladenosine (m6A), a post transcriptional RNA modification3. Elevated levels of circRNAs and abnormal m6A modification significantly correlate with clinical diagnosis and development of Alzheimer’s disease, respectively4, 5. Alzheimer’s disease and related “tauopathies” are pathologically defined by various forms of tau aggregates in the brains of affected individuals. We have previously reported that tau‐induced dysfunction of lamin, a nuclear scaffold protein, causes nuclear polymorphisms including invaginations and blebs 6, 7. We have also found that polyadenylated RNA accumulates within nuclear invaginations in the context of tauopathy and that genetic and pharmacologic reduction of RNA export reduce RNA accumulation within invaginations and suppresses tau neurotoxicity8. While dysfunction of the nuclear envelope and consequent aberrant RNA export mediate tau‐induced neurotoxicity, the identity of RNAs that accumulate within nuclear blebs, the role of m6A in circRNA accumulation, and their relationship with aging and tauopathy are currently unknown. Based on the association between nuclear polymorphism and RNA export, alongside the global enrichment of circRNAs and disrupted RNA methylation in Alzheimer’s disease brains, I hypothesize that m6A‐dependent accumulation of circRNAs in tauopathies sequester complementary RNAs and RNA binding proteins into large inclusions that trigger RNA export via nuclear blebbing.

Methods

RNA‐seq, digital PCR, TUNEL, electron microscopy and immunofluorescence.

Results

I find that circRNAs accumulate in brains of a Drosophila model of tauopathy and that RNAi‐mediated reduction of mbl, which is particularly enriched in its circular form in the brain, significantly suppresses tau‐induced neurotoxicity. RNAi‐mediated reduction of an m6A writer and reader significantly reduces circMbl biogenesis and neuronal death in tau transgenic Drosophila. I find that circMbl lines the nuclear blebs that contain large inclusions in brains of tau transgenic Drosophila.

Conclusions

Tau‐induced accumulation of circRNA is mediated by aberrant m6A modification. Presence of circMbl‐lined nuclear buds suggests a potential role of nuclear blebs in circRNA nuclear export.
The Effects of Melatonin on Cognition and Αβ load in an Alzheimer’s Disease Mouse Model
Authors: Miliotou Eleni , Riley‐DiPaolo Lexi , Stavnezer Amy Jo
Published: December 2021

Background

The neurohormone melatonin is released from the pineal gland and is an endogenous antioxidant and free radical scavenger that regulates sleep/wake cycle and circadian rhythm. Decline of nocturnal melatonin secretion with severe sleep disturbance has been consistently reported in Alzheimer’s Disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. However, exogenous melatonin supplementation can reduce Αβ neurotoxicity and formation while improving cognitive performance and sleep quality.

Method

In this study, the long‐term influence of melatonin on behavioral and neuropathological changes were evaluated in the 5xFAD mouse model, when treatment was started in adolescence. After ∼20 weeks of orally administered melatonin (10 mg/kg), Morris water maze behaviour was assessed in 54 mice, followed by Aβ42 quantification in hippocampal tissue.

Result

Overall, transgenic mice showed impaired learning, and a significant increase in Aβ42 plaque load, replicating previous results with this strain. Melatonin marginally improved learning by decreasing distance travelled in the Morris water maze and slightly decreasing Aβ42 concentration in the hippocampus. These results support the hypothesis that long‐term melatonin supplementation may be beneficial at early stages of the disease process and should be explored more fully.

Conclusion

Abnormal accumulation of neurotoxic forms of Αβ occur in brain regions involved in the regulation of sleep in AD patients and the disturbed regulation of sleep that occurs early in the disease may contribute to cognitive decline by decreasing Αβ clearance. In contrast to conventional antioxidants, melatonin crosses the blood‐brain barrier, is relatively devoid of toxicity, and constitutes a straight‐forward potential therapeutic candidate in AD treatment.
Identification of Akt activators for the treatment of Alzheimer’s disease: An In‐silico approach
Authors: Kumar B Harish , Prasada K Shama , Pai Sreedhar Ranganth
Published: December 2021

Background

In Alzheimer’s disease (AD), Aβ directly downregulates the activity of the Akt pathway by decreasing insulin receptor, IGF, IGFR and PI3K levels leading to reduced phosphorylation of Akt. The diminished inhibitory influence of Akt results in the deranged activity of GSK‐3β, Forkhead, BAD, mTOR pathways, which leads to increased tau phosphorylation, oxidative stress, and cell death. Many researchers have proven that activating the Akt pathway can ameliorate cognitive defects like learning and memory in AD and protect neurons from cell death.

Method

Maestro platform from Schrodinger Inc. was used for shape‐based screening by using already know activators like SC79, inositol‐(1,3,4,5)‐tetrakisphosphate, Baicalin, Puerarin, and Chlorogenic acid as a template to screen Asinex and enamine CNS databases. Later, pharmacophore models like receptor‐ligand complex (1UNQ PBD) pharmacophore, Receptor cavity‐based pharmacophore, and multiple ligand‐based pharmacophores were built and used to screen the same databases. Lastly, the Fingerprint‐based 2D QSAR model was built using known active ligands and inactive ligands. Seventeen QSAR models which predicted the activity without error were used to screen databases. Ligands that came out positive in a maximum number of all models were selected and then docked with PH domain of the Akt1, which is the only PDB(1UNQ) with PH domain in an active conformation.

Result

Due to the lack of crystal structure of Akt1 protein in active conformation, ligand‐based approach was used. After all the approaches mentioned in the methodology, we selected Asinex CNS ‐275 and Enamine CNS‐76 lead molecules and docked them with PH domain of Akt1. After docking, each molecule pose was analyzed and molecules that interact with the critical residues like Arg 23, Arg 25, Lys 14, Arg 86, Asn 53 essential for activation were selected. Using all these approaches from the library of 2,00,000 compounds, we have narrowed it down to 13 lead molecules.

Conclusion

Using an in silico‐based approach, we identified lead molecules that may act as Akt activators and help alleviate the harmful effect of tau hyperphosphorylation. However, an in vivo experiment is needed for supporting this claim.
Event‐Related Delta Oscillations are Diminished in Parkinson’s Disease with Different Cognitive Status: A Longitudinal EEG Study
Authors: Gündüz Duygu Hünerli , Çolakoğlu Beril Dönmez , Güntekin Bahar , Yener Gorsev
Published: December 2021

Background

The neurophysiological differences between Parkinson’s disease mild cognitive impairment (PD‐MCI) and cognitively normal Parkinson’s disease (PD‐CN) have been poorly understood, covering limited electrophysiological interpretation. Delta event‐related oscillations (ERO) are recognized as a useful electrophysiological indicator of cognitive decline (Güntekin et al., 2018; Yener et al., 2019). The aim of the present study was to investigate baseline and follow‐up delta EROs of individuals with PD‐CN and PD‐MCI in comparison with healthy controls (HC).

Methods

Thirty one PD‐CN, 23 PD‐MCI, and 30 demographically‐matched HC were included. All examinations were applied to participants in a time period of 12.50±0.80 months. A comprehensive neuropsychological test battery was used for the PD‐MCI classification (Litvan et al., 2012). EEGs were recorded during visual oddball paradigm. The maximum peak‐to‐peak event‐related delta (0.5‐3.5 Hz) responses of the groups to the target stimulus were measured. Repeated measures ANOVA included Group (3 levels: HC, PD‐CN, PD‐MCI) as a between‐subject factor, Time (2 levels: baseline, follow‐up), and Anterior‐Posterior Electrode Location [AP (6 levels: frontal, central, temporal, temporoparietal, parietal, occipital)] as a within‐subject factor.

Results

There was an interaction effect for Group x Time on event‐related delta responses [F(2,81)=3.650, p=0.030], indicating follow‐up delta EROs were reduced only in PD‐CN group (Figure 1). Further analyses revealed that there was an interaction effect for AP x Group on baseline delta responses [F(10,405)=11.629, p<0.001], and follow‐up delta [F(10,405)=9.569, p<0.001] responses. At baseline, PD‐MCI had reduced delta EROs in all electrode locations except occipital than both PD‐CN and HC, while PD‐CN had reduced delta responses than HC over frontal and central areas (for all; p<0.030) (Figure 2). At follow‐up, higher delta responses were detected in HC compared to both PD‐CN and PD‐MCI over all locations except occipital area (for all; p<0.001) (Figure3).

Conclusions

The current study revealed that longitudinal delta ERO reduction was apparent only in PD‐CN group. Individuals with PD‐MCI demonstrated decline at both baseline and follow‐up assessments. These findings may be interpreted as diminished delta EROs can be a successful indicator of subtle cognitive alterations in PD‐CN before neuropsychological assessments.
Effect of Aerobic Exercise Training on Mood and Cognition in Adults at Risk for Alzheimer’s Disease
Authors: Brach Talia L. , Gaitán Julian M. , Okonkwo Ozioma C.
Published: December 2021

Background

Aerobic exercise is positively associated with mood and cognition, but its effect on protection against decline in mood and cognition in late‐middle aged adults at risk for Alzheimers disease (AD) is unknown.

Method

23 late‐middle aged adults from a cohort enriched with family history of AD and the APOE4 allele were randomized into Usual or Enhanced Physical Activity (PA) groups. Enhanced PA completed a progressive 26‐week supervised aerobic exercise program at 70‐80% heart rate reserve for 50 minutes, 3 times per week to achieve or exceed public health guidelines. Usual PA only received materials regarding active lifestyle. The Profile of Mood States (POMS) assessed total mood disturbance (TMD), characterized by fatigue, depression, tension, confusion, anger, and vigor. The California Verbal Learning Test II (CVLT‐II) measured verbal learning and memory via the total, short, and long delayed recall, and learning slope. The Delis‐Kaplan Executive Function System (DKEFS) assessed executive function via the trail‐making, color‐word interference (CWI), verbal fluency, and tower subtests. Graded treadmill exercise testing assessed cardiorespiratory fitness (CRF), and triaxial accelerometry measured moderate‐to‐vigorous physical activity (MVPA). Independent samples t‐tests between groups and Pearson’s correlations were conducted on baseline to post‐intervention change scores.

Result

Fatigue decreased significantly in Enhanced PA compared to Usual PA (‐1.18 ± 4.05 vs 2.92 ± 2.68, p= .009), and the decreases in TMD (‐7.64 ± 17.78 vs 6.00 ± 16.42, p=.07) and tension (‐2.00 ± 4.41 vs 0.67 ± 2.39, p= .08) among Enhanced PA were nearly significant. Changes in other mood subcategories and in cognition were not different between groups (all p’s> .15), except improved executive function on the DKEFS CWI as previously published (Gaitán et al. 2019). Correlations of mood with verbal learning and memory or executive function were not significant (all r’s < .17, all p’s> .44). Correlations of mood or cognition with CRF or MVPA were not significant (p’s> .175), except between DKEFS CWI and CRF as previously published (Gaitán et al. 2019).

Conclusion

Aerobic exercise training may protect against fatigue in adults at risk for AD. However, change in mood was not associated with cognitive changes in this cohort.
Cellular proteomic profiling using proximity labeling by TurboID in microglial and neuronal cell lines
Authors: Sunna Sydney N , Rayaprolu Sruti , Bowen Christine A , Bagchi Pritha , Seyfried Nicholas T. , Rangaraju Srikant
Published: December 2021

Background

Despite advancements in systems‐level analyses to Alzheimer's disease, global proteomic profiling of brain homogenate cannot resolve molecular changes occurring in distinct cell types. Proximity labeling of proteins by the biotin‐ligase TurboID, coupled with mass spectrometry (MS) analyses of biotinylated proteins, can resolve cell‐type specific proteomes from brain without cell type enrichment. Before in‐vivo applications, it is important to identify proteins preferentially labeled by TurboID in mammalian cells, and directly compare biotinylated proteomes with whole cell proteomes. We determined whether TurboID‐labeled proteomes in microglial (BV2) and neuronal (N2A) cell lines are representative of total cellular proteomes under homeostatic and activated conditions.

Method

We generated N2A and BV2 cell lines that stably express TurboID fused to a nuclear export sequence. Cells incubated in 200μM biotin and 1μg/mL lipopolysaccharide (LPS) for 48hrs to mimic inflammatory stress. Western blotting and label‐free quantitation MS identified biotinylated proteins in whole‐cell‐lysates and streptavidin‐enriched preparations.

Result

Western‐blot confirmed robust biotinylation in TurboID N2A and BV2 cell‐lines. LFQ‐MS quantified >3,000 proteins in inputs and >2,400 proteins in biotinylated proteomes from both cell types. In BV2 and N2A inputs, TurboID expression and biotinylation didn't impact the proteome, evidenced by few differentially‐expressed proteins and absence of cellular respiration measures in mitochondrial stress‐tests, comparing TurboID and non‐TurboID cells. Of all the proteins quantified in BV2 and N2A TurboID inputs, ∼50% were also quantified in the biotinylated proteomes in both cell types. Compared to input proteomes of both cell types, the biotinylated proteome was enriched in vesicular, ribosomal, mitochondrial, cytoplasmic and cytoskeletal proteins. The biotinylated proteome successfully differentiated BV2 and N2A cells. LPS stimulation altered levels of 759 proteins in BV2 cells in the input proteome, of which the top increased (e.g. Irg1, Oasl1, Cyb5a) and decreased (e.g. Mrc1, Mgl2, Arg1) proteins showed concordant changes in the biotinylated BV2 proteome.

Conclusion

Our analyses show that proteomic labeling by TurboID in microglial and neuronal cell lines doesn't disrupt cellular function. The resulting biotinylated proteomes are reasonably representative of the whole‐cell proteome and captures the most robust alterations induced by immune stimuli in microglia. These foundational data will guide future applications of proximity‐labeling for cell‐type‐specific proteomics in‐vivo.
Alcohol use disorder and mild cognitive impairment: Shared neural signatures during an episodic memory task
Authors: Barlis Katie L , Bustos Nicholas , Warner Graham , Nowling Duncan , Cosentino Emily , Benitez Andreana , Flanagan Julianne , Mintzer Jacobo E , Joseph Jane E
Published: December 2021

Background

Alcohol Use Disorder (AUD) has previously been identified as a risk factor for Alzheimer’s disease and cognitive impairment. However, the mechanisms underlying the connection between AUD and Alzheimer’s disease remain unclear. Episodic memory is impaired in both AUD and mild cognitive impairment (MCI), and is associated with the transition from MCI to Alzheimer’s disease. This study used functional magnetic resonance imaging (fMRI) to compare activation during encoding of an episodic memory task in mid‐life AUD subjects to subjects with mild cognitive impairment (MCI), a prodromal stage of Alzheimer’s disease.

Method

Participants included 14 AUD subjects age 45 to 65 (M = 56 years), and 14 MCI subjects age 45 to 85 (M = 67 years). Healthy control subjects were matched to AUD and MCI groups by age and sex. Images were acquired on a 3T Siemens Prisma (3 mm3 voxels, TR = 1.1s, TE = 30 ms). During the fMRI task, participants viewed pictures of faces and objects and were instructed to remember these pictures for later. Activation maps were produced through general linear model analysis using cluster correction thresholding (Z>3.3, p = .05) in FSL (version 6.0).

Results

AUD and MCI groups showed reduced extent of BOLD activation during encoding versus baseline (Figure 1). Compared to matched controls, AUD and MCI subjects showed less medial temporal lobe (amygdala), brainstem, and thalamus activation. Additionally, AUD subjects did not exhibit the frontal activation that was present in MCI and matched controls.

Conclusions

Given the small sample sizes, caution should be taken in interpreting these results. Nonetheless, these preliminary findings suggest that mid‐life AUD subjects show similar patterns of reduced activation during episodic encoding to those with MCI. Reduced medial temporal lobe activation may represent vulnerability to Alzheimer’s disease, which is present in both MCI and AUD. Explanations for the differences in frontal activation might include compensatory mechanisms in MCI or executive dysfunction in AUD. Future analyses will aim to disentangle these effects by examining the relationship between BOLD signal activation and behavioral outcomes.
Starvation and activity dependent modulation of salt taste behavior in Drosophila
Authors: Kaushik Shivam , Limas Diego E Rincon , Kain Pinky
Published: December 2021

Background

Sodium present in NaCl is a fundamental nutrient required for many physiological processes but high salt consumption in western world is contributing health risk to all age individuals. Although high salt detection pathways have been studied in great detail, the mechanisms that regulate high salt consumption in animals are largely unknown. To understand how pre‐exposure to high NaCl diet modulates subsequent feeding behavior, we looked into the neural mechanisms of high NaCl consumption in adult Drosophila.

Method

We used Neuro‐Genetics, imaging and behavioral assays to determine how flies respond to high NaCl exposure.

Result

We studied the neural mechanism by which flies modify their acceptance of high salt as a function of diet, where a long‐term high‐salt exposure increases taste sensitivities of pharyngeal LSO (Labral sense organ) neurons and enhances high salt intake. We discovered that exposing flies to high NaCl diet (200mM NaCl in fly food) for three days modify their feeding responses to high levels of salt. High NaCl fed flies show decline in high salt aversion under starvation. Genetic suppression of LSO pharyngeal neurons in high NaCl fed flies inhibits excessive salt intake. We found that this modulation requires functional LSO neurons and starvation state, and that multiple independent taste receptor neurons and pathways are involved in this process. Silencing any one of multiple LSO neuronal types inhibits excessive salt intake.

Conclusion

Our data support the idea that high dietary salt modulates and reshapes salt and other taste curves to promote over consumption of food in flies. Our study suggest flies can adapt to the amount of salt ingested over several days, indicating the presence of a critical mechanism to reset the salt appetite and related neural circuits. Identification of new molecular sensors for salt and related neural controls such as hormones, neuropeptides, and neurotransmitters may yield insights into the coordination of processes in the nervous system.
Racial considerations of age and white matter hyperintensities
Authors: Misiura Maria , Igwe Kay C , Wharton Whitney , Verble Danielle D , Turner Jessica A , Dotson Vonetta , Brickman Adam M. , Hu William T
Published: December 2021

Background

White matter hyperintensities (WMH) are regions that appear very bright on Fluid‐Attenuated Inversion Recovery (FLAIR) magnetic resonance images (MRI). They are generally attributed to cerebrovascular disease (CVD) and confer a greater risk for Alzheimer’s Disease (AD). Black individuals (BIs) typically have greater WMH burden than their white counterparts and have a two‐fold greater risk of developing AD in their lifetimes. For this project, we wanted to determine whether WMH were related to diagnostic category, and if this relationship differed according to race.

Method

Cohort 1 included middle‐aged cognitively normal black and white individuals (N=50). Cohort 2 included older black and white individuals on the dementia continuum (NC=47, MCI=10, AD=3). Data collection included a neuropsychological battery and magnetic resonance imaging (MRI). Fluid attenuated inversion recovery (FLAIR) MRI data were analyzed to obtain lobe specific WMH volumes (frontal, temporal, occipital, parietal, cerebellar). Because of age and diagnostic differences, we analyzed each cohort separately. We constructed multivariate linear models for each cohort with WMH volumes as the outcome variable, age, race, gender, and diagnostic category (Cohort 2 only) as independent variables. We included age X race, and race X diagnosis (Cohort 2 only) interaction terms.

Result

There were no racial or diagnostic differences in regional WMH volume. However, in Cohort 1, there was a significant race X age interaction in the parietal lobe such that in BIs, as age increased, WMH volume also increased (B=, t(3,46)=2.07, p =0.04). In Cohort 2, this relationship extended to the temporal (B=0.09, t(3,55)=2.95, p=0.04), and occipital lobes (B=0.09, t(3,55)=2.99, p=0.04). There was no such relationship observed in WIs.

Conclusion

Age conferred greater WMH volume in BIs than in WIs in cognitively normal individuals and individuals on the dementia spectrum. The relationship we observed between WMH and AD could be seen as additive rather than causative, as we observed only a relationship between age and WMH volume, not diagnostic category. The differential findings across race suggest that age has a greater effect on BIs on CVD and brain health. These findings, although not causal, should encourage AD researchers to collect more diverse samples, and consider underlying CVD in data analyses.
Investigating the mechanisms of brain innate immune activation following bacterial infection in Drosophila
Authors: Mainali Sameekshya , Harper Luren , Bolling Jordan , Berry Kaitlin , Kitchens Elizabeth , Chtarbanova Stanislava
Published: December 2021

Background

Central Nervous System (CNS) infections caused by bacteria and resulting immune responses could underlie neurodegenerative diseases such as Alzheimer’s disease (AD). Neuroinflammation is a well‐known component of AD furthering disease pathology and progression. However, the mechanisms by which infectious agents contribute to neuroinflammatory reactions and neurodegeneration are not fully understood. The model organism Drosophila melanogaster shares conserved mechanisms of innate immune activation and neural development with mammals, and thus represents an excellent system for the investigation of pathogen‐associated neurodegeneration. Previous work in flies has shown that bacterial brain injection results in age‐dependent neurodegeneration and impaired locomotor activity. Furthermore, these phenotypes depend on NF‐κB in both glia and neurons, suggesting that long‐term inflammatory processes take place in both cell types. However, a detailed picture of innate immune pathway activation in the Drosophila brain following bacterial infection is lacking. Here, we investigate the activation in the brain of the two Drosophila NF‐ κB pathways, Toll and Immune deficiency (IMD), in response to infection with Gram‐positive and Gram‐negative bacteria, respectively.

Method

Fly brains are infected by pricking heads with a thin, metal needle, dipped into concentrated pellets of overnight cultured bacterial solutions. Activation of NF‐κB pathways in brain cell types is assayed using immunohistochemistry and confocal microscopy of flies carrying fluorescent NF‐κB reporters. The dependence of canonical NF‐κB pathway components is assayed using quantitative reverse‐transcription PCR (RT‐qPCR) using RNA isolated from dissected brains of control‐injected or bacterially injected flies.

Result

Our preliminary results show that brain bacterial injection leads to the activation of GFP reporters for both Toll and IMD pathways in multiple glial subtypes in the fly brain 24h following infection. We have optimized a protocol for brain‐specific gene expression of NF‐κB targets and are currently examining the response to bacterial challenge of multiple mutants for components of the Toll and IMD pathways.

Conclusion

We have established a Drosophila model of bacterial brain infection that allows studying the long‐term consequences of pathogen‐mediated neuroinflammatory reactions associated with neurodegeneration. Our system could be used to identify the processes of immediate protection as well as the long‐term effects of brain infection.
Astroglial ER protein Membralin is an essential neuroinflammation regulator
Authors: Jiang LuLin , Li Xiaoguang , Myers Stephanie , Huang Timothy , Kaufman Randal J.
Published: December 2021

Background

The endoplasmic reticulum (ER) is the primary organelle for synthesizing membrane proteins, secretory proteins, and lipids. Disturbance of ER homeostasis in peripheral immune cells is associated with inflammatory responses. Glial cells, including astrocytes and microglia, are the immune cells in the brains, becoming reactive/gliosis during the progression of Alzheimer’s disease (AD). However, whether and how glial protein homeostasis mediate the reactive state of glial cells is yet to be investigated. We previously identified a novel ER‐associated degradation component, membralin/TMEM259, as an important mediator in neurodegeneration. Loss of membralin impaired the turnover of nicastrin protein, which increased gamma‐secretase complex formation and activity. Knockdown membralin in the TgCRND8‐AD mice exaggerated beta‐amyloid‐associated neuronal damage. Moreover, selective deletion of membralin in astrocytes decreased excitatory amino acid transporter 2 (EAAT2) expression and induced excitotoxicity. Elevating membralin levels in SOD1 G93A
ALS mouse model can significantly extend the lifespan of the animals.

Method

Transcriptomic profiles of the cortex tissues and histology were analyzed in the astrocyte‐conditional membralin knockout animals. Primary astrocyte ER morphology was examined by electron microscopy analysis. We crossed the transgenic membralin animals with 5xFAD animals and assessed the AD pathology.

Result

Astroglial membralin knockout animals develop strong neuroinflammation phenotypes. Loss of membralin in astrocytes disrupts ER homeostasis and nuclear envelop integrity, and induces senescence‐like phenotypes. In contrast, the elevation of membralin in astrocytes can alleviate the induction of C3+ astrocytes upon c1q/IL1a/TNFa treatment. Further, elevating membralin levels in 5XFAD mouse model can significantly reduce the AD pathologies.

Conclusion

Membralin is critical in mediating the reactive states of astrocytes. Modulation of astroglial ER homeostasis can be a promising target to regulate neuroinflammation for AD therapy.
Investigating epigenetic loci implicated in Alzheimer’s disease via the CRISPR‐Cas9 system
Authors: Schrauben Michael , Smith Adam R. , Washer Sam , Dempster Emma , Lunnon Katie
Published: December 2021

Background

Differentially methylated positions (DMPs) identified by Epigenome‐wide association studies (EWAS) of Alzheimer’s disease (AD) were modified by the CRISPR‐Cas9 system to investigate the role of epigenetic alterations in AD pathogenesis.

Method

Cell lines of neuronal phenotype (SH‐SY5Y) and microglial phenotype (IHM‐SV40) were used to explore the functional consequence of loci demethylation, where removal of the methyl groups was achieved by the modified CRISPR‐dCas9 system. Lentiviral delivery of the dCas9‐TET1CD demethylase tool and guide RNA (gRNA) constructs targeting the CpG sites associated with differential expression of ANK1 and BIN1 in AD was validated by fluorescence‐activated cell sorting (FACS). Bisulphite pyrosequencing was applied to confirm the DNA methylation edit.

Result

High transduction efficiencies were observed during FACS of SH‐SY5Y cells transduced with CRISPR‐dCas9 constructs targeting the BIN1 locus and IHM‐SV40 cells with constructs against the ANK1 locus. Methylation analysis of these target regions in the modified cell lines demonstrated a reduction in methylation when compared to the untreated control cells. This modification was maintained over a three‐week period.

Conclusion

Delivery and activity of our CRISPR‐dCas9 fusion constructs was demonstrated in cell line models of AD. In the future, we intend to profile the epigenome and transcriptome of these modified cell lines to identify any off‐target effects of the CRISPR‐dCas9 system and determine differences in mRNA transcript variant levels of our target genes.
Assessment of genetic modulators in cerebrospinal fluid levels of YKL‐40, and C‐reactive protein: An association with Alzheimer disease
Authors: Parveen Kayenat
Published: December 2021

Background

Alzheimer's disease (AD) is one of the most complex progressive neurological diseases that cause memory loss and cognitive deterioration owing to the death of brain cells. Some potential biomarkers have been discovered in cerebrospinal fluid (CSF) which could help in the prediction of AD. These biomarkers are either associated with amyloid pathology or tau pathology. But some of the biomarkers are also associated with neuro‐Inflammation, which is the third potential event associated with AD pathogenesis[i]. The levels of YKL‐40 and C‐Reactive Protein(CRP) in CSF are the biomarkers, which are found to be associated with AD[ii]. In this study, we are investigating more about the roles of these biomarkers in AD pathogenesis.

Method

The analysis was performed on the DELCODE dataset (n=311). The findings were validated in ADNI(n=277) and requested the independent cohort ACE (n=543) to contribute to our study in meta‐analysis. The Z‐score is produced and utilized as a phenotype with sex, age, 3 Principal Component Analysis, and diagnosis as covariates in linear regression. Furtheron, we used DELCODE, ADNI, and ACE summary statistics to run random and fixed‐effect models for the meta‐analysis.

Result

Following the study, we discovered the genetic variants of YKL‐40, and CRP with genome‐wide significant P‐values (5 x 10‐8) and evidence of connection in all datasets. In these biomarkers, however, including diagnosis as a covariate plays important role. All of the biomarkers' lambda values indicate that there is no inflation in the analysis(λ = 1.0). There was no discernible difference in age or gender. According to the meta‐analysis the genome‐wide significant loci linked with YKL‐40 appear to be in the region of chr1 and chr3; and for CRP in chr6 and chr19.

Conclusion

Our findings imply that the important SNPs linked to these biomarkers are found in protein‐coding areas and are connected with inflammation. In the CSF of people with AD, levels of YKL40, and CRP are higher than in healthy people. Innate immune responses and neuro‐inflammatory signaling pathways are both affected by these proteins. Taken together, these findings emphasize the importance of comprehending the complexities of AD.
Cerebrospinal fluid levels of fatty acid binding protein 3 associate with brain amyloidosis in cognitively healthy individuals at risk of developing Alzheimer’s disease
Authors: Dhiman Kunal , Martins Ralph N , Gupta Veer Bala
Published: December 2021

Background

An accurate diagnosis of preclinical Alzheimer’s disease (AD) can be made by developing classifier algorithms that include biomarkers commensurate with different pathological changes. Amyloid positivity forms the key criterion of identifying cognitively healthy individuals having either preclinical AD pathological change or preclinical AD. Therefore, biomarkers that not only associate with a specific pathophysiology, but also with brain amyloidosis should be included in diagnostic classifiers. Here, we assessed the association of baseline levels of cerebrospinal fluid (CSF) fatty acid binding protein 3 (FABP3) – a biomarker of neurodegeneration associated with disrupted lipid metabolism, with brain amyloidosis at baseline, as well as change in amyloidosis, in cognitively healthy individuals.

Method

Fatty acid binding protein 3 levels were measured in CSF samples of 142 cognitively healthy participants from the Australian Imaging, Biomarkers and Lifestyle study of ageing (AIBL), using a commercial electrochemiluminescence immunoassay on a Meso Scale Discovery platform. Participants were aged >60 years, and their brain amyloid‐β (Aβ) load was assessed via positron emission tomography (PET) using different tracers.

Result

Association of CSF FABP3 levels with baseline brain Aβ load as measured by standardized uptake value ratio (SUVR), as well as change in Aβ load at follow‐up was assessed using linear regression analyses. The average follow‐up time interval was 1.7 years. FABP3 levels were positively associated with baseline brain Aβ load as measured by SUVR (standardised β = 0.22, p = 0.009) and also with the change in brain Aβ load i.e., change in SUVR at follow‐up (standardised β = 0.32, p = 0.004).

Conclusion

Results indicate that changes in amyloid pathology align with changes in lipid metabolism. Since FABP3 is a biomarker of neurodegeneration, indicative of disrupted lipid metabolism and associated with amyloidosis, it can be used to facilitate screening or diagnosis of preclinical AD.
Data from single nuclei RNA‐sequencing reveals a prodromal gene network response in excitatory neurons of a humanized rat Alzheimer’s disease model
Authors: Tipton Allison E , George Jonique , Ratner Marcia , Farb David , Russek Shelley
Published: December 2021

Background

Despite significant investment into treatments for AD, little progress has been made in bringing new therapeutics to market. Recent failure of monoclonal antibodies targeting Ab plaques to slow disease progression in phase 3 clinical trials, suggests now more than ever it is imperative to identify early disease signatures before symptom onset whose modification may alter disease trajectory. Our collaborating lab recently identified early dysfunction in the transgenic tri‐synaptic hippocampal circuit using in vivo recordings during awake immobility in the TgF344‐AD rat model (doi: 10.1101/2021.05.10.443512). This dysfunction emerges between 6‐9 months, prior to when TgF344‐AD rat shows overt pathology (including Ab plaques, frank neuronal loss, and endogenous tau pathology) at 16 and 26 mo, but not at 6 mo (Towne, 2013).

Methods

The molecular basis of this trisynpatic circuit dysfunction was explored using 10x Genomics’ v3 gene expression assay to perform snRNA‐seq on freshly dissected hippocampi from 6 mo male and female littermate pairs of Tg and Wt rats (n=16). ∼2000 cells/subject were collected, and cDNA libraries were sequenced (NovaSeq 600) to a depth of ∼120k reads/nuclei. Cell Ranger Pipeline demultiplexed and aligned reads to rat pre‐mRNA reference and generated gene count matrices. A final dataset of 33,079 high quality nuclei were clustered (Louvain graph‐based), and cell‐type specific marke used to determine identity. Identity was collapsed into broad neuronal and glial cell types for differential gene expression (DEG) across genotype (Hurdle Model).

Results

DEGs revealed profound alterations between genotype in cell types important in the trisynaptic circuit, including dentate granule cells (DGCs), and excitatory neurons (ExcNs). (FDR cutoff=0.05, 1111 (DGCs) and 1070 (ExcN’s) DEGs between genotypes). Pathway enrichment revealed dysregulation relevant to memory function: including decreased synaptogenesis, neurite branching, proliferation, and microtubule and cytoskeleton dynamics. DEG’s were enriched for neurological signs including epilepsy, seizures, hyperactivity, and dementia.

Conclusion

Prior to development of overt pathology or memory deficits, TgF344‐AD rats display transcriptomic alterations in their excitatory neurons, suggestive of impaired ability to form new connections and susceptibility to aberrant, hyperactivity. Electrophysiological detection, followed by intervention targeting these early alterations in gene expression may prevent progression of disease to a symptomatic stage.
The short term continuous darkness affects brain and behavior in a sex‐dependent manner: A comparative study in C57BL/6 male and female mice
Authors: Singh Dhyanendra
Published: December 2021

Background

Standard light‐dark cycle (12h light and 12h dark) has an imperative role in keeping experimental animals healthy and adaptive. If it gets perturbed, it can negatively impact the brain, behavior, and Physiology. The circadian clock (central clock) of hypothalamic suprachiasmatic nuclei received light signals from the environment. Subsequently, in collaboration with the peripheral clocks of the tissues, the central clock transformed these light signals into oscillations in behavior and physiology to maintain homeostasis in coping with environmental fluctuations or stressors. To understand how light‐dark cycle disruption affects the brain and, as a result, behavior, it's critical to simultaneously look at the molecular and behavioral changes. Further, sex is one of the inevitable variables neglected in neurobiology and behavioral study even after the advancement of science in the 20th century

Method

In the current study, we evaluated the influence of the altered light‐dark cycle on the brain (prefrontal cortex), stress response, and behavior of C57BL/6 male and female mice by exposing them to the continuous darkness (0h light and 24h dark) for three weeks

Result

Our results show that constant darkness leads to the change in behavior (in the open field test), the prefrontal cortex (pro‐ and anti‐inflammatory genes and neurotrophic factors), and stress response (stress hormones) in a sex‐dependent manner

Conclusion

The present study laid the foundation for understanding how the altered light‐dark cycle perturbed behavior, brain, and stress response in a sex‐dependent way.
Intra‐ and inter‐rater reliability of brain's activation area measurement on imagej: An easy and low‐cost method for cognitive and neurophysiological studies
Authors: Monteiro‐Junior Renato Sobral , Santos Iaggo Raphael David , Machado Frederico Sander ,   Research and Study Group on Neuroscience, Exercise, Health and Sport ‐ GENESEs
Published: December 2021

Background

Neuroimage is an important resource in cognitive neuroscience1 but its analysis is complex and requires reliable and affordable procedures. The present study sought to determine the intra‐ and inter‐rater reliability of brain´s activation area of neuroimages obtained derived from an integrated EEG system equipment.

Method

The study follows the Declaration of Helsinki. Fifteen cerebral cortex images were obtained through Neurometry v6 (Bioevolution®, Brazilian National Health Surveillance Agency registration 81403519002)2,3 from 15 individuals above 60 years, who consented to participate of the study. This device consists of a 19‐channel EEG (10‐20 system). Amplitude of brain waves (delta, theta, alpha, beta, and gamma) are automatically processed, filtered and analyzed with Math Library MtxVec, LAPACK and Math Kernel BLAS with codes Streaming SIMD (SSE2, SSE3 and SSE4)2. The neuroimages display different colors according to the amplitude of brain waves: brown/blue (low activation), green/yellow (moderate activation), and orange/red (high activation)2. Images retrieved were exported and analyzed with the ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) in 3 phases: 1st An experienced researcher (RSMJ) standardized Broadmann´s area as the region of interest (ROI); 2nd The researcher specified the brain´s activation area in accordance with colored regions (brown, blue, green, yellow, orange, and red) in the Broadmann´s area 10 (BA10) covering 1,391 pixels. Colors were covered using 2 ImageJ tools (polygon and freehand selections). In order to calculate the colored area of interest, pixels quantification was performed with a 400% magnification filter (Figure 1). The procedure was repeated 24h later for the intra‐rater reliability; 3rd An inexperienced assessor (IRDS) independently performed the same procedure of the 2nd phase. Intraclass Correlation Coefficients (ICC3,1 and ICC3,k)4 were calculated to determine the intra‐ and inter‐rater reproducibility. Typical Error of Measurement (TEM=SD∑difference between measures÷√2)5 was also calculated to quantify typical deviations from the right measure.

Results

Intra‐ and inter‐rater reliability were excellent (ICC=0.99, p<0.01) and good (ICC=0.82, p<0.01), respectively (Table 1). TEM varied from 4 to 17%.

Conclusion

Quantification of the brain´s activation area of neuroimages derived from Neurometry v6 using the ImageJ ROI is highly reliable, independently of the assessor´s experience.
Evaluation and optimization of different cf‐RNA extraction methods for Alzheimer‐related genes detection
Authors: Le Hoang Anh P , Tran Tai T , Cao Minh Thien H , Le Mai Thao T , Pham Bao H , Ha Huong T T
Published: December 2021

Background

Cell‐free RNA (cf‐RNA) in plasma has become a fascinating less‐invasive diagnostic approach for Alzheimer’s Disease (AD) since brain‐originated mRNA and microRNA can enter the bloodstream through the blood‐brain barrier. Studying cf‐RNA expression in blood samples collected from AD‐patients and at‐risks patients allows further understanding of disease pathogenesis, contributes to the development of early diagnosis and prognosis methods. However, contaminants risks, low yield, and integrity can alter final cf‐RNA quality and concentration, challenging their application. This study aims to evaluate and optimize different cf‐RNA extraction protocols, as well as to detect the AD‐related transcripts in the final sample.

Method

We assessed different cf‐RNA extraction methods and evaluated hemolysis ‐ the main contamination threat to the final product. Blood samples were collected from healthy volunteers into designated collection tubes to compare their effects on hemolysis and RNA products. Plasma cf‐RNA was extracted using the Trizol and Zymo cf‐RNA kits, with two modifications to the Trizol protocol: addition of glycogen and with the use of Rneasy mini kit to increase cf‐RNA yield. Among 30 blood samples were collected, 12 were treated with Zymo, six with Trizol, eight with Trizol w/glycogen and four with Rneasy. cf‐RNA was then converted to cDNA and examined with PCR and qPCR.

Result

Norgen tubes showed a lower hemolysis level compared to Streck tubes. The yield of cf‐RNA from the Zymo kit was about 5‐18 µg/ml. The Trizol protocol resulted in almost no detectable cf‐RNA. The addition of glycogen significantly increased the yield to a comparable level with the Zymo kit (4‐15 µg/ml). No genomic DNA contamination was detected. Despite high cf‐RNA yield from the modified Trizol protocol, the housekeeping gene Beta‐actin (ACTB) or other AD‐related transcripts (CNTF and MAG) could not be detected. Using cDNA obtained from the Zymo kit, ACTB was reliably detected, but AD‐related transcripts’ results varied. cDNA concentration was high‐correlated with the melt curve analysis of ACTB qPCR, suggesting Zymo kit obtained cDNA had high integrity.

Conclusion

Overall, the Zymo kit is superior to the Trizol‐based protocols in both cf‐RNA yield and integrity. The final sample showed detectable levels of ACTB and AD‐related transcripts.
Dissecting a neuron‐to‐liver crosstalk to modulate lipid metabolism in lisosomal storage diseases
Authors: Garcia‐Macia Marina , Bolaños Juan Pedro
Published: December 2021

Background

Neuronal ceroid lipofuscinoses (NCL), known as Batten disease, are the most common of the rare neurodegenerative disorders in children. To date, defects in thirteen different genes have been identified in NCL patients. Despite the genetic heterogeneity, Batten diseases are grouped together based on clinical similarities and broadly uniform neuropathological features, including accumulation of lipofuscin in lysosomes, as well as profound neurodegeneration and widespread gliosis. Amongst these, the incidence of Cln7 disease, caused by mutation in MFSD8 gene, is the highest in southern and Mediterranean Europe. CLN7/MFSD8 encodes a lysosomal membrane glycoprotein with unknown function. Lysosomes are the only organelles able to hydrolyse triacylglycerols, which fuels the mitochondria for energy generation. The autophagic machinery provides TGs to the lysosomes through a process known as lipophagy. Although defective autophagy has been related with Cln7 disease, Cln7 role in lipid metabolism is unknown, particularly in lipophagy. Here, we hypothesized that disruption of lipophagy links neuronal death and Cln7‐mediated Batten disease.

Method

To address this, we have studied lipophagy in Cln7 knockout (Cln7‐KO) mice and investigated whether Cln7 loss in the hypothalamus disrupts liver lipophagy. We have obtained experimental data from different techniques in Cln7‐KO mice fibroblasts, liver, brown fat and brain, to stablish a connection between the brain and the metabolism of the peripheral tissues.

Result

Thus, our data show that Cln7 deficiency in the hypothalamus damages liver lipophagy resulting in fat accumulation. Ongoing work is being developed to validate these observations using robust metabolic and in vivo uncoupling approaches.

Conclusion

Cln7 deficiency causes neuronal damages which seem to produce lipophagy impairment in the periphery tissues.
In Vitro and In Vivo Pharmacological Comparison of Mu‐Opioid Receptor Activity of the Kratom (Mitragyna speciosa) Alkaloid Mitragynine and its Metabolite 7‐Hydroxymitragynine
Authors: Calvache Maria P Guerrero , Obeng Samuel , Leon Francisco , Gamez‐Jimenez Lea R , Patel Avi , Ho Nicholas P , Crowley Morgan L , Pallares Victoria , Mottinelli Marco , McCurdy Christopher R , McMahon Lance R , Hiranita Takato
Published: December 2021

Background

The µ‐opioid receptor (MOR) pharmacology of mitragynine and its 7‐hydroxy metabolite have not been fully established.

Method

For both we assessed binding affinity and efficacy in vitro, as well as discriminative stimulus and antinociceptive effects in rats.

Result

Both mitragynine (MG) and 7‐hydroxymitragynine (7‐MG) had higher binding affinities at human MOR (Ki values 77.9 and 709 nM, respectively) than at the human k‐ (KOR) or d‐opioid receptors (DOR). MG was identified as a MOR antagonist while 7‐MG was identified as a MOR partial agonist (Emax=41%) using the [35S]GTPγS functional assay. In Sprague Dawley rats trained to discriminate 3.2 mg/kg morphine from vehicle (i.p.), MG produced a maximum of 72% morphine‐lever responding. Whereas morphine produced a maximum of 65% MG‐lever responding in a separate group of rats trained to discriminate 32 mg/kg MG from vehicle (i.p.), other MOR agonists (i.p.) produced high percentages of morphine‐lever and MG‐lever responding (respectively): 7‐MG (100% and 98%), fentanyl (100% and 80%), buprenorphine (100% and 79%), and nalbuphine (99% and 98%). MG (up to 56 mg/kg, i.p.) did not produce significant antinociception, whereas 7‐MG (17.8 mg/kg, i.p.) produced robust antinociception in the hotplate assay with the temperature set at 52 °C. Naltrexone (0.032 mg/kg, i.p.) antagonized the discriminative stimulus, rate‐decreasing, and antinociceptive effects of morphine and 7‐MG. Conversly, naltrexone (0.032 mg/kg, i.p.) antagonized the discriminative stimulus effects of MG, but not its rate‐decreasing effects. Pretreatment with MG (5.6 mg/kg, i.p.) potentiated the discriminative‐stimulus effects of morphine but antagonized the antinociceptive effects of morphine.

Conclusion

Striking differences in MOR efficacy in vitro are consistent with 7‐MG having greater MOR efficacy than MG in vivo. MG exerts both MOR antagonist and agonist activity depending on the MOR efficacy required.